Novel compounds useful in the treatment of cardiovascular diseases

Abstract

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

Description

Technical Field

[0001] This invention provides novel compounds and pharmaceutical compositions thereof for the treatment of cardiovascular diseases, as well as methods of using them.

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. nonprovisional patent application No. 16 / 600,476, filed October 12, 2019, which is incorporated herein by reference in its entirety.

[0004] This application claims the benefit of U.S. Nonprovisional Patent Application No. 16 / 429,015, filed June 2, 2019, which is incorporated herein by reference in its entirety.

[0005] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 755,516, filed November 4, 2018, which is incorporated herein by reference in its entirety. Background Technology

[0006] According to the 2019 American Heart Association statistics on heart disease and stroke (Benjamin et al., 2019), cardiovascular disease is the leading cause of death in the United States. These diseases were found in 48% of the 121 million U.S. citizens, or adult population, aged 20 and older. All of these cardiovascular diseases are associated with cellular hypoxia. Cellular hypoxia is defined as a relative inadequacy of oxygen availability or utilization in living cells compared to normal physiological conditions.

[0007] By 2035, the annual cost of cardiovascular disease in the United States will reach $1.1 trillion. Despite significant progress in the management of cardiovascular disease, these same diseases remain the leading cause of death in the United States and must also be considered a global problem. In 2016, approximately 17.6 million people worldwide died from cardiovascular disease, a 14.5% increase from 2006.

[0008] Healthcare professionals are somewhat proud of the significant decline in cardiovascular disease mortality rates in the United States in recent years. However, those who once died acutely from these diseases are now chronically ill patients requiring long-term management. The challenge for society as a whole is to design low-cost management strategies to address this growing pandemic.

[0009] When oxygen supply is scarce, cellular metabolism, and therefore life itself, is jeopardized. Living cells respond to changes in the amount of available environmental oxygen with extremely sensitive mechanisms that reprogram pathways of gene expression. The nature of the response is specific to each cell's condition and environment. Hypoxia-inducible factor (HIF) has been found in organisms ranging from primitive worms to humans; everywhere, oxygen delivery is a crucial variable in biological life, and there are currently many pharmaceutical development programs dedicated to altering the physiology of HIF (Semenza, 2019). HIF provides us with important insights into a key component of hypoxia-mediated gene expression, but the scope of human cellular responses to oxygen extends beyond the regulation of HIF expression.

[0010] Hypoxia is associated with inadequate perfusion and is noted in cardiovascular disease and other conditions related to reduced blood flow to perfused organs. Reperfusion is associated with the return of blood flow. Reperfusion is generally desirable, but prolonged local ischemia and subsequent blood return can lead to local inflammation and cell damage due to the toxic byproducts of oxidative metabolism. There is a way to mitigate this process. Repeated, transient hypoperfusion and reperfusion can increase tolerance to future ischemic events. This phenomenon is known as conditioning. There has been a strong search for effective treatments for the adverse consequences of non-fatal oxygen delivery and severe perfusion reduction, and current treatment options are limited.

[0011] Most compounds currently developed clinically for managing hypoxia-related gene expression inhibit prolyl hydroxylase. Prolyl hydroxylase causes the targeted degradation of hypoxia-inducible factors (HIFs), therefore prolyl hydroxylase inhibitors prevent HIF degradation and actually enhance HIF-mediated events. Prolyl hydroxylase inhibitors are compounds that enhance cellular adaptive responses to hypoxia on a systemic basis via HIF. HIF enhancement by prolyl hydroxylase inhibitors can induce the kidneys to synthesize additional erythropoietin, an endogenous molecule that stimulates erythrocyte development for therapeutic purposes in the case of anemia. HIF enhancement also leads to angiogenesis, resulting in more blood vessels growing into hypoxic areas, which may be beneficial in cases such as coronary artery disease leading to myocardial ischemia and related cardiac dysfunction. Disadvantageously, the same holistic approach can simultaneously increase arterial supply to dormant malignancies and / or alter cellular metabolism to conserve oxygen in the presence of oxygen. The pathophysiological complexity of hypoxia-related disease states is easily underestimated. To develop safe and effective medicines, highly selective approaches to modulate hypoxia-mediated events will be needed. This will involve delivery within a limited, organ-specific space or triggering activity under appropriate pathological conditions.

[0012] Increased PGE2 production was achieved by transactivating NF-κB p65, HMG I(Y), and Sp1 in cultured human umbilical vein endothelial cells via hypoxia-induced transcriptional regulation of cyclooxygenase-2 (COX-2) expression. (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 insufficient to drive human adaptation to hypoxia, and that NF-κB is another important mediator of hypoxia-driven gene transactivation in the vascular endothelium.

[0013] Ischemic cardiovascular disease is related to insufficient blood flow and also to hypoxia. Ischemic preconditioning is part of the therapeutic effect attributed to the compounds of this invention. Hypoxia increases endothelial expression of COX-2. COX-2, as a chaperone in nitric oxide (NO) synthesis, is crucial for ischemic preconditioning (Li et al., 2007). Both are necessary to achieve the cardioprotective effect conferred later in ischemic preconditioning (Guo et al., 2012).

[0014] The SLC14 (solute carrier 14) family of urea transporter genes regulates the transmembrane transport of urea. UT-B (urea transporter B, a product of the SLC14A1 gene) promotes the transmembrane transport of urea, water, and urea analogs. (Shayakul, Clemencon, & Hediger, 2013) UT-B is widely expressed, including in the heart, vascular endothelium, and erythrocytes. UT-B-deficient mice exhibit 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 the equilibrium is slow, and UT-B promotes rapid urea excretion from erythrocytes. (Sands, 1999) Urea is generally considered a waste product, and it carries nitrogen from amino acid breakdown to the recycling process. Renal failure is associated with decreased nitric oxide synthase (NOS) activity. However, NOS activity is not reduced in rats with normal renal function when BUN levels rise to uremia levels. (Xiao, Erdely, Wagner, & Baylis, 2001) Urea may have cardioprotective properties in certain situations. (Wang et al., 1999)

[0015] Membrane UT-B is abundant in human vascular endothelium from cultures derived from various locations and appears to be involved in the regulation of nitric oxide (NO) synthesis (Wagner, Klein, Sands, & Baylis, 2002). UT-B in the vascular endothelium is normally used to remove urea from the cell membrane. Inhibition of UT-B in the vascular endothelium leads to the accumulation of urea within the cell. This is believed to result in feedback inhibition of arginase (the enzyme that converts L-arginine to urea), which enhances the activity and expression of the L-arginine substitution pathway, in this case, endothelial NOS (eNOS) that enables increased NO production (Sun et al., 2016). Interestingly, it illustrates how the presence of urea enhances NO production.

[0016] Hypoxia increases UT-B expression in hypoxic vascular endothelium. The inventors discovered that the messenger RNA of the UT-B gene (SLC14A1) was significantly upregulated in human vascular endothelial cells under hypoxic (1% oxygen) cell cultures. Under these conditions, the upregulation of UT-B expression draws urea from endothelial cells and removes the feedback inhibition of arginase that causes increased eNOS activity, thus reducing the net vasodilatory effect of nitrate administration. A source of urea or urea analogues should maintain effective intracellular urea substrate levels to overcome UT-B upregulation and enable L-arginine to participate in eNOS production of NO, thereby increasing vasodilation in response to hypoxia and dilating adjacent vascular smooth muscle.

[0017] It would be beneficial to discover compounds that enhance vasodilatory release of NO under hypoxic conditions, while enabling ischemic preconditioning in a targeted (e.g., local) manner, thereby leading to the treatment and prevention of major adverse cardiac events in cardiovascular disease. Summary of the Invention

[0018] Therefore, in one aspect, the present invention provides novel compounds or pharmaceutically acceptable salts thereof that respond to hypoxic regions of cells.

[0019] 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.

[0020] In another aspect, the present invention provides a novel method for treating diseases mediated by cellular hypoxia, comprising: administering to a mammal in need 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 method for preparing at least one of the compounds of the present invention.

[0022] In another aspect, the present invention provides novel compounds or pharmaceutically acceptable salts for therapeutic purposes.

[0023] In another aspect, the present invention provides the use of novel compounds for the manufacture of medicaments for treating diseases mediated by cellular hypoxia.

[0024] These and other objectives have been achieved through the inventors’ discoveries, which will become apparent during the following detailed description. The inventors’ discoveries are intended to provide a therapeutic response focused on hypoxic regions of cells by the claimed compounds or pharmaceutically acceptable salts thereof. Detailed Implementation

[0025] All references cited in this article are incorporated herein by reference in their entirety.

[0026] In addition to urea or urea analogs (e.g., amides or hydroxyacetamides), the compounds of this invention are also designed to release nitrates at the endothelial surface. They are small molecules having a central isosorbide moiety attached to a urea moiety (Formulas I and IV), an amide moiety (Formulas II and V), or a hydroxyacetamide moiety (Formulas III and VI) and a relative nitrate group (-NO3). Ethanolamides offer the advantage of relatively high permeability across UT-B and low toxicity. (Zhao, Sonawane, Levin, & Yang, 2007) It is anticipated that the compounds of this invention will utilize the passive movement of urea and urea analogs across the cell membrane into vascular endothelial cells. Under hypoxic conditions in the vascular endothelium, the expression of the UT-B gene increases. UT-B-mediated transport (removal) of urea and urea analogs occurs under hypoxia, which appears to limit the activity of eNOS and NO synthesis. Therefore, intracellular urea substitution would enhance the effect of nitrates supplied to the vascular endothelium. It is expected that, under hypoxic conditions, the combination of urea and / or urea analogs with nitrates will promote a range of events, including vasodilation and ischemic preconditioning.

[0027] The compounds of this invention are characterized by their ability to be metabolized to NO via aldehyde dehydrogenase-2 or converted to nitrite and subsequently reduced by a series of enzymes, including xanthine oxidase, to form the nitrate group of NO. Alternatively, intracellular L-arginine is oxidized to NO by nitric oxide synthase. Historically, NO has been known as an endothelial-derived relaxant factor, which induces the formation of cyclic guanosine monophosphate (cGMP) by activating the NO receptor soluble guanylate cyclase. cGMP binds to and enhances the activity of protein kinase G, promoting calcium removal from smooth muscle cells, thereby reducing vascular tone because reduced intracellular calcium impairs the contractility of smooth muscle.

[0028] NO is also essential for ischemic preconditioning, although the exact intracellular mechanism of this effect is not as well understood as the vasodilatory mechanism. COX-2 expression is a necessary component of ischemic preconditioning, and COX-2 expression is induced by hypoxia via a mechanism dependent on the NF-κB transactivation system. Furthermore, as previously reported, endothelial nitric oxide synthase appears to be slightly downregulated when hypoxia induces COX-2 upregulation (Schmedtje, Jr. et al., 1997). Therefore, the mechanism of increasing NO production through COX-2 induction under hypoxic conditions has therapeutic value in the vascular endothelium.

[0029] Therefore, in one aspect, the compounds of the present invention will enhance the formation and release of vasodilatory NO, while enabling ischemic pretreatment in a targeted manner.

[0030] In another aspect, the compounds of the present invention provide more substrates to overcome the consequences of hypoxia-induced UT-B expression and the transport of associated urea and / or analogues out of the cell and across the cell membrane.

[0031] In another aspect, since the compounds of the present invention are not overall modulators of HIF- (hypoxia-inducible factor) and its downstream effects, their therapeutic response will be concentrated in the region 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, unlike therapeutic agents that nonselectively mimic the downstream effects of hypoxia and have side effects.

[0032] In another aspect, the present invention provides novel compounds of formulas I, II, III, IV, V and / or VI:

[0033]

[0034] in:

[0035] R 1 It does not exist;

[0036] Alternatively, R 1 Selected from (R) 1 The right-hand portion is attached to the isosorbide moiety: (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2C(=O)O, and CH2C(=O)NH; and,

[0037] R 2 Selected from (R) 2The right-hand portion 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;

[0038] Or its pharmaceutically acceptable salt.

[0039] In another respect, the compound is of formula I or IV, or a pharmaceutically acceptable salt thereof.

[0040] In another respect, the compound is of formula I or IV, and R 1 It does not exist, or its pharmaceutically acceptable salt.

[0041] In another respect, the compound is of formula I or IV, and R 1 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O and (CH2)3NH, or pharmaceutically acceptable salts thereof.

[0042] In another respect, the compound is of formula II or V, or a pharmaceutically acceptable salt thereof.

[0043] In another respect, 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 pharmaceutically acceptable salts thereof.

[0044] In another respect, the compound is of formula III or VI, or a pharmaceutically acceptable salt thereof.

[0045] In another respect, the compound is of formula III or VI, and R 1 It does not exist, or its pharmaceutically acceptable salt.

[0046] In another respect, the compound is of formula III or VI, and R 1 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O and (CH2)3NH, or pharmaceutically acceptable salts thereof.

[0047] In another aspect, the present invention provides novel pharmaceutical compositions comprising: a pharmaceutically acceptable carrier and a therapeutically effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0048] In another aspect, the present invention provides a novel method for treating diseases mediated by cellular hypoxia, comprising: administering to a patient in need a therapeutically effective amount of at least one compound of the present invention or a pharmaceutically acceptable salt thereof.

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

[0050] In another aspect, cardiovascular diseases are selected from: coronary artery disease, myocardial infarction, heart failure, arrhythmia, cardiac electrophysiological disorders, congenital cardiovascular abnormalities, developmental cardiovascular abnormalities, inflammatory cardiomyopathy, Kawasaki disease, infectious cardiomyopathy, sudden death / cardiac arrest, atherosclerosis, cardiovascular diseases of atherosclerosis, valvular heart disease, venous insufficiency, cardiac thrombosis, vascular thrombosis, thromboembolism, peripheral artery disease, aortic aneurysm, aortic dissection, vascular aneurysm, vascular dissection, stroke, systemic hypertension, and pulmonary hypertension.

[0051] In another aspect, the present invention provides compounds of the present invention for use in therapeutics.

[0052] In another aspect, the present invention provides the use of the present invention in manufacturing a medicament for treating diseases mediated by cellular hypoxia.

[0053] In another aspect, the present invention provides the use of the present invention in manufacturing a pharmaceutical agent for treating cardiovascular diseases.

[0054] The invention may be implemented in other specific forms without departing from the spirit or essential attributes of the invention. The invention encompasses all combinations of the aspects of the invention mentioned herein. It should be understood that any and all embodiments of the invention may be combined with any other embodiments to describe additional embodiments. It should also be understood that each individual element of an embodiment is intended to be considered independently as its own. Furthermore, any element of an embodiment is intended to be combined with any and all other elements from any embodiment to describe additional embodiments.

[0055] definition

[0056] Unless otherwise stated, the instances provided in the definition of this application are non-inclusive. They include, but are not limited to, the described instances.

[0057] Cellular hypoxia refers to hypoxia at the level of a single cell, and is not necessarily related to a lack of oxygen at the level of a whole organism or the environment.

[0058] The compounds described herein may have an asymmetric center, a geometric center (e.g., a double bond), or both. Unless a specific stereochemical or isomeric form is specifically indicated, all chiral, diastereomeric, racemic, and geometric isomeric forms of the structure are intended. Compounds of the present invention containing asymmetrically substituted atoms can be isolated in optically active or racemic form. How to prepare the optically active form is well known in the art, such as by the degradation of the racemic form, by synthesis from optically active starting materials, or by using chiral auxiliaries. Geometric isomers of alkenes, 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 be present and can be isolated as mixtures of isomers or as separate isomers. All methods for preparing the compounds of the present invention and for preparing intermediates therein are considered part of the present invention. All tautomers of the compounds shown or described are also considered part of the present invention.

[0059] "Mammals" and "patients" encompass warm-blooded mammals (e.g., humans and domestic animals) that are typically under medical care. Examples include cats, dogs, horses, cattle, and humans, as well as humans only.

[0060] "Treating" encompasses treating disease states in mammals and includes: (a) preventing the development of a disease state in mammals, particularly when such mammals are susceptible to a disease state but have not yet been diagnosed with the disease; (b) suppressing a disease state, such as inhibiting its development; and / or (c) alleviating a disease state, such as inducing the remission of the disease state until a desired endpoint is reached. Treatment also includes improving the symptoms of the disease (e.g., reducing pain or discomfort), where such improvement may or may not directly affect the disease (e.g., cause, transmission, expression, etc.).

[0061] "Pharmaceutically acceptable salt" refers to a derivative of the disclosed compound, wherein the parent compound is modified by preparing its acid or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral acid or organic acid salts of basic residues such as amines; and bases or organic salts of acidic residues such as carboxylic acids. Pharmaceutically acceptable salts include conventionally non-toxic salts or quaternary ammonium salts of parent compounds formed from, for example, non-toxic inorganic or organic acids. For example, such conventionally non-toxic salts include those derived from inorganic and organic acids selected from: 1,2-ethanedisulfonic acid, 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetate, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, hydroxyacetyl-p-aminobenzoic acid, hexylresorcinol acid, etc. Hypoglycine, hydrobromic acid, hydrochloric acid, hydroiodic acid, hydroxymaleic acid, hydroxynaphthyl carboxylic acid, hydroxyethyl sulfonic acid, lactic acid, lactobionic acid, dodecyl sulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, naphthalenesulfonic acid, nitric acid, oxalic acid, dihydroxynaphthyl acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, aminosulfonic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannic acid, tartaric acid, and toluenesulfonic acid.

[0062] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing a basic or acidic moiety. Generally, such salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of a suitable base or acid in water, in an organic solvent, or in a mixture of both; generally, 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.

[0063] "Therapeutic effective amount" includes the amount of the compounds of the present invention that are effective when administered alone or in combination for the treatment of the indications listed herein. "Therapeutic effective amount" also includes the amount of a combination of the claimed compounds that are effective in treating the desired indication. The combination of compounds may be a synergistic combination. The synergistic effect described (Chou & Talalay, 1984) occurs when, when administered in combination, the effect of the compounds is greater than the additive effect of the compounds when administered alone as a single agent. Generally, the synergistic effect is most clearly exhibited at the second-best concentration of the compounds. Compared to individual components, the synergistic effect may be lower cytotoxicity, increased activity, or some other beneficial effect of the combination.

[0064] It is expected that the compounds of the present invention will have the activities described herein.

[0065] Preparations and dosage

[0066] In this invention, one or more compounds of the invention can be administered in any suitable manner (e.g., enteric or parenteral). Examples of administration methods include oral and transdermal administration. Those skilled in the art will recognize that the routes of administration for the compounds of the invention can vary significantly. Sustained-release compositions may be advantageous, in addition to other oral administration methods. 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. Biodegradable, non-biodegradable, biodegradable, and non-biodegradable administration systems may also be used, including drug-eluting structures, such as stents, placed via catheters, which can deliver the compounds of the invention directly to the vessel wall.

[0067] If a solid composition is prepared in tablet form, the main active ingredient can be mixed with a pharmaceutical mediator, examples of which include silica, starch, lactose, magnesium stearate, and talc. Tablets can be coated with sucrose or another suitable substance, or can be treated to have sustained or delayed activity, and thus continuously release a predetermined amount of the active ingredient. Gelatin capsules can be obtained by mixing the active ingredient with a diluent and incorporating the resulting mixture into soft or hard gelatin capsules. Syrups or elixirs may contain the active ingredient along with sweeteners (typically calorie-free), preservatives (e.g., methylparaben and / or propylparaben), flavoring agents, and suitable pigments. Water-dispersible powders or granules may contain the active ingredient mixed with dispersants or wetting agents or suspending agents such as polyvinylpyrrolidone, and with sweeteners or flavoring agents. Suppositories can be used for rectal administration, prepared with a binder (e.g., cocoa butter and / or polyethylene glycol) that melts at rectal temperatures. Parenteral administration may be performed 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 may also be formulated into microcapsules or microspheres, optionally with one or more carriers or additives. The active ingredient may also be present in the form of complexes with cyclodextrins such as α-, β-, or γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, and / or methyl-β-cyclodextrin.

[0068] The daily dosage of the compounds of the present invention will vary from person to person and may be determined to some extent according to the severity of the disease being treated. The dosage of the compounds of the present invention will also vary depending on the compound being applied. Examples of dosages of the compounds of the present invention include approximately 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 100 mg / kg of mammalian body weight. The compounds may be administered over a period of time in a single dose or in many smaller doses. The duration of compound administration varies from person to person and can continue until the desired result (i.e., reduction of body fat or prevention of body fat gain) is achieved. Therefore, depending on the subject being treated, the desired result, and the subject's response rate to the treatment according to the invention, the therapy can last from one day to several weeks, months, or even years.

[0069] Possible examples of the tablets of the present invention are as follows.

[0070]

[0071]

[0072] Possible examples of the capsules of the present invention are as follows.

[0073]

[0074] In the capsules described above, the active ingredients have a suitable particle size. Crystalline lactose and microcrystalline cellulose are mixed evenly with each other, sieved, and then magnesium stearate is incorporated. The final mixture is then filled into appropriately sized hard gelatin capsules.

[0075] Possible examples of the injection solution of the present invention are as follows.

[0076]

[0077] Other features of the invention will become apparent during the following description of exemplary embodiments, which are provided for the purpose of illustrating the invention and are not intended to limit the invention.

[0078] Synthesis Example

[0079] The compounds of the present invention can be synthesized using the methods described below in conjunction with synthetic methods known in the field of synthetic organic chemistry, or variations thereof as understood by one of ordinary skill in the art. Preferred methods include, but are not limited to, those described below. Reactions are carried out in solvents suitable for the reagents and materials used and applicable to influencing the transformation. Those skilled in the art of organic synthesis will understand that the functional groups present on the molecule should conform to the proposed transformation. Sometimes judgments need to be made to modify the order of synthetic steps or to choose one particular process scheme over another in order to obtain the desired compounds of the present invention. It will also be appreciated that another major consideration in the planning of any synthetic route in the art is the prudent choice of protecting groups used to protect the reactive functional groups present in the compounds described in the present invention. Authoritative discussions of many alternatives described 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.

[0080] Synthetic Examples 1-34 represent procedures that can be used to prepare the compounds of the present invention. Synthetic Examples 1-17 utilize the known compounds isosorbide-2-mononitrate (5-hydroxy-1,4:3,6-didehydr-D-glucitol 2-nitrate) and 5-amino-isosorbide-2-mononitrate (5-amino-1,4:3,6-didehydr-D-glucitol 2-nitrate), and Synthetic Examples 18-34 utilize isosorbide-5-mononitrate (2-hydroxy-1,4:3,6-didehydr-D-glucitol 5-nitrate) and 2-amino-isosorbide-5-mononitrate (2-amino-1,4:3,6-didehydr-D-glucitol 5-nitrate) as starting materials. In specific embodiments of the invention, Synthetic Examples 1-17 are used for Formulas I, II, and III, and Synthetic Examples 18-34 are used for Formulas IV, V, and VI.

[0081] Synthesis Example 1

[0082]

[0083] 5-amino-isosorbitol-2-mononitrate (1) solution in 1N HCl was treated with potassium isocyanate for 10-12 hours at room temperature with stirring to obtain the 2-ureido derivative (2) after routine post-treatment via extraction.

[0084] Synthesis Example 2

[0085]

[0086] 5-Amino-isosorbitol-2-mononitrate ester (1) can be treated with Nt-BOC-glycine (t-BOC = tert-butyl-oxycarbonyl) in dichloromethane in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and dimethyl-aminopyridine (DMAP). After stirring overnight at ambient temperature, the t-BOC amide (3) can be separated by conventional methods. Subsequent treatment with trifluoroacetic acid (3) provides a deprotected amino acid adduct (4), which, upon treatment with potassium isocyanate and dilute HCl solution as previously described, yields a ureoglycine adduct (5).

[0087] Synthesis Example 3

[0088]

[0089] In the presence of (N-pyrrolidinyl)pyrimidine, treatment of isosorbide-2-mononitrate (6) with 1-cyclohexyl-3-(2-morpholinyl)carbodiimide (CMC) in dichloromethane (DCM) yields a protected amino acid adduct (7). Removal of the protecting group with TFA (trifluoroacetate) provides an amino compound (8). Subsequent treatment with potassium isocyanate and dilute HCl solution at 0°C to ambient temperature produces a urea compound (9).

[0090] Synthesis Example 4

[0091]

[0092] In the presence of [Cp*Ir(Pro)Cl] (Pro = proline group) (Cp = cyclopentadienyl group), treatment of 5-amino-isosorbitol-2-mononitrate (1) with t-BOC-aminoethanol or t-BOC-3-aminopropanol in toluene or water yields a protected diamino adduct (10). As previously described, removal of the protecting group with TFA yields a primary amine (11), which can be converted to urea (12a, 12b) in dilute hydrochloric acid solution using potassium cyanate.

[0093] Synthesis Example 5

[0094]

[0095] Isosorbide-2-mononitrate (6) can be deprotonated in tetrahydrofuran (THF) with sodium hydride or lithium diisopropylamino, and then treated with t-BOC-aminobromoethane or t-BOC-3-aminopropylbromo to give an ether (12). Deprotection of the t-BOC group with TFA will give a primary amine (13), and subsequent treatment with potassium isocyanate as previously described will give a urea (14).

[0096] Synthesis Example 6

[0097]

[0098] Treatment of 5-amino-isosorbitol-2-mononitrate (1) with ethyl cyanoacetate in THF or DCM produces urea (15). Further reaction with anhydrous ammonia in methanol produces ureocarboxamide (16).

[0099] Synthesis Example 7

[0100]

[0101] Treatment of isosorbide-2-mononitrate (6) with tert-butyl cyanoacetate in acetonitrile in the presence of N-methylimidazole yields a carbamate (17). The tert-butyl group can be removed in an ether with HCl or with TFA to give a carboxylic acid (18). Acid amidation with ammonia in the presence of DCC provides a carboxamide (19).

[0102] Synthesis Example 8

[0103]

[0104] Treatment of 5-amino-isosorbitol-2-mononitrate (1) or isosorbitol-2-mononitrate (6) with malonamide in the presence of DCC / DMAP or CMC / (N-pyrrolidinyl)pyrimidine can produce malonamide (20) or malon-ester-amide group (21), respectively.

[0105] Synthesis Example 9

[0106]

[0107] Treatment of 5-amino-isosorbitol-2-mononitrate (1) with phosgene and pyridine at approximately 0 °C yields carbamoyl chloride (22a, X = NH). The reaction of this carbamoyl chloride with glycineamide at low temperature in the presence of DMAP in a DCM yields ureocarboxamide (23). Alternatively, carbamoyl chloride (22) (X = NH) can be treated with hydroxyacetamide in the presence of DMAP in a DCM to yield carbonate (24) with terminal formamide groups. If isosorbitol-2-mononitrate (6) is used as the starting material for this sequence, the phosgene reaction at low temperature yields carbonyl chloride (22, X = O), and if this intermediate is treated with glycineamide or hydroxyacetamide under the previously described conditions, carbamoyl ester (25) or carbonate (26) can be prepared, respectively.

[0108] Synthesis Example 10

[0109]

[0110] Treatment of 5-amino-isosorbitol-2-mononitrate (1) with 3-hydroxypropionamide or 4-hydroxybutyramide in toluene at high temperature in the presence of a cobalt catalyst and molecular sieve can produce formamide-amines (27) and (28), respectively.

[0111] Synthesis Example 11

[0112]

[0113] Isosorbide-2-mononitrate (6) can be treated with sodium hydride in THF or with diisopropylaminolithium in THF and then alkylated with alkylate with 3-bromopropionamide or 4-bromobutyramide to produce formamido ethers (29) and (30), respectively.

[0114] Synthesis Example 12

[0115]

[0116] Treatment of 2-amino-isosorbitol-5-mononitrate ester (1) with acetoxyacetyl chloride in the presence of triethylamine in THF produces an amide adduct (31). Hydrolysis of the acetate ester with sodium hydroxide solution produces a hydroxyamide (32).

[0117] Synthesis Example 13

[0118]

[0119] Treatment of isosorbide-2-mononitrate (6) with acetoxyacetyl chloride in the presence of triethylamine in THF produces an ester adduct (33). Hydrolysis of acetate with dibutyltin oxide produces a glycolic acid adduct (34).

[0120] Synthesis Example 14

[0121]

[0122] N-acetoxyacetylglycine can be prepared by treating benzylglycine with acetoxyacetyl chloride in the presence of triethylamine, followed by hydrogenolysis of benzoyl ester in the presence of a palladium catalyst. Treatment of 5-amino-isosorbitol-2-mononitrate ester with acetoxyacetylglycine in DCM in the presence of DCC (1) yields an acetylated amide adduct (35). The removal of acetate can be accomplished by reaction with KOH or trimethyltin hydroxide in methanol solution (36).

[0123] Synthesis Example 15

[0124]

[0125] Treatment of isosorbide-2-mononitrate (6) with acetoxyacetylglycine in DCM in the presence of DCC / DMP yields an acetylated ester adduct (37). The acetate can be removed by reaction with trimethyltin hydroxide (38).

[0126] Synthesis Example 16

[0127]

[0128] Treatment of 5-amino-isosorbitol-2-mononitrate (1) with Nt-BOC-aminoethanol or Nt-BOC-3-aminopropanol in toluene at high temperature in the presence of a cobalt catalyst and molecular sieves provides a protected amino compound (39). The protecting group can be removed using TFA, yielding an unprotected primary amine (40). The reaction of the amine (40) with acetoxyacetyl chloride in the presence of trimethylamine provides an amide (41), which, upon further treatment with KOH in methanol, yields hydroxyacetamides (42 and 43).

[0129] Synthesis Example 17

[0130]

[0131] Deprotonation of isosorbide-2-mononitrate (6) in THF with sodium hydride or LDA followed by the addition of Nt-BOC-aminoethyl bromide or Nt-BOC-3-aminopropyl bromide yields a protected ether (44). Deprotection of the amino group using TFA yields a primary amine (45), which, upon treatment with ethoxyacetyl chloride, yields an amide (46). Hydrolysis of the acetate in MeOH with KOH yields hydroxyacetamide (47 and 48).

[0132] Synthesis Example 18

[0133]

[0134] 2-amino-isosorbitol-5-mononitrate (49) in 1N HCl solution can be treated with potassium isocyanate at room temperature for 10-12 hours with stirring to obtain 2-ureido derivative (50) after routine post-treatment via extraction.

[0135] Synthesis Example 19

[0136]

[0137] 2-Amino-isosorbitol-5-mononitrate ester (49) can be treated with Nt-BOC-glycine (t-BOC = tert-butyl-oxycarbonyl) in dichloromethane in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and dimethyl-aminopyridine (DMAP). After stirring overnight at ambient temperature, the t-BOC amide can be separated by conventional methods (51). Subsequent treatment with trifluoroacetic acid (51) provides a deprotected amino acid adduct (52), which, upon treatment with potassium isocyanate and dilute HCl solution as previously described, yields a ureoglycine adduct (53).

[0138] Synthesis Example 20

[0139]

[0140] Treatment of isosorbide-5-mononitrate (54) with 1-cyclohexyl-3-(2-morpholino)carbodiimide (CMC) in dichloromethane (DCM) in the presence of (N-pyrrolidinyl)pyrimidine yields a protected amino acid adduct (55). Removal of the protecting group with TFA (trifluoroacetate) provides an amino compound (56). Subsequent treatment with potassium isocyanate and dilute HCl solution at 0°C to ambient temperature yields a urea compound (57).

[0141] Synthesis Example 21

[0142]

[0143] In the presence of [Cp*Ir(Pro)Cl] (Pro = proline group) (Cp = cyclopentadienyl group), treatment of 2-amino-isosorbitol-5-mononitrate (49) with t-BOC-aminoethanol or t-BOC-3-aminopropanol yields a protected diamino adduct (58) in toluene or water. As previously described, removal of the protecting group with TFA yields a primary amine (59), which can be converted to urea in dilute hydrochloric acid solution using potassium cyanate (60a, 60b).

[0144] Synthesis Example 22

[0145]

[0146] Isosorbide-5-mononitrate (54) can be deprotonated in tetrahydrofuran (THF) with sodium hydride or lithium diisopropylamino, and then treated with t-BOC-aminobromoethane or t-BOC-3-aminopropylbromide to give an ether (60). Deprotection of the t-BOC group with TFA gives a primary amine (61), and subsequent treatment with potassium isocyanate as previously described gives a urea (62).

[0147] Synthesis Example 23

[0148]

[0149] Treatment of 2-amino-isosorbitol-5-mononitrate (49) with ethyl cyanoacetate in THF or DCM yields urea (63). Further reaction with anhydrous ammonia in methanol yields ureocarboxamide (64).

[0150] Synthesis Example 24

[0151]

[0152] Treatment of isosorbide-5-mononitrate with tert-butyl cyanoacetate in acetonitrile (54) in the presence of N-methylimidazole yields a carbamate (65). The tert-butyl group can be removed in an ether with HCl or with TFA to give a carboxylic acid (66). Acid amidation with ammonia in the presence of DCC provides a carboxamide (67).

[0153] Synthesis Example 25

[0154]

[0155] Treatment of 2-amino-isosorbitol-5-mononitrate (49) or isosorbitol-5-mononitrate (54) with malonamide in the presence of DCC / DMAP CMC / (N-pyrrolidinyl)pyrimidine can produce malonamide (68) or malon-ester-amide (69), respectively.

[0156] Synthesis Example 26

[0157]

[0158] Treatment of 2-amino-isosorbitol-5-mononitrate (49) with phosgene and pyridine at approximately 0 °C yields carbamoyl chloride (70a, X = NH). The reaction of this carbamoyl chloride with glycineamide at low temperature in the presence of DMAP in a DCM yields ureocarboxamide (71). Alternatively, carbamoyl chloride (70a, X = NH) can be treated with hydroxyacetamide in the presence of DMAP in a DCM to yield carbonates (72) with terminal formamide groups. If isosorbitol-5-mononitrate (54) is used as the starting material for this sequence, the phosgene reaction at low temperature can produce carbonyl chloride (70b, X = O), and if this intermediate is treated with glycineamide or hydroxyacetamide under the previously described conditions, carbamoyl ester (73) or carbonate (74) can be prepared, respectively.

[0159] Synthesis Example 27

[0160]

[0161] Treatment of 2-amino-isosorbitol-5-mononitrate (49) with 3-hydroxypropionamide or 4-hydroxybutyramide in toluene at high temperature in the presence of a cobalt catalyst and molecular sieve can produce formamide-amines (75) and (76), respectively.

[0162] Synthesis Example 28

[0163]

[0164] Isosorbide-5-mononitrate (6) can be treated with sodium hydride in THF or with diisopropylaminolithium in THF and then alkylated with alkylate with 3-bromopropionamide or 4-bromobutyramide to produce formamide-ethers (77) and (78), respectively.

[0165] Synthesis Example 29

[0166]

[0167] Treatment of 2-amino-isosorbitol-5-mononitrate ester with acetoxyacetyl chloride in the presence of triethylamine in THF (49) yields an amide adduct (79). Hydrolysis of the acetate ester with sodium hydroxide solution yields a hydroxyamide (80).

[0168] Synthesis Example 30

[0169]

[0170] Treatment of isosorbide-5-mononitrate (54) with acetoxyacetyl chloride in the presence of triethylamine in THF produces an ester adduct (81). Hydrolysis of acetate with dibutyltin oxide produces a glycolic acid adduct (82).

[0171] Synthesis Example 31

[0172]

[0173] N-acetoxyacetylglycine can be prepared by treating benzylglycine with acetoxyacetyl chloride in the presence of triethylamine, followed by hydrogenolysis of benzoyl ester in the presence of a palladium catalyst. Treatment of 2-amino-isosorbitol-5-mononitrate ester (49) with acetoxyacetylglycine in DCM in the presence of DCC yields an acetylated amide adduct (83). The removal of acetate ester (84) can be accomplished by reaction with KOH or trimethyltin hydroxide in methanol solution.

[0174] Synthesis Example 32

[0175]

[0176] Treatment of isosorbide-5-mononitrate (6) with acetoxyacetylglycine in DCM in the presence of DCC / DMP yields an acetylated ester adduct (85). The acetate can be removed by reaction with trimethyltin hydroxide (86).

[0177] Synthesis Example 33

[0178]

[0179] Treatment of 2-amino-isosorbitol-5-mononitrate (49) with Nt-BOC-aminoethanol or Nt-BOC-3-aminopropanol in toluene at high temperature in the presence of a cobalt catalyst and molecular sieves provides a protected amino compound (87). The protecting group can be removed using TFA, yielding an unprotected primary amine (88). The reaction of the amine (88) with acetoxyacetyl chloride in the presence of trimethylamine provides an amide (89), which can be further treated with KOH in methanol to produce hydroxyacetamide (90 and 91).

[0180] Synthesis Example 34

[0181]

[0182] Deprotonation of isosorbide-5-mononitrate (54) in THF with sodium hydride or LDA followed by the addition of Nt-BOC-aminoethyl bromide or Nt-BOC-3-aminopropyl bromide yields a protected ether (92). Deprotection of the amino group using TFA yields a primary amine (93), which, upon treatment with ethoxyacetyl chloride, yields an amide (94). Hydrolysis of the acetate in MeOH with KOH yields hydroxyacetamides (95 and 96).

[0183] biology

[0184] The compounds of this invention have two main indications. The first indication is as an antianginal agent. It is desired that the compounds, acting as coronary vasodilators, improve exercise capacity in cases of coronary artery disease. The second indication is for secondary prevention of cardiovascular disease, and this application seeks vasodilatory and ischemic pretreatment.

[0185] The experiments described herein were conducted to test whether the compounds of the present invention are suitable for at least two primary indications. These experiments aimed to determine the effects of the compounds of the present invention and hypoxia on the production of reactive oxygen species (ROS) and markers of inflammation and ischemic pretreatment, as well as mediators of vascular smooth muscle relaxation, in vitro using human aortic endothelial cells (HAECs) and peripheral blood mononuclear cells (PBMCs). Primary HAECs isolated from normal healthy adults were available from Thermo Fisher Scientific (Grand Island, NY) and cultured in medium 200. Peripheral blood mononuclear cells were used as part of the in vitro determination of the effects of the compounds of the present invention, as described below.

[0186] Cell culture can be performed in a humidified cell culture incubator at Biospherix (Lacona, NY), which dynamically controls oxygen and carbon dioxide in a closed environment, covering all aspects of cell manipulation. Culture media with a pH of 7.3 can be prepared. Incubator conditions can be either normoxic (21% O2, 5% CO2) or anoxic (1% O2, 5% CO2, balance N2) in a humidified incubator with an internal temperature of 37°C (using a controlled incubator with CO2 / O2 monitoring and a CO2 / N2 gas source). The culture medium is equilibrated to ambient gas conditions overnight before exposure to cells. As early as possible, and in some cases in primary cultures, cells will be incubated with the experimental gas mixture for varying times. A closed ambient chamber with a glove-type cell access device can be used to prevent reoxygenation. Regular analysis of the cell culture medium using pH, pCO2, and pO2 electrodes ensures a controlled environment.

[0187] HAECs can be exposed to normoxic (20% oxygen) or hypoxic (1% oxygen) conditions 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) with or without the compounds of this invention. Intracellular levels of ROS, such as superoxide anion and hydrogen peroxide, can be measured using ethidium hydrogen fluorescein (DHE) and 2',7'-dichlorofluorescein (DCF) fluorescence assays. DNA-binding activity of pro-inflammatory transcription factors (NF-κB and AP-1) in HAECs 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 HAECs cultured in normoxic versus hypoxic conditions under lipopolysaccharide (LPS) stimulation. (Folco, Sukhova, Quillard, & Libby, 2014) Other biomarkers (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 HAECs. mRNA and protein expression levels can be measured by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Following stimulation of HAECs in primary cultures, ELISA can be used to analyze selection mediators of relevant endothelial responses, such as PGE2. A comprehensive assessment of metabolism can be performed using a combination of high-resolution breath assays and Seahorse extracellular flux assays to indicate the contribution of mitochondria to glycolysis in patient cells, using established protocols.

[0188] The activation levels of relevant transcription factors can be determined by measuring the DNA-binding activity of nuclear extracts isolated from HAECs in EMSA. HAECs can be treated as instructed above, and then the cells are incubated with lysis buffer (10 mM Tris-HCl, pH 8.0, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, 100 μM phenylmethylsulfonyl fluoride, 0.1% NP-40) on ice for 5 min, followed by centrifugation at 600 × g for 4 min at 4 °C to collect the nuclei. The nuclear particles are then washed with lysis buffer without NP-40, incubated on ice for 10 min in nuclear extract buffer (20 mM Tris-HCl, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol), and centrifuged at 18,300 × g for 15 min at 4 °C. The supernatant containing the nuclear extract can be immediately frozen on dry ice and transferred to -80°C until analysis. The binding reaction can be performed using 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 of poly[dI-dC]. After adding the reagents, the mixture can be incubated at room temperature for 25 minutes. Then, a biotin-labeled specific oligonucleotide probe and / or an unlabeled control can be added, and the binding mixture can be incubated at room temperature for 25 minutes. Competitive studies can be performed by adding a molar excess of unlabeled oligonucleotides to the binding reaction. The protein-DNA complex can be obtained by electrophoresis on a non-denaturing 5% polyacrylamide gel at 150 V for 3 hours using 0.25×TBE buffer (50 mM Tris-Cl, 45 mM boric acid, 0.5 mM EDTA, pH 8.4). This is a variant of the LightShift chemiluminescent EMSA kit (Thermo Fisher Scientific, Rockford, Illinois).

[0189] Quantitative real-time polymerase chain reaction (QRT-PCR) can be used for assays. A fluorescent 5'-nuclease assay with probes and primers (Applied Biosystems, Foster City, CA) can be used for gene expression analysis. HAECs can be processed as instructed above, and total RNA can be isolated from HAECs using the RNeasy mini kit (Qiagen, Valencia, CA). 1 μg of total RNA can be reverse transcribed in 20 μL of 5 mM MgCl2, 10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1 mM dNTP, 1 unit / μL recombinant RNasin, 15 units / μg avian myeloblastomavirus (AMV) reverse transcriptase, and 0.5 μg random hexamer at 25°C for 15 min, 42°C for 45 min, and 99°C for 5 min. Individual genes can be amplified 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 for the first cycle, repeated 45 times) on the Applied Biosystems 7300 Real-Time PCR System. Gene expression assays for human TNF-α, IL-4, IL-6, IL-10, IL-12, IL-13, IFN-γ, COX-2, eNOS, MCP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) can be used as specific probes and primers for PCR amplification. The threshold cycle (CT) can be determined from each well using Applied Biosystems Sequencing Software, which represents the fractional number of cycles in which the amplified target gene reaches a fixed threshold.

[0190] Intracellular levels of ROS can be measured using DHE and DCF fluorescence staining with a fluorescence microscope. HAECs can be treated as instructed above, and DHE or carboxy-H2DCF-DA (Invitrogen Corp., Carlsbad, CA) can be added to cells at a concentration of 5 μM in PBS for 30 minutes in a humidified cell culture incubator at 37°C with 5% CO2 / 95% air. HAECs can be washed with PBS and examined under a fluorescence microscope. The DHE or DCF fluorescence intensity of the acquired digital images can be quantified accordingly.

[0191] The effect of the compounds of this invention on hypoxia-mediated NO release from endothelial cells can be measured by separating cells using membrane-permeable 4,5-diaminofluorescein diacetate (DAF-2 diacetate), which leads to the accumulation of intracellular DAF-2 in membrane-impermeable cells. 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)

[0192] Parallel studies can be used to test the effects of the compounds of this invention on cellular ROS homeostasis and metabolism in cellular metabolic assays. Plated cells from different experimental groups can be studied on a Seahorse extracellular flux analyzer. Oxygen consumption rate (mitochondrial function) and extracellular acidification rate (glycolysis) can be measured using established methods under basal conditions, maximal respiration conditions, and under conditions of suppressed mitochondrial bioenergy. (Dai et al., 2016) Parallel subsets of cells can be placed in an Orbororos high-resolution respiration chamber to simultaneously measure cellular ROS and metabolism using established protocols. (Alleman et al., 2016)

[0193] Peripheral blood mononuclear cells and serum can be obtained from human volunteers. Patients can serve as their own controls in clinical trials designed to test the compounds of this invention in patients with ischemic cardiomyopathy (including a history of myocardial infarction) and for secondary prevention indications. This study is limited to 5 × 8 mL blood samples per draw, using BD Vacutainer CPT and heparin sodium (Franklin Lake, New Jersey), from which 2 mL of plasma-derived serum and 8 × 10 mL of serum per tube can be expected to be drawn. 6 PBMCs contain heparin sodium anticoagulant and FICOLL Hypaque density liquid, separated by a polyester gel barrier. The tubes can be centrifuged at 1500g for 15 minutes at room temperature. Plasma can be obtained as the supernatant above the mononuclear cells and platelet layer at the 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 coagulate at room temperature for 4 hours, followed by freezing the coagulated plasma overnight at -20°C, thawing, and centrifuging the remaining serum at 5000g for at least 5 minutes. The serum can be filtered, aliquoted, and frozen at -20°C. PBMCs can be centrifuged at 400g for 10-15 minutes at 18°C-20°C, washed twice in phosphate-buffered saline, and then centrifuged at 8×10⁻⁶. 6 Cells were resuspended in 2 mL of AIM V medium (LifeTech) and then divided into 1 × 10⁻⁶ cells. 6Aliquots of cells. Cultures were performed in a humidified cell culture incubator at Biospherix (Lacona, NY), which dynamically controls oxygen and carbon dioxide in a closed environment, covering all aspects of cell manipulation.

[0194] DHE / DCF fluorescence assay, real-time RT-PCR, ELISA, EMSA, and reporter gene assays can be used 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). The products of eNOS and COX-2, compared to controls, confirm the therapeutic value of the compounds of this invention. Toxicity can be assessed using Seahorse technology through the metabolism of the compounds of this invention.

[0195] Tables 1-6 show the structures of the compounds of the present invention that can be synthesized as described above.

[0196] Table 1

[0197]

[0198] Example # <![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]]>

[0199] Table 2

[0200]

[0201] Example # <![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]]>

[0202] Table 3

[0203]

[0204]

[0205]

[0206] Table 4

[0207]

[0208] Example # <![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]]>

[0209] Table 5

[0210]

[0211]

[0212]

[0213] Table 6

[0214]

[0215] Example # <![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]]>

[0216] In view of the foregoing teachings, many modifications and variations of the present invention are possible. Therefore, it should be understood that, within the scope of the appended claims, the invention may be practiced in ways different from those specifically described herein.

[0217] References

[0218] Alleman, RJ, Tsang, AM, Ryan, TE, Patterson, 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.

[0219] 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.

[0220] 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.

[0221] 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 rat ischemia / reperfusion models. *Cardiovascular Drugs and Therapy*, 30, 559-566.

[0222] Folco, EJ, Sukhova, GK, Quillard, T., & Libby, P. (2014). Moderate hypoxia potentiates interleukin-1 beta production in activated human macrophages. Circulation Research, 115, 875-883.

[0223] Gennaro, AR & Remington, JP (1990). Remington Pharmaceutical Sciences. In (18th edition, p. 1445). Easton, PA: Mack.

[0224] Greene, TW & Wuts, PGM (1991). Protective groups in inorganic synthesis. (2nd ed.) New York: Wiley.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] Li, Q., Guo, Y., Tan, W., Ou, Q., Wu, WJ, Sturza, D. et al. (2007). Cardioprotection supported by inducible nitric oxide synthase gene therapy is mediated by cyclooxygenase-2 via a nuclear factor-κB dependent pathway. *Circulation*, 116, 1577-1584.

[0229] Li, X., Chen, G, Yang, B. (2012) studied the physiology of urea transporter in knockout mice. Frontiers in Physiology, 3:217.

[0230] Sands, JM (1999). Regulation of Renal Urea Transporters. Journal of the American Society of Nephrology, 10, 635-646.

[0231] 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.

[0232] Semenza, GL (2019). Pharmacologic Targeting of Hypoxia-Inducible Factors. Annual Review of Pharmacology and Toxicology, 59, 379-403.

[0233] Shayakul, C., Clemencon, B., & Hediger, MA (2013). The urea transporter family (SLC14): physiological, pathological and structural aspects. Molecular Aspects of Medicine, 34, 313-322.

[0234] Sun, Y., Lau, CW, Jia, Y., Li, Y., Wang, W., Ran, J., Li, F., Huang, Y., Zhou, H., Yang, B. (2016) Functional inhibition of ureatransporter UT-B enhances endothelial-dependent vasodilation and lowers blood pressure via L-arginine-endothelial nitric oxide synthase-nitric oxide pathway. Scientific Reports 6, 18697.

[0235] 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.

[0236] 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.

[0237] 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. *American Journal of Physiology - Renal Physiology* 280, F996-F1000.

[0238] Xu, Q., Ji, YS, & Schmedtje, JF, Jr. (2000). Sp1 increases expression of cyclooxygenase-2 in hypoxic vascular endothelium - Implications for the mechanisms of aortic aneurysm and heart failure. Journal of Biochemistry, 275, 24583-24589.

[0239] Zhao, D., Sonawane, ND, Levin, MH, & Yang, B. (2007). Comparative transport efficiencies of urea analogues through urea transporter UT-B. Acta Biochimica et Biophysica Acta (BBA)-Biomembranes, 1768, 1815-1821.

Claims

1. A compound of formula III or VI for the treatment of cardiovascular diseases: III VI in: R 1 It does not exist; Alternatively, R 1 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O, (CH2)3NH, CH2C(=O)O and CH2C(=O)NH; or pharmaceutically acceptable salts thereof.

2. The compound according to claim 1, wherein: R 1 It does not exist; Or its pharmaceutically acceptable salt.

3. The compound according to claim 1, wherein: R 1 Selected from: (CH2)2O, (CH2)2NH, (CH2)3O, and (CH2)3NH; Or its pharmaceutically acceptable salt.

4. The compound according to claim 1, wherein: R 1 It is CH2C(=O)O, or a pharmaceutically acceptable salt thereof.

5. The compound according to claim 1, wherein: R 1 It 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. The pharmaceutical composition according to claim 6, wherein the cardiovascular disease is selected from: coronary artery disease, heart failure, arrhythmia, cardiac electrophysiological disorders, congenital cardiovascular abnormalities, developmental cardiovascular abnormalities, inflammatory cardiomyopathy, Kawasaki disease, infectious cardiomyopathy, sudden death / cardiac arrest, valvular heart disease, venous insufficiency, cardiac thrombosis, peripheral artery disease, aortic aneurysm, vascular aneurysm, vascular dissection, stroke, systemic hypertension, and pulmonary hypertension.

8. The pharmaceutical composition according to claim 6, wherein the cardiovascular disease is selected from: coronary artery disease, heart failure, arrhythmia, cardiac electrophysiological disorders, congenital cardiovascular abnormalities, developmental cardiovascular abnormalities, inflammatory cardiomyopathy, Kawasaki disease, infectious cardiomyopathy, sudden death / cardiac arrest, valvular heart disease, venous insufficiency, vascular thrombosis, peripheral artery disease, aortic aneurysm, vascular aneurysm, vascular dissection, stroke, systemic hypertension, and pulmonary hypertension.

9. The pharmaceutical composition according to claim 6, wherein the cardiovascular disease is selected from: coronary artery disease, heart failure, arrhythmia, cardiac electrophysiological disorders, congenital cardiovascular abnormalities, developmental cardiovascular abnormalities, inflammatory cardiomyopathy, Kawasaki disease, infectious cardiomyopathy, sudden death / cardiac arrest, valvular heart disease, venous insufficiency, thromboembolism, peripheral artery disease, aortic aneurysm, vascular aneurysm, vascular dissection, stroke, systemic hypertension, and pulmonary hypertension.

10. The pharmaceutical composition according to any one of claims 7 to 9, wherein the coronary artery disease is myocardial infarction or atherosclerosis.

11. The pharmaceutical composition according to any one of claims 7 to 9, wherein the vascular dissection is aortic dissection.

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