Treatment of congestive heart failure

Abstract

The present invention relates to a method of treatment for improving cardiac function in subjects with congestive heart failure, and in particular for preventing or reducing the decline in cardiac function that occurs after myocardial ischemia. The method utilizes compounds that selectively inhibit prolyl hydroxylase domain-containing protein 1 (PHD1). Some embodiments relate to combination therapies for treating heart failure with reduced ejection fraction (HFrEF) or heart failure with preserved ejection fraction (HFpEF).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 299,383, filed January 13, 2022, and U.S. Provisional Application No. 63 / 306,994, filed February 4, 2022, each of which is incorporated by reference in its entirety herein.

[0002] The present invention relates to the compound 3-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino-]-3-methylbutyric acid for use in treating heart failure, particularly congestive heart failure, in a subject in need thereof. [Background technology]

[0003] Congestive heart failure (CHF) is a clinical syndrome defined as chronic myocardial contractile dysfunction resulting in inadequate cardiac output. CHF can occur as a symptom of underlying conditions such as systemic or pulmonary hypertension, or as a sequela of or in association with cardiac disease such as myocardial infarction (MI), arteriosclerosis, cardiomyopathy, congenital heart disease, or rheumatic fever. CHF affects more than 20 million people worldwide, with approximately 5.3 million Americans diagnosed with the syndrome. The prevalence of CHF increases exponentially with age, affecting 6-10% of people aged 65 years or older. Half of patients diagnosed with CHF die within four years, and more than half of patients diagnosed with severe CHF die within one year. It is estimated that there are 278,000 CHF-related deaths annually in the United States. (See, e.g., Roger et al. (2011) Circulation 123:e18-e209; Remme et al. (2001) Eur Heart J 22:1527-1560; Stewart et al. (2001) Eur J Heart Fail 3:315-322.)

[0004] Current treatments for CHF usually involve lifestyle modifications such as reducing salt in the diet and incorporating daily exercise. Medications used in CHF include angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) to lower blood pressure, beta-blockers to reduce heart rate, and diuretics to control fluid retention. Invasive procedures may also be performed, including cardiac resynchronization therapy (CRT) for patients with wide QRS complexes on the ECG, implantable cardioverter defibrillators (ICDs) for patients with low ejection fraction, and heart transplants if all other measures fail to control symptoms.

[0005] Recent papers have suggested a role for hypoxia-inducible factors (HIFs) in congestive heart disease. (See, e.g., Semenza (2000) Adv Exp Med Biol 475:123-130.) HIFs are heterodimeric nuclear proteins (HIFα / β) that mediate activation of cellular defense mechanisms during hypoxia. Under normoxic conditions, HIF subunits are constitutively expressed, but HIFα subunits are targeted for proteasome-mediated degradation by prolyl hydroxylation carried out by prolyl hydroxylase domain-containing proteins (PHD1, 2, and 3, also known as HPH (HIF prolyl hydroxylase)-3, 2, and 1, and EGLN-2, 1, and 3, respectively). (Fong and Takeda(2008) Cell Death and Differentiation.15:635-641, Bernhardt et al.(2007) Methods in Enzymology.435:221-245.)

[0006] Articles disclosing methods of treating CHF using HIF contradictorily include both activation of HIF, particularly by inhibiting PHD proteins (International Publication No. WO2003 / 049686, Philipp et al. (2006) Eur J Heart Fail 8(4):347-354, Bao et al. (2010) J Cardiovasc Pharmacol 56(2):147-155), and inhibition of HIF (US Published Application No. US2010 / 008908). Each article provides data showing changes in cardiac function associated with the methods disclosed therein. Support for each method relies on data provided by inhibition of PHD proteins, the former using inhibitors of all three PHDs (pan-inhibitors) and the latter using a PHD3-specific knockout. However, studies have shown that the three PHD isoforms function differently in regulating HIF and may have other regulatory roles unrelated to HIF.

[0007] Many studies have been conducted to better define the role of each PHD isoform. Many of these studies have been performed using genetically engineered knockout or knockdown animals for each of the PHD genes, or using siRNA, shRNA, or RNAi for single isoforms to inhibit or reduce gene expression. With regard to PHD1, studies have suggested that inhibition of this protein is therapeutically beneficial for treating myocyte degeneration (U.S. Pat. No. 7,858,593), protecting myofibrils against ischemia (Aragones et al. (2008) Nat. Genet. 40:170-180), protecting against hepatic ischemia-reperfusion (Schneider et al. (2010) Gastroenterology 138(3):1143-1154), and treating colitis and other forms of inflammatory bowel disease (Tambuwala et al. (2010) Gastroenterology 139:2093-2101). Regarding PHD2, studies suggest contradictory roles in cardioprotection: in one study, somatic inactivation of PHD2 caused polycythemia and CHF, whereas in another, cardiomyocyte-specific PHD2 knockout improved cardiac function after myocardial infarction. (See Minamishima et al. (2008) Blood 111:3236-3244, and Holscher et al. (2011) J Biol Chem 286(13):11185-11194). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2003 / 049686 [Patent Document 2] U.S. Pat. No. 7,858,593 [Non-patent literature]

[0009] [Non-Patent Document 3] Roger et al.(2011)Circulation 123:e18-e209

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

Non-licensed Document 14

Non-licensed Document 15

[0010] The present inventors have discovered that a compound of formula I [ka] It has been discovered that the compound 3-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino-]-3-methylbutyric acid, which selectively inhibits prolyl hydroxylase domain-containing protein 1 (PHD1), having the structure: is surprisingly effective in improving cardiac function in subjects with congestive heart failure, and in particular in preventing or reducing the decline in cardiac function that occurs after myocardial ischemia. For clarity, 3-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino-]-3-methylbutyric acid is also referred to herein as the "compound of Formula I," or as "Formula I" or "Formula 1." Experimental results in animal models suggest that the use of this compound surprisingly does not result in a significant increase in hematocrit or circulating hemoglobin levels or circulating erythropoietin levels, a property that is more advantageous than the use of non-selective PHD inhibitors (i.e., compounds that inhibit all three PHD isoforms) in the treatment of CHF. Thus, the present invention, in one embodiment, provides a method of treating congestive heart failure in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby treating congestive heart failure. The subject in need thereof has congestive heart failure or is at risk of having congestive heart failure.

[0011] In another embodiment, the present invention provides a method for improving cardiac function in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby improving cardiac function. In various embodiments, the index of cardiac function may be selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, end systolic volume, end diastolic volume, and cardiac contractility.

[0012] In another embodiment, the present invention provides a method for preventing or reducing a decline in cardiac function in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby preventing or reducing a decline in cardiac function. In various embodiments, the index of cardiac function may be selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, end-systolic volume, end-diastolic volume, and cardiac contractility.

[0013] In another embodiment, the present invention provides a method for reducing cardiac damage in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby reducing cardiac damage. In various embodiments, cardiac damage may be selected from the group consisting of infarct size resulting from myocardial infarction, cardiac hypertrophy, and peri-infarct angiogenesis.

[0014] In one embodiment, the compound of formula I is administered to a subject having or at risk of having congestive heart failure.In certain embodiments, the compound of formula I is administered to a subject after myocardial infarction in the subject.In other embodiments, the compound of formula I is administered to a subject after a diagnosis of atherosclerosis, cardiomyopathy, congenital heart disease, or rheumatic fever in the subject.In one embodiment, the compound of formula I is administered to prevent congestive heart failure after a myocardial ischemic event.

[0015] In various embodiments, the method comprises administering the compound of formula I periodically for an extended period of time. The frequency of administration can be, for example, once every 12, 24, 36, 48, 60, or 72 hours, once a day, once every 2, 3, 4, 5, or 6 days, or once a week, and the duration of treatment can be one or more days, one or more weeks, one or more months, or one or more years. This method can be used long-term to improve cardiac function without the complications of polycythemia and fluid overload. The compound of formula I is non-erythropoietic, making it an ideal candidate for the treatment of chronic diseases such as CHF, where increased erythropoietin is undesirable.

[0016] The present invention also provides compounds of formula I for use in the treatment of congestive heart failure. In various embodiments, the present invention provides compounds of formula I for improving cardiac function, preventing or reducing the decline in cardiac function, and preventing cardiac damage. The compounds can be used chronically and can improve cardiac function without the complications of hyperviscosity syndrome and fluid overload associated with polycythemia.

[0017] In the above-described embodiments, the compounds of formula I can be used alone or in combination with inhibitors of sodium-glucose cotransporter 2 (SGLT2). SGLT2 inhibitors, also known as gliflozins, for use in the present invention include, but are not limited to, canagliflozin, dapagliflozin, and empagliflozin.

[0018] In any embodiment of the method, the compound of formula I can be administered in combination with other medications, such as ACE inhibitors, ARBs, beta blockers, and diuretics. [Brief description of the drawings]

[0019] [Figure 1A]Figure 1 shows the effect of the compound of formula I at 6 and 20 mg / kg on cardiac function in an animal model of congestive heart failure after myocardial infarction as measured by changes in ejection fraction. Mean ± SE, n = 12. * p < 0.05 vs. vehicle, ** p < 0.01 vs. vehicle, *** p < 0.001 vs. vehicle. [Figure 1B] Figure 1 shows the effect of the compound of formula I at 6 and 20 mg / kg on cardiac function in an animal model of congestive heart failure after myocardial infarction as measured by changes in relaxation time. Mean ± SE, n = 12. * p < 0.05 vs. vehicle, ** p < 0.01 vs. vehicle, *** p < 0.001 vs. vehicle. [Figure 1C] Figure 1 shows the effect of the compound of formula I at 6 and 20 mg / kg on cardiac function in an animal model of congestive heart failure after myocardial infarction as measured by changes in stroke work. Mean ± SE, n = 12. * p < 0.05 vs. vehicle, ** p < 0.01 vs. vehicle, *** p < 0.001 vs. vehicle. [Figure 2A] Figure 1 shows the effect of the compound of formula I at 6 and 20 mg / kg on cardiac function following delayed treatment of congestive heart failure after myocardial infarction in an animal model, as measured by change in ejection fraction. Mean ± SE, n = 11-12. *p<0.05 vs. vehicle, **p<0.01 vs. vehicle, ***p<0.001 vs. vehicle, ###p<0.001 vs. baseline. [Figure 2B] Figure 1 shows the effect of the compound of formula I at 6 and 20 mg / kg on cardiac function following delayed treatment of congestive heart failure after myocardial infarction in an animal model, as measured by change in end systolic volume. Mean ± SE, n=11-12. *p<0.05 vs. vehicle, **p<0.01 vs. vehicle, ***p<0.001 vs. vehicle, ###p<0.001 vs. baseline. [Figure 3A]1 shows that chronic treatment with the compound of formula I at 6 and 20 mg / kg did not produce erythrocytic effects in an animal model of post-myocardial infarction congestive heart failure as measured by changes in hemoglobin. Mean ± SE, n = 11-12. [Figure 3B] FIG. 3 shows that chronic treatment with the compound of formula I at 6 and 20 mg / kg did not produce erythrocytic effects in an animal model of post-myocardial infarction congestive heart failure as measured by changes in hematocrit (FIG. 3). Mean ± SE, n=11-12. [Figure 4A] Figure 1 shows the effect of daily administration of the compound of formula I at 2, 6 and 20 mg / kg on cardiac function in an animal model of post-myocardial infarction congestive heart failure as measured by changes in ejection fraction. Mean ± SE, n = 12. * p < 0.05 vs. vehicle, ** p < 0.01 vs. vehicle, *** p < 0.001 vs. vehicle. [Figure 4B] Figure 1 shows the effect of daily administration of the compound of formula I at 2, 6 and 20 mg / kg on cardiac function in an animal model of congestive heart failure after myocardial infarction as measured by changes in end-systolic volume. Mean ± SE, n = 12. * p < 0.05 vs. vehicle, ** p < 0.01 vs. vehicle, *** p < 0.001 vs. vehicle. [Figure 4C] Figure 1 shows the effect of daily administration of the compound of formula I at 2, 6 and 20 mg / kg on cardiac function in an animal model of congestive heart failure after myocardial infarction as measured by changes in stroke work. Mean ± SE, n = 12. * p < 0.05 vs vehicle, ** p < 0.01 vs vehicle, *** p < 0.001 vs vehicle. [Diagram 5] 1 is a graph showing the dose-dependent reducing effect of a compound of formula I on infarct size following acute myocardial ischemia. [Figure 6A] 1 is a graph showing that the compound of formula I improved left ventricular function (LVDP) after global ischemia. [Figure 6B] 1 is a graph showing that the compound of formula I improved left ventricular function (LVEP) after global ischemia. [Figure 6C] 1 is a graph showing that the compound of formula I improved coronary blood flow after global ischemia. [Figure 7A] 1 is a graph showing pressure-volume relationships. [Figure 7B] 1 shows a graph depicting the heart weight / body weight ratio. [Figure 8A] Graph showing ejection fraction 28 days after administration (QOD). [Figure 8B] 1 is a graph showing workload 28 days after administration (QOD). [Figure 8C] 1 is a graph showing stroke volume 28 days after administration (QOD). [Figure 8D] 1 is a graph showing cardiac output. [Figure 9] Graph showing cardiac histology findings 28 days after administration (QOD). [Figure 10A] FIG. 10B shows that the dose response of the compound of formula I on cardiac function in congestive heart failure (CHF) was examined by inducing CHF after myocardial ischemia to determine the minimal effective dose (MED) of 2 mg / kg. [Figure 10B] The dose response of the compound of formula I on cardiac function in congestive heart failure (CHF) was examined by inducing CHF after myocardial ischemia as shown in FIG. 10B to determine the minimal effective dose (MED) of 2 mg / kg. [Figure 11A] FIG. 1 shows the effect of duration of administration of a compound of formula I on cardiac function in congestive heart failure (CHF) by induction of CHF after myocardial infarction. [Figure 11B] FIG. 1 shows the effect of duration of administration of a compound of formula I on cardiac function in congestive heart failure (CHF) by induction of CHF after myocardial infarction. [Figure 11C] 13 is a graph showing the effect on collagen region. [Figure 12] 1 is a graph showing ejection fraction, illustrating progression of heart failure with preserved ejection fraction. [Figure 13A] 13 is a graph showing improvement in expansion function. [Figure 13B]1 is a graph showing improvement in left atrial weight. [Figure 13C] 1 is a graph showing improvement in relaxation time. [Figure 13D] 1 is a graph showing an improvement in the minimum value of dP / dt. [Figure 14A] 1 is a graph showing mean blood pressure. [Figure 14B] 1 is a graph showing diastolic blood pressure. [Figure 14C] 1 is a graph showing systolic blood pressure. [Figure 15A] 1 is a graph showing the ejection fraction of monotherapy of the compound of formula I and an exemplary SGLT2i (empagliflozin). [Figure 15B] FIG. 1 is a graph showing end systolic volume of monotherapy of the compound of formula I and an exemplary SGLT2i (empagliflozin). [Figure 15C] 1 is a graph showing end-diastolic volume of the compound of formula I and an exemplary SGLT2i (empagliflozin). [Figure 16A] 1 is a graph showing the ejection fraction of the compound of formula I in combination with empagliflozin compared to monotherapy. [Figure 16B] FIG. 1 is a graph showing end-systolic volume when the compound of formula I is used in combination with empagliflozin compared to monotherapy. [Figure 17A] 1 is a graph showing coronary perfusion pressure after treatment with the compound of formula. [Figure 17B] 1 is a graph showing coronary resistance following treatment with the compound of formula. [Figure 18] FIG. 1 shows the PK profile of the compound of formula I in rats (high oral bioavailability and low CL). [Figure 19] 1 shows the preliminary PK profile of the compound of Formula I in dogs. [Figure 20] 1 shows the PK profile of the compound of formula I in monkeys. [Figure 21] FIG. 1 shows LV relaxation following treatment with a compound of formula I and an exemplary SGLT2i (empagliflozin) compared to monotherapy. [Figure 22] FIG. 1 shows LV minimum pressure after treatment with a compound of formula I and an exemplary SGLT2i (empagliflozin) compared to monotherapy. [Figure 23] FIG. 1 shows end-diastolic pressure after treatment with a compound of formula I and an exemplary SGLT2i (empagliflozin) compared to monotherapy. [Figure 24A] FIG. 1 is a graph showing LV relaxation time illustrating that the combination of the compound of formula I with empagliflozin demonstrated additive effects compared to monotherapy in treating HFpEF with improvement in left ventricular filling pressures (diastolic and end diastolic pressures). [Figure 24B] FIG. 1 is a graph showing LV diastolic pressure, illustrating that the combination of the compound of formula I with empagliflozin demonstrated additive efficacy compared to monotherapy in treating HFpEF with improvement in left ventricular filling pressures (diastolic and end diastolic pressures). [Figure 24C] FIG. 1 is a graph showing end-diastolic pressure, illustrating that the combination of the compound of formula I with empagliflozin demonstrated additive efficacy compared to monotherapy in treating HFpEF with improvement in left ventricular filling pressures (diastolic and end-diastolic pressures). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular compounds, compositions, methodologies, procedures, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention, and is not intended to limit the scope of the invention, which is defined in the appended claims.

[0021] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All publications cited herein are incorporated by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that may be used in connection with the present invention.

[0023] The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology, and pharmacology within the skill of the art. Such techniques are explained fully in the literature (see, e.g., Gennaro, AR, ed. (1990) Remington's Pharmaceutical Sciences, 18th Edition). th ed., Mack Publishing Co., Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc., DMWeir, and CC Blackwell, eds. (1986) Handbook of Experimental Immunology, Vols. I-IV, Blackwell Scientific Publications, Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Vols. I-III, Cold Spring Harbor Laboratory Press, Ausubel, FMet al., eds. (1999) Short Protocols in Molecular Biology, 4 th(1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag; see Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press; Newton & Graham eds. (1997) PCR (Introduction to Biotechniques Series), 2nd ed., Springer Verlag).

[0024] The terms "PHD", "prolyl hydroxylase domain-containing protein", "HIF prolyl hydroxylase", "HPH" and "HIF PH" refer to members of the Egl-Nine (EGLN) gene family described by Taylor (2001, Gene 275:125-132) and characterized by Aravind and Koonin (2001, Genome Biol 2:RESEARCH 0007), Epstein et al. (2001, Cell 107:43-54), and Bruick and McKnight (2001, Science 294:1337-1340). The term "PHD1" refers to prolyl hydroxylase domain-containing protein-1. This protein is also called EIT6, HIF-PH1, HIFPH1, EGLN2, and HPH-3. PHD1 proteins include, but are not limited to, human PHD1 (GenBank Accession No. NP_444274.1), human EGLN2 isoform 1 (GenBank Accession No. CAC42510, Taylor (supra)), human EGLN2 isoform 3 (GenBank Accession No. NP_542770), mouse EGLN2 (GenBank Accession No. CAC42516), and rat EGLN2 (GenBank Accession No. AAO46039). PHD2 (also known as EGLN-1) includes, but is not limited to, human EGLN1 (GenBank Accession No. AAG33965, Dupuy et al. (2000) Genomics 69:348-54), mouse EGLN1 (GenBank Accession No. CAC42515), and rat EGLN1 (GenBank Accession No. P59722). PHD3 (also known as EGLN-3) includes, but is not limited to, human EGLN3 (GenBank Accession No. CAC42511, Taylor (supra)), mouse EGLN3 (GenBank Accession No. CAC42517), and rat EGLN3 (SM-20) (GenBank Accession No. AAA19321).In other embodiments of the invention, EGLN can include Caenorhabditis elegans EGL-9 (GenBank Accession No. AAD56365) and the Drosophila melanogaster CG1114 gene product (GenBank Accession No. AAF52050). All GenBank sequences associated with the indicated accession numbers are incorporated herein by reference in their entirety.

[0025] In addition to those provided above for the PHD1 protein, a further GenBank accession number associated with the human PHD1 gene is NM_053046.3 (mRNA), which is incorporated herein by reference in its entirety.

[0026] The terms "disorder," "disease," and "condition" are used inclusively herein to refer to any state that deviates from normal.

[0027] The terms "treating," "treatment," and the like are used herein to mean administering therapy to a patient in need thereof.

[0028] An "effective amount" of a compound is an amount sufficient to produce a desired result in a treated subject, for example, an amount sufficient to treat congestive heart failure, improve cardiac function, prevent or reduce the decline of cardiac function, or reduce cardiac damage. The effective amount may vary depending on, among other things, the particular compound, the nature or severity of the condition being treated, the age, weight, etc., of the subject being treated, the route of administration or formulation of the compound, and the administration regimen. The effective amount can be easily determined by those skilled in the medical arts.

[0029] The term "ester" refers to a compound of formula I in which the carboxylic acid is in the form -COOR, where R is alkyl, substituted alkyl, aryl, or substituted aryl. For example, esters of formula I include compounds in which R is alkyl. Esters of formula I can be provided via esterification of the hydroxyl group of the carboxylic acid and / or via esterification of the carboxylic acid moiety, for example, with a suitable reagent such as an acyl halide or anhydride. Such methods are well known in the art.

[0030] The term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts of Formula I, which are derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc. The pharmaceutically acceptable salts of the present invention can be provided by compounds of Formula I at the hydroxyl group at the C4 position of the isoquinoline and / or at the carboxylic acid moiety by methods well known in the art.

[0031] The term "stereoisomer" or "stereoisomers" refers to compounds that differ in the chirality of one or more stereocenters. Stereoisomers include enantiomers (non-superimposable mirror images of compounds) and diastereomers (compounds that have two or more stereoisomeric centers that are non-mirror images of one another and differ in one or more stereoisomeric centers between the two stereoisomers). The compounds of the present invention can exist as a mixture of stereoisomers or as a single stereoisomer.

[0032] The term "tautomer" refers to tautomers of compounds that differ in the location of a proton, such as enol, keto, imine enamine tautomers, or tautomers of heteroaryl groups that contain ring atoms bonded to both the ring NH and the ring =N moieties, such as pyrazole, imidazole, benzimidazole, triazole, tetrazole, etc.

[0033] The term "prodrug" as used herein refers to a compound of formula I that can be converted in vivo to a carboxylate group and / or cleaved from the amide N atom and / or cleaved from the C4 hydroxy to provide the active drug, a pharma- ceutically acceptable salt thereof, or a biologically active metabolite thereof. Suitable groups are well known in the art and include, in particular, for the carboxylic acid moiety, prodrugs selected from esters including, but not limited to, those derived from alkyl alcohols, substituted alkyl alcohols, hydroxy-substituted aryls, and heteroaryls, etc.

[0034] The term "excipient" as used herein means an inert or inactive substance used in the manufacture of a pharmaceutical or other tablet, including, but not limited to, substances used as binders, disintegrants, coating agents, compression / encapsulation aids, creams or lotions, lubricants, parenterals, sweeteners or flavoring agents, suspending / gelling agents, or wet granulation agents. Binders include, for example, carbopol, povidone, xanthan gum, etc.; coating agents include, for example, cellulose acetate phthalate, ethylcellulose, gellan gum, maltodextrin, etc.; compression / encapsulation materials include, for example, calcium carbonate, glucose, fructose dc, honey dc, lactose (anhydrous or monohydrate, optionally combined with aspartame, cellulose, or microcrystalline cellulose), starch dc, sucrose, etc.; disintegrants include, for example, croscarmellose sodium, gellan gum, sodium starch glycolate, etc.; creams and lotions include, for example, maltodextrin, carrageenan, etc.; lubricants include, for example, magnesium stearate, Examples of suitable oral preparations include glycerol, stearic acid, sodium stearyl fumarate, chewable tablet materials include dextrose, fructose dc, lactose (monohydrate, optionally combined with aspartame or cellulose), parenteral agents include mannitol, povidone, plasticizers include dibutyl sebacate, polyvinyl acetate phthalate, suspending / gelling agents include carrageenan, sodium starch glycolate, xanthan gum, sweeteners include dextrose, fructose dc, sorbitol, sucrose dc, and wet granulating agents include calcium carbonate, maltodextrin, microcrystalline cellulose, and the like.

[0035] With respect to the ability of the compounds of formula I to inhibit PHD1, the term "selectively" as used herein is intended to refer to a compound that is at least about 5 times more potent at inhibiting the activity of PHD1 than it is at inhibiting the activity of PHD2 and / or the activity of PHD3. Such compounds are referred to interchangeably herein as "PHD1 selective inhibitors," "PHD1 selective inhibitor compounds," and "compounds that selectively inhibit PHD1." "Inhibiting PHD1" or "inhibiting the activity of PHD1" means reducing, eliminating, or attenuating the enzymatic activity of the PHD1 protein. The selectivity of a compound is measured by the IC 50 Compounds that selectively inhibit PHD1 activity can be determined by determining the IC 50 Lower IC for PHD1 than 50 The compound exhibits the following properties:

[0036] The term "alkyl" refers to saturated monovalent hydrocarbyl groups having 1 to 10 carbon atoms, more specifically 1 to 5 carbon atoms, and even more specifically 1 to 3 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and the like. The term "C1-C4 alkyl" refers to alkyl having 1 to 4 carbon atoms, including methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, and the like.

[0037] The term "aryl" or "Ar" refers to a monovalent aromatic carbocyclic group of 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-1,4-benzoxazin-3(4H)-one-7-yl, etc.), provided that the point of attachment is the aryl group. Preferred aryls include phenyl and naphthyl.

[0038] The term "carboxyl" refers to --COOH or its salts.

[0039] The term "carboxylester" refers to the groups -C(O)O-alkyl, -C(O)O-alkenyl, -C(O)O-alkynyl, -C(O)O-cycloalkyl, -C(O)O-aryl, -C(O)O-heteroaryl, and -C(O)O-heterocyclic.

[0040] The term "cyano" refers to the group --CN.

[0041] The term "hydroxy" or "hydroxyl" refers to the group --OH.

[0042] The term "heteroaryl" refers to an aromatic ring of 1-15 carbon atoms, preferably 1-10 carbon atoms, and 1-4 heteroatoms in the ring selected from the group consisting of oxygen, nitrogen, and sulfur. Such heteroaryl groups can have a single ring (e.g., pyridinyl, furyl, or thienyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), provided that the point of attachment is through the ring containing the heteroatom and the ring is aromatic. The nitrogen and / or sulfur ring atoms can be optionally oxidized to provide N-oxide or sulfoxide, and sulfone derivatives. Examples of heteroaryl include, but are not limited to, pyridinyl, pyrimidinyl, pyrrolyl, pyrazolyl, indolyl, thiophenyl, thienyl, and furyl.

[0043] Cardiac function parameters End-diastolic volume (EDV) is the amount of blood in the ventricle at the end of filling, i.e., at the end of diastole, and end-systolic volume (ESV) is the amount of blood in the ventricle at the end of contraction, i.e., at the end of systole. ESV is a clinical measure of the adequacy of cardiac output. ESV can be measured using 2-dimensional echocardiography, magnetic resonance imaging (MRI), and computed tomography (CT). Stroke volume (SV) is the amount of blood output from the heart during one phase of the cardiac cycle and is calculated by subtracting ESV from EDV.

[0044] Ejection fraction (EF) is the percentage of blood pumped out of a filled ventricle with each heartbeat. Ejection fraction is usually measured in the left ventricle (LV) and is usually in the range of 55-70%. A reduced EF indicates weakening of the heart muscle and can occur when the heart muscle is damaged by myocardial infarction, myocardial disease (cardiomyopathy), or other causes. An EF below 40% suggests heart failure, and an EF below 35% increases the risk of sudden cardiac arrest. EF can be calculated by dividing SV by EDV and can be measured by imaging techniques such as cardiac ultrasound, cardiac catheterization, MRI, CT, and nuclear medicine (multi-gated acquisition (MUGA), also known as nuclear stress testing).

[0045] Cardiac output (CO) is the volume of blood ejected from the ventricles per minute. In a normal resting adult, CO is 4-8 liters per minute. CO can be measured using thermodilution techniques or calculated by multiplying SV by heart rate.

[0046] Stroke work (SW) is the amount of work done by the ventricle in pumping blood into the aorta. Stroke work can be estimated by multiplying SV by mean aortic pressure (MAP).

[0047] Cardiac contractility is the inotropic state of the myocardium and is the primary determinant of cardiac output and an important factor in cardiac compensation. The maximum rate of rise in left ventricular pressure (dP / dt max) is an index of the initial velocity of myocardial contraction and is used clinically to characterize the contractile ability of the heart. Normal LV dP / dt has a maximum value of approximately 1600 mmHg / sec, but is often below 1200 mmHg / sec in patients with myocardial impairment. Relaxation time, measured by the LV relaxation time constant (Tau), is used to evaluate LV diastolic function and is a sensitive sign of LV dysfunction in the setting of chronic heart failure. The normal range is 4-85 ms, but it can be prolonged to over 90 ms in various diseases that result in impaired myocardial relaxation. Tau can be calculated by dividing the LV pressure (P) by -dP / dt. The end-systolic pressure-volume relationship (ESPVR) represents the maximum pressure that the ventricle can develop at any given LV volume. The slope of the ESPVR represents the end-systolic elastance, which is an index of myocardial contractility. The ESPVR is relatively insensitive to changes in preload, afterload, and heart rate. This makes it a better indicator of systolic function than other hemodynamic parameters such as ejection fraction, cardiac output, and stroke volume. ESPVR becomes steeper and shifts to the left as inotropy (the force of contraction) increases. ESPVR becomes flatter and shifts to the right as inotropy decreases.

[0048] Damage to the myocardium can result from an acute insult, such as a myocardial infarction, or from a chronic insult, such as chronic overloading of the heart due to hypertension or arteriosclerosis. Chronic damage can and often does occur after a myocardial infarction due to reduced pumping function of the damaged heart. Under physical stress, the myocardium can thicken (hypertrophy), resulting in smaller heart chambers. Healthy cardiac hypertrophy (physiologic hypertrophy) is the normal response to healthy exercise, resulting in increased cardiac muscle mass and pumping power. Unhealthy cardiac hypertrophy (pathologic hypertrophy) is a response to stress or disease, resulting in hypertrophy of cardiomyocytes, hyperplasia of non-muscular myocardial components, and reduced pumping power. In pathologic hypertrophy, the mass of the heart can increase by up to 150%. As used herein, "cardiac hypertrophy" refers to pathologic hypertrophy.

[0049] method The method of the present invention comprises reacting a compound of formula I [ka] The present inventors have advantageously discovered that compounds of formula I can have beneficial therapeutic effects in improving cardiac function in subjects with congestive heart failure without the concomitant side effects resulting from the inhibition of other PHD isoforms. In particular, compounds of formula I do not significantly increase circulating erythropoietin, hemoglobin levels, or hematocrit in treated subjects. Thus, the present method has the great advantage that it can be used long-term to improve cardiac function without causing the hyperviscosity syndrome caused by erythrocytosis or cardiac complications associated with fluid overload.

[0050] In one embodiment, the present invention provides a method of treating congestive heart failure in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby treating congestive heart failure. Treating congestive heart failure may include, among others, improving cardiac function (e.g., as determined by an improvement or positive change in one or more well-known cardiac function parameters), preventing or reducing the decline of cardiac function (e.g., as determined by a prevention or reduction of the decline or lack of significant negative change in one or more well-known cardiac function parameters), or reducing cardiac damage (e.g., as determined by the effect on one or more well-known cardiac damage markers). Some examples of cardiac function parameters and cardiac damage markers are described herein, but as a competent medical practitioner can easily recognize, there are many more that are well-known and suitable for use in evaluating the effectiveness of treatment.

[0051] In another embodiment, the present invention provides a method for improving cardiac function in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby improving cardiac function. In various embodiments, the indicator of cardiac function can be selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, end systolic volume, end diastolic volume, and cardiac contractility. In some embodiments, the indicator of cardiac function can be selected from the group consisting of ejection fraction, stroke work, stroke volume, and cardiac output.

[0052] In another embodiment, the present invention provides a method for preventing or reducing the decline of cardiac function in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby preventing or reducing the decline of cardiac function. In various embodiments, the indicator of cardiac function may be selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, end systolic volume, end diastolic volume, and cardiac contractility. In some embodiments, the indicator of cardiac function may be selected from the group consisting of ejection fraction, stroke work, stroke volume, and cardiac output.

[0053] Cardiac function can be measured by any method known to those skilled in the art. The method of the present invention can be administered to a subject whenever the subject's cardiac function is outside the normal range, typically below or significantly below the normal range, such as when any one or more of EDV, ESV, SV, EF, CO, and SW are below the typical or normal range. It is within the ability of a medical practitioner of ordinary skill to determine whether a subject has cardiac function outside the normal range to the extent that treatment with the methods and compounds described herein would be beneficial. For example, the normal range of human left ventricular cardiac function is as follows for the parameters listed herein: [Table 2]

[0054] In another embodiment, the present invention provides a method for reducing cardiac damage in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, thereby reducing cardiac damage. In various embodiments, cardiac damage may be selected from the group consisting of infarct size (resulting from myocardial infarction), cardiac hypertrophy (e.g., as measured by cardiomyocyte diameter), and peri-infarct angiogenesis.

[0055] In one embodiment, the compound of formula I is administered to a subject having or at risk of having congestive heart failure. In a particular embodiment, the compound is administered to the subject after a myocardial infarction in the subject. In one embodiment, the compound is administered to prevent congestive heart failure after a myocardial ischemic event in the subject. In another embodiment, the compound is administered to the subject after a diagnosis of atherosclerosis, cardiomyopathy, congenital heart disease, or rheumatic fever in the subject.

[0056] In various embodiments, the method includes administering the compound of formula I periodically for an extended period of time. The frequency of administration in periodic administration can be, for example, once every 12, 24, 36, 48, 60, or 72 hours, once a day, once every 2, 3, 4, 5, or 6 days, or once a week. The duration of treatment can be one day or more, one week or more, one month or more, or one year or more. The extended treatment period usually means administering the compound two or more times.

[0057] This method can be advantageously used long term to improve cardiac function without polycythemia and fluid overload. In certain embodiments, a method is provided for treating congestive heart failure in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, wherein administration of the compound does not produce a concomitant significant increase in circulating hemoglobin concentration in the subject. In certain embodiments, a method is provided for treating congestive heart failure in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, wherein administration of the compound does not produce a concomitant significant increase in circulating erythropoietin concentration in the subject. In certain embodiments, a method is provided for treating congestive heart failure in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I, wherein administration of the compound does not produce a concomitant significant increase in hematocrit in the subject.

[0058] subject In various embodiments, the subject is an animal, particularly an animal of a mammalian species, including rats, rabbits, cows, sheep, pigs, mice, horses, and primates, hi a most preferred embodiment, the subject is a human.

[0059] In general, subjects suitable for carrying out the methods of the present invention (i.e., subjects in need of treatment for congestive heart failure, subjects in need of improvement of cardiac function, subjects in need of prevention or reduction of decline in cardiac function, or subjects in need of reduction of cardiac damage) include any subject having or at risk of having congestive heart failure. Suitable subjects include any mammalian subject, particularly human subjects. In various embodiments, the subject has congestive heart failure. In certain embodiments, the subject has or is at risk of having congestive heart failure due to myocardial infarction in the subject. In other embodiments, the subject has or is at risk of having congestive heart failure as a result of cardiac damage. In these embodiments, the cardiac damage can be selected from the group consisting of infarct size, cardiac hypertrophy, and peri-infarct angiogenesis. In other embodiments, the subject has or is at risk of having congestive heart failure due to coronary artery disease, hypertension, atherosclerosis, cardiomyopathy, congenital heart disease, or rheumatic fever.

[0060] Generally, subjects suitable for carrying out the method of the present invention include any subject with reduced cardiac function. In various embodiments, the indicator of cardiac function may be selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, and cardiac contractility.

[0061] compound The method of the present invention comprises reacting a compound of formula I [ka] The present invention utilizes compounds of the formula (I) which are capable of selectively inhibiting PHD1 over other isoforms, such as the PHD2 and / or PHD3 enzymes. As a result of this selectivity profile, the compounds are typically non-erythropoietic at the doses required to practice the invention, making them ideal candidates for chronic diseases such as congestive heart failure where increased erythropoietin is undesirable.

[0062] As used herein, references to "a compound of Formula I" are generally not limited to the free acid form shown in Formula I, but also include pharma- ceutically acceptable salts, esters, tautomers and prodrugs of that compound, where use of such forms is appropriate or preferred.

[0063] HIF-PH assay The selectivity profile of the compounds of formula I can be determined, for example, using any assay familiar to those of skill in the art and commonly available, such as the HIF-PH assay below.

[0064] Ketoglutarate α-[1- 14 C]-sodium salt, α-ketoglutarate sodium salt, and HPLC purified peptides can be obtained commercially, for example, from Perkin-Elmer (Wellesley MA), Sigma-Aldrich, and SynPep Corp. (Dublin CA), respectively. The peptides used in the assay can be fragments of HIFα as described above, or as disclosed in International Publication WO 2005 / 118836, which is incorporated herein by reference. HIF-PH, such as PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed, for example, in insect Hi5 cells and partially purified, for example, through a SP ion exchange chromatography column. Enzyme activity can be measured using the assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304), 14 The assay reaction was performed in 50 mM HEPES (pH 7.4), 100 μM α-ketoglutarate sodium salt, 0.30 μCi / mL ketoglutarate-α-[1- 14 C-sodium salt, 40 μM FeSO4, 1 mM ascorbate, and 1541.8 units / mL catalase. The reaction is initiated by the addition of HIF-PH enzyme. The IC of the compound for each enzyme is 50(the compound concentration that inhibits enzyme activity by 50%) can be determined.

[0065] The peptide-dependent turnover was calculated by subtracting the turnover in the absence of peptide from the turnover in the presence of the substrate peptide. Inhibition rate (%) and IC 50 is calculated using the peptide-dependent turnover rate at a given inhibitor concentration. The IC 50 The calculation of values ​​is performed using the software GraFit (Erithacus Software Ltd., Surrey UK). IC of test compounds for PHD1 and for PHD2 and / or PHD3 50 can be readily determined using the methods described above and can be used to assess the selectivity of compounds for PHD1 inhibition.

[0066] The compounds of formula I are at least about 5-fold more active in inhibiting the PHD1 enzyme than the PHD2 enzyme, i.e., IC 50 ,IC for PHD2 50 (i.e., IC 50 PHD2 / IC 50 PHD1) is greater than or equal to about 5. By way of example, the selectivity for compounds of formula I is shown below in Table 1. The selectivity of compounds of formula I as demonstrated in additional experiments is shown below in Table 1A. [Table 1] [Table 1A]

[0067] The compounds used in the methods of the present invention are advantageous over compounds previously disclosed in the art. In particular, the use of PHD1 selective inhibitor compounds in the treatment of congestive heart failure improves cardiac function without significantly increasing hematocrit or hemoglobin. Congestive heart failure associated with somatic inactivation of PHD2 has been attributed to hyperviscosity syndrome and fluid overload due to polycythemia. (Minamishima et al. (2008) Blood 111:3236-3244.) The use of non-specific HIF prolyl hydroxylase inhibitors, while showing improved cardiac function, has shown increased circulating erythropoietin, resulting in polycythemia, limiting their usefulness as long-term therapy. (Bao et al. (2010) J Cardiovasc Pharmacol 56(2):147-155.) Thus, the present invention provides substantial advantages over methods currently available in the art.

[0068] Pharmaceutical Formulations and Routes of Administration The composition of the present invention can be delivered as a pharmaceutical composition or medicament directly or with a suitable carrier or excipient, as is well known in the art. The treatment method of the present invention can include administering an effective amount of the compound of the present invention to a subject in need thereof. In a preferred embodiment, the subject is a mammalian subject, and in a most preferred embodiment, the subject is a human subject.

[0069] The effective amount of such compounds, compositions or agents can be easily determined by routine experimentation, as can the most effective and convenient route of administration and the most suitable formulation. A variety of formulations and drug delivery systems are available in the art. See, for example, Gennaro, AR, ed. (1995) Remington's Pharmaceutical Sciences, supra.

[0070] Suitable routes of administration can include, for example, oral, rectal, topical, nasal, pulmonary, intestinal, and parenteral administration. Primary routes of parenteral administration include intravenous, intramuscular, and subcutaneous administration. Secondary routes of administration include intraperitoneal, intraarterial, intraarticular, intracardiac, intrathoracic, intradermal, intralesional, intrapleural, intrathecal, intrauterine, and intraventricular administration. The physical, chemical, and biological properties of the drug, as well as the indication to be treated, determine the type of formulation and route of administration to be used, as well as whether local or systemic administration is desired.

[0071] Pharmaceutical dosage forms of the compounds of the invention can be provided in immediate release, controlled release, sustained release, or targeted drug delivery systems. Commonly used dosage forms include, for example, solutions and suspensions, (micro)emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols, lyophilized preparations, and the like. Depending on the route of administration used, special equipment may be required to apply or administer the drug, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks. Pharmaceutical dosage forms often consist of a drug, excipient(s), and a container / closure system. One or more excipients, also called inactive ingredients, can be added to the compounds of the invention to improve or facilitate the manufacture, stability, administration, and safety of the drug, and can provide a means to achieve a desired drug release profile. Thus, the type of excipient(s) added to the drug may depend on various factors, such as, for example, the physical and chemical properties of the drug, the route of administration, and the manufacturing procedure. Pharmaceutically acceptable excipients are available in the art and include those described in various pharmacopeias (see, e.g., the United States Pharmacopoeia (USP), the Japanese Pharmacopoeia (JP), the European Pharmacopoeia (EP), the British Pharmacopoeia (BP), the U.S. Food and Drug Administration (www.fda.gov), Center for Drug Evaluation and Research (CEDR) publications such as the Inactive Ingredient Guide (1996), Ash and Ash, Eds. (2002) Handbook of Pharmaceutical Additives, Synapse Information Resources, Inc., Endicott NY, etc.).

[0072] Pharmaceutical dosage forms of the compounds of the present invention can be prepared by any of the methods known in the art, such as, for example, conventional mixing, sieving, dissolving, melting, granulating, dragee making, tabletting, suspending, extruding, spray drying, shaping, emulsifying, (nano / micro)encapsulating, encapsulating, or lyophilizing processes. As mentioned above, the compositions of the present invention can contain one or more physiologically acceptable inactive ingredients that facilitate processing of the active molecules into preparations for pharmaceutical use.

[0073] The appropriate formulation depends on the desired route of administration. For intravenous injection, the composition can be formulated in an aqueous solution, using physiologically compatible buffers, such as phosphates, histidine, or citrates, as necessary, for example, to adjust the pH of the formulation, and isotonicity agents, such as sodium chloride or dextrose. For transmucosal or nasal administration, semi-solids, liquid formulations, or patches may be preferred, and may optionally include a penetration enhancer. Such penetrants are generally known in the art. For oral administration, the compound can be formulated in liquid or solid dosage forms, and immediate release or controlled / sustained release formulations. Suitable dosage forms for oral ingestion by a subject include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions, and emulsions. The compound can be formulated in rectal compositions, such as suppositories or retention enemas, containing conventional suppository bases, such as cocoa butter or other glycerides.

[0074] Solid oral dosage forms can be obtained using excipients that may include fillers, disintegrants, binders (dry and wet), dissolution retarders, lubricants, glidants, antiadherents, cation exchange resins, wetting agents, antioxidants, preservatives, colorants, and flavoring agents. These excipients can be of synthetic or natural origin. Examples of such excipients include cellulose derivatives, citric acid, dicalcium phosphate, gelatin, magnesium carbonate, magnesium / sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinylpyrrolidone, silicates, silicic acid dioxide, sodium benzoate, sorbitol, starch, stearic acid or its salts, sugars (i.e., glucose, sucrose, lactose, etc.), talc, tragacanth mucilage, vegetable oils (hydrogenated), and waxes. Ethanol and water can serve as granulation aids. In some cases, it is desirable to coat the tablets with, for example, a taste-masking film, a gastric acid-resistant film, or a release-retarding film. Natural and synthetic polymers are often used in combination with colorants, sugars, and organic solvents or water to coat tablets, resulting in dragees. When capsules are preferred over tablets, the drug powder, suspension, or solution can be delivered in a compatible hard or soft shell capsule.

[0075] In one embodiment, the compounds of the present invention can be administered locally via skin patches, semi-solid formulations, or liquid formulations, such as gels, (micro)emulsions, ointments, solutions, (nano / micro)suspensions, or foams. Penetration of drugs into the skin and underlying tissues can be controlled, for example, by using penetration enhancers; appropriate selection and combination of lipophilic, hydrophilic, and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, emulsifiers; by pH adjustment; and by the use of complexing agents. Other techniques, such as iontophoresis, may also be used to control the skin penetration of the compounds of the present invention. Transdermal or topical administration is preferred, for example, when topical administration with minimal systemic exposure is desired.

[0076] For administration by inhalation, i.e., administration to the nose, the compounds for use according to the present invention are conveniently delivered in the form of solution, suspension, emulsion, or semisolid aerosol from pressurized packs, or nebulizers, usually using propellants such as halogenated carbons derived from methane and ethane, carbon dioxide, or any other suitable gas. For topical aerosols, hydrocarbons such as butane, isobutene, and pentane are useful. For pressurized aerosols, the appropriate dosage form can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, can be formulated for use in inhalers and insufflators. These usually contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0077] Compositions formulated for parenteral administration by injection are usually sterile and can be provided in unit dosage form, for example, in ampoules, syringes, injection pens, or multi-dose containers, the latter usually including preservatives. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles and can include formulating agents such as buffers, isotonicity agents, viscosity enhancing agents, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents, and preservatives. Depending on the injection site, the vehicle can include water, synthetic or vegetable oils, and / or organic cosolvents. In certain cases, such as lyophilized products and concentrates, parenteral formulations are reconstituted or diluted prior to administration. Depot formulations providing controlled or sustained release of the compounds of the invention can include injectable suspensions of nano / microparticles, or nano / micro or non-micronized crystals. Polymers such as poly(lactic acid), poly(glycolic acid), or copolymers thereof, can function as controlled / sustained release matrices, in addition to others well known in the art. Other depot delivery systems may come in the form of implants and pumps that require an incision.

[0078] Carriers suitable for intravenous injection of the compounds of the present invention are well known in the art, and include, for example, aqueous solutions containing a base such as sodium hydroxide to form an ionized compound, sucrose or sodium chloride as an isotonicity agent, and buffers such as phosphate or histidine. Cosolvents such as polyethylene glycol may be added. These aqueous systems are effective in dissolving the compounds of the present invention and have low toxicity when administered systemically. The proportions of the components of the solution system can be changed significantly without compromising the solubility and toxicity properties. In addition, the identity of the components can be changed. For example, low toxicity surfactants such as polysorbates or poloxamers can be used, as can polyethylene glycol or other cosolvents, biocompatible polymers such as polyvinylpyrrolidone can be added, and other sugars and polyols can be used instead of dextrose.

[0079] The therapeutically effective amount can be initially estimated using various techniques known in the art. Initial doses used in animal studies can be based on effective concentrations established in cell culture assays. For example, data obtained from animal studies and cell culture assays can be used to determine a suitable dose range for humans. In certain embodiments, the compounds of the present disclosure are formulated for oral administration. Exemplary doses of the compounds of the present disclosure in pharmaceutical formulations for oral administration are about 0.01 mg / kg to about 60 mg / kg of subject's body weight, particularly 0.5 to about 20 mg / kg of subject's body weight. In some embodiments, the pharmaceutical formulation comprises about 0.7 to about 6.0 mg / kg of subject's body weight, or alternatively, about 1.0 to about 3.0 mg / kg of subject's body weight. A typical dosing regimen for oral administration is to administer the pharmaceutical formulation for oral administration once every 12, 24, 48, or 72 hours, once a day, once every 2, 3, 4, 5, or 6 days, or once a week.

[0080] An effective amount or therapeutically effective amount or dose of an agent, e.g., a compound of the invention, refers to the amount of the agent or compound that results in amelioration of symptoms or prolongation of survival in a subject. The toxicity and therapeutic efficacy of such molecules can be determined, for example, by LD 50 (the dose lethal to 50% of the population), and ED 50 The LD 2 ratio can be measured by standard pharmaceutical procedures in cell culture or experimental animals to determine the dose that is therapeutically effective in 50% of the population. The ratio of toxic to therapeutic effects is the therapeutic index, and the LD 2 ratio is the dose that is therapeutically effective in 50% of the population. 50 / ED 50 Agents that exhibit large therapeutic indices are preferred.

[0081] An effective amount, or therapeutically effective amount, is that amount of a compound or pharmaceutical composition that will elicit the biological or medical response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, physician, or other clinician. The dosage may be administered with little or no toxicity, and may be administered without ED 50 The circulating concentration range includes: 0.1 to 0.5% by weight of the compound of formula (I) and / or 0.1 to 0.5% by weight of the compound of formula (I). The dosage may vary within this range depending on the dosage form employed and / or the route of administration utilized. The exact formulation, route of administration, dosage, and dosage interval should be selected according to methods known in the art, taking into account the characteristics of the subject's condition.

[0082] Dosage and interval can be adjusted individually to provide plasma levels of the active moiety sufficient to achieve the desired effect, i.e., the minimum effective concentration (MEC). The MEC varies for each compound, but can be estimated, for example, from in vitro data and animal experiments. The dosage required to achieve the MEC will depend on individual characteristics and route of administration. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to the plasma concentration.

[0083] The amount of compound or composition administered can depend on a variety of factors, including the sex, age, and weight of the subject being treated, as well as the severity of the disease, the manner of administration, and the judgment of the prescribing physician.

[0084] The composition may be provided in a pack or dispenser device that contains one or more unit dosage forms containing active ingredient as needed.Such a pack or device may be made of metal or plastic foil such as blister pack, or glass and rubber stopper such as vial.The pack or dispenser device may be provided with instructions for administration.Also, the composition containing the compound of the present invention that is formulated in a suitable pharmaceutical carrier may be prepared, placed in a suitable container, and labeled for the treatment of the indicated condition.

[0085] These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in light of the disclosure herein and are specifically contemplated. EXAMPLES

[0086] The present invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the invention. The present invention is not limited in scope by the exemplified embodiments, which are intended only as illustrations of single aspects of the invention. Any functionally equivalent methods are within the scope of the invention.

[0087] Example 1. Effect of a compound of formula I on cardiac function in an animal model of congestive heart failure. The effect of the compound of formula I on cardiac function in congestive heart failure (CHF) was investigated by inducing CHF after myocardial infarction (MI). Three groups of rats (n=12) were subjected to MI by ligating the left coronary artery for 30 minutes followed by reperfusion. In the sham group, rats underwent a similar operation except that the left coronary artery was not occluded. Treatment with the compound of formula I was started 2 days after reperfusion. In the MI group, the compound was orally administered at vehicle, 6 mg / kg, or 20 mg / kg every other day from day 2 to day 26 after MI. In the sham group, rats were administered vehicle on the same schedule. On day 28 after MI, cardiac function, heart weight, and complete blood count (CBC) were measured. Histomorphometric analysis of cardiac sections was performed to quantify collagen deposition by picrosirius red staining (PSR), remodeling by hematoxylin and eosin staining, and vessel number by immunohistochemistry for smooth muscle α-actin.

[0088] As shown in Figure 1, treatment with the compound of formula I significantly improved ejection fraction (Figure 1A), relaxation time (Figure 1B), and stroke work (Figure 1C) compared to vehicle-treated MI animals. Treatment with the compound of formula I also significantly improved cardiac hypertrophy, stroke volume, cardiac output, end-systolic volume, end-systolic pressure, end-diastolic pressure, and arterial elastance compared to vehicle-treated animals. Treatment with the compound of formula I also normalized the slope of the ESPVR. Histomorphometric analysis of the left ventricle showed that thinning of the ventricular wall was prevented and peri-infarct hypervascularization was significantly reduced. Treatment with the compound of formula I did not result in a significant increase in circulating hemoglobin levels at the same time, nor did it significantly affect other CBC parameters.

[0089] Similar experiments were also performed where rats were given lower doses more frequently: vehicle, 2 mg / kg, 6 mg / kg, or 20 mg / kg of compound were orally administered daily from day 2 to day 26 post-MI. These results show that lower doses given at more frequent intervals are as effective as higher doses given at less frequent intervals.

[0090] These results show that the administration of the compound of formula I effectively treats congestive heart failure.The administration of the compound of formula I every day or every other day for 28 days significantly improves cardiac function in congestive heart failure after MI, and significantly improves ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, cardiac hypertrophy, end systolic volume and cardiac contractility.The improvement of cardiac function and treatment of congestive heart failure are achieved without increasing blood hemoglobin.

[0091] Example 2. Effect of delayed administration of a compound of formula I on cardiac function in an animal model of congestive heart failure. The effect of delayed treatment of the compound of formula I on cardiac function in congestive heart failure (CHF) was examined by inducing CHF after MI. Four groups of rats (n=11-12) were subjected to MI by left coronary artery ligation for 30 minutes followed by reperfusion. In the sham group, rats underwent a similar procedure except that the left coronary artery was not occluded. Cardiac function was measured in one group on day 28 (baseline) after MI. In the remaining MI groups, vehicle or test article administration was initiated on day 28. Rats were orally administered vehicle, 6 mg / kg, or 20 mg / kg of the compound every other day from day 28 to day 54 after MI. On day 56 after MI, cardiac function, heart weight, and complete blood count (CBC) were measured.

[0092] As shown in Figure 2, delayed treatment with the compound of formula I significantly improved the ejection fraction (Figure 2A) and end systolic volume (Figure 2B) at 56 days post-MI compared to vehicle-treated MI animals. Delayed administration of the compound of formula I also improved cardiac hypertrophy, dPdt max , relaxation time, and end-diastolic volume. Treatment with the compound of formula I did not significantly affect hematocrit (FIG. 3A) and hemoglobin (FIG. 3B) levels, indicating that long-term treatment of congestive heart failure with the compound of formula I is not complicated by polycythemia-induced hyperviscosity syndrome and fluid overload.

[0093] These results indicate that when administered at a more advanced stage of congestive heart failure, the compound of formula I effectively improves cardiac function. Administration of the compound of formula I, starting 28 days after myocardial infarction and administered every other day for 28 days, significantly improved cardiac function, including improved ejection fraction, end systolic volume, relaxation time, cardiac hypertrophy, and cardiac contractility.

[0094] Example 4. Effect of a compound of formula I on cardiac function in an animal model of congestive heart failure - daily administration. The effect of the compound of formula I on cardiac function in congestive heart failure (CHF) was investigated by inducing CHF after myocardial infarction (MI). Four groups of rats (n=12) were subjected to MI by ligating the left coronary artery for 30 minutes followed by reperfusion. In the sham group, rats underwent a similar operation except that the left coronary artery was not occluded. Treatment with the compound of formula I was started 2 days after reperfusion. In the MI group, the rats were orally administered vehicle, 2 mg / kg, 6 mg / kg, or 20 mg / kg of the compound daily from day 2 to day 26 after MI. In the sham group, the rats were administered vehicle on the same schedule. On day 28 after MI, cardiac function, heart weight, and complete blood count (CBC) were measured.

[0095] Daily administration of the compound of formula I significantly improved ejection fraction (FIG. 4A), systolic volume (FIG. 4B), and stroke work (FIG. 4C) compared to vehicle-treated MI animals. Daily administration of the compound of formula I significantly improved cardiac hypertrophy, stroke volume, cardiac output, relaxation time, dPdt compared to vehicle-treated animals. max , end-diastolic pressure, and arterial elastance were also significantly improved. Daily treatment with the compound of formula I did not result in a concomitant significant increase in circulating hemoglobin levels, nor did it significantly affect other CBC parameters.

[0096] These results show that the administration of the compound of formula I effectively treats congestive heart failure. Daily administration of the compound of formula I for 28 days significantly improves cardiac function in congestive heart failure after MI, and significantly improves ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, cardiac hypertrophy, end systolic volume and cardiac contractility. The improvement of cardiac function and treatment of congestive heart failure are achieved without increasing blood hemoglobin.

[0097] Example 5. Compound of formula I 3-[(1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carbonyl)-amino]-3-methyl-butyric acid [ka]

[0098] This compound was prepared according to the method disclosed in US Patent 9,409,892. Briefly, to 1-cyano-4-hydroxy-7-phenoxy-isoquinoline-3-carboxylic acid methyl ester (100 mg, 0.31 mmol) and 3-amino-3-methyl-butyric acid (Oakwood (97 mg, 0.94 mmol) in DMF (3 mL)) in DMF (3 mL) was added solid NaOMe (68 mg, 1.25 mmol). The resulting mixture was heated in an oil bath at 150-160 °C for 3 h. The reaction mixture was diluted with water (100 mL) and acidified to pH = 3-4 with 1N hydrochloric acid. The precipitate was collected and dried. The crude residue was purified by silica gel chromatography eluting with 10 → 50% EtOAc / CH2Cl2. The fractions containing the product were collected and concentrated. It was triturated with MeOH (2 mL). The solid was collected and dried in vacuum to give 51 mg (0.13 mmol) of the title compound in 41% yield. LC-MS ESI-: 404.06 (M-1) - .I C 50 PHD2 / IC 50 PHD1=23.

[0099] Example 6. Acute Cardioprotection

[0100] The compound of formula I dose-dependently reduced infarct size following acute myocardial ischemia, as shown in FIG.

[0101] The compound of formula I improved left ventricular function and coronary flow after global ischemia, as shown in Figures 6A, 6B, and 6C.

[0102] Example 7. Activity in preclinical heart failure models Efficacy testing of the compound of formula I demonstrated significant improvements across multiple cardiac parameters following injury. Experiments were performed as described below.

[0103] A) Rat model of ischemic heart failure with reduced ejection fraction (HFrEF)

[0104] Preclinical trial design: Male Sprague Dawley rats The left coronary artery is ligated for 30 minutes, followed by reperfusion. Recovery after 28 days of ligation Cardiac function 28 days after ischemia-reperfusion using a Millar pressure-volume catheter inserted into the left ventricle via the carotid artery Pressure-volume loops are recorded over several cardiac cycles and cardiac function parameters are derived.

[0105] Protocol and findings: [Table 3]

[0106] result: The compound of Formula I as monotherapy inhibited cardiac hypertrophy after 28 days of administration (QOD), as shown in Figures 7A and 7B.

[0107] The compound of formula I as monotherapy significantly improved cardiac function after 28 days of dosing (QOD), as shown in Figures 8A (ejection fraction, EF), 8B (stroke work), 8C (stroke volume), and 8D (cardiac output).

[0108] The compound of formula I maintained left ventricular wall thickness and reduced collagen deposition at 28 days of administration (QOD), as shown in FIG.

[0109] The dose response of the compound of formula I on cardiac function in congestive heart failure (CHF) was examined by inducing CHF after myocardial ischemia, as shown in Figures 10A and 10B, to determine the minimal effective dose (MED) of 2 mg / kg.

[0110] The effect of administration duration of a compound of formula I on cardiac function in congestive heart failure (CHF) was examined by inducing CHF after myocardial infarction to determine the minimum treatment duration, as shown in Figures 11A, 11B, and 11C.

[0111] B) ZSF1 model of heart failure with preserved ejection fraction (HFpEF)

[0112] This study measured the effect of a compound of formula I on cardiac function in male obese ZSF1 rats after 16 weeks of treatment.

[0113] Lean ZSF1 rats were used as controls.

[0114] Obese ZSF1 rats were developed by crossing diabetic Zucker fatty rats with spontaneously hypertensive heart failure (SHHF) rats.

[0115] Obese ZSF1 rats exhibited the following: High blood pressure Renal dysfunction obesity type 2 diabetes Insulin resistance, hyperinsulinemia Hypertriglyceridemia Hypercholesterolemia.

[0116] These comorbidities led to the development of heart failure with preserved ejection fraction in ZSF1 rats, as shown in FIG.

[0117] Compound of Formula I monotherapy improved diastolic function across multiple parameters, as shown in Figures 13A (end-diastolic pressure), 13B (left atrial mass), 13C (relaxation time), and 13D (dp / dt minimum).

[0118] Formula I compound monotherapy normalized blood pressure as shown in Figures 14A (mean blood pressure), 14B (diastolic blood pressure), and 14C (systolic blood pressure).

[0119] Example 8. Combined activity of the compound of formula I and an SGLT2i in preclinical heart failure models A) Rat model of ischemic heart failure with reduced ejection fraction (HFrEF) The compound of formula I and the exemplary SGLT2i, empagliflozin, each showed similar efficacy as monotherapy in improving cardiac function in HFrEF, as shown in Figures 15A (ejection fraction), 15B (end-systolic volume), and 15C (end-diastolic volume).

[0120] The combination of the compound of formula I and empagliflozin showed additive effects in treating myocardial ischemia-induced heart failure, as shown in Figures 16A and 16B.

[0121] The findings that myocardial ischemia-induced heart failure was treated with a combination of the compound of formula I and empagliflozin, resulting in a 40-50% improvement in ejection fraction and end systolic volume, compared to the similar approximately 30% improvement observed when each treatment was used alone, indicating a significant additive effect of combining these therapies.

[0122] B) ZSF1 model of heart failure with preserved ejection fraction (HFpEF) The compound of formula I and the exemplary SGLT2i, empagliflozin, each showed similar efficacy as monotherapy in improving cardiac function in HFpEF, as shown in FIG. 21 (LV relaxation), FIG. 22 (LV nadir pressure), and FIG. 23 (end-diastolic pressure).

[0123] Also, as shown in Figure 1, the combination of the compound of formula I with empagliflozin demonstrated additive effects in the treatment of HFpEF. The improvement in left ventricular filling pressures (minimum diastolic pressure and end diastolic pressure) was more pronounced with the combination therapy than with the monotherapy.

[0124] Example 9. Pharmacology Compounds of formula I induced coronary vasodilation in isolated heart preparations, as shown in Figures 17A and 17B.

[0125] Example 10. Pharmacokinetics (PK), Absorption, Distribution, Metabolism, and Excretion (ADME) The PK profile of the compound of formula I in rats (high oral bioavailability and low oral bioavailability CL) was studied and the results are shown in FIG. 18 and summarized in the table below. [Table 4]

[0126] Oral bioavailability was estimated to be approximately 78%.

[0127] The compound of formula I exhibited a low CL of 22 mL / hr / kg, lower than the hepatic blowdown rate.

[0128] Vss was higher than the plasma volume, suggesting possible distribution to tissue compartments.

[0129] The preliminary PK profile of the compound of Formula I in dogs was investigated, and the results are shown in FIG. 19 and summarized in the table below. [Table 5]

[0130] Oral bioavailability in dogs was estimated to be approximately 25%.

[0131] The clearance in dogs was higher than that in rats (approximately 5-10 times the CL value in rats).

[0132] Vss was higher than the plasma volume, suggesting possible distribution to tissue compartments.

[0133] The PK profile of the compound of formula I in monkeys was examined and the results are shown in Figure 20 and summarized in the table below. Of note, this compound had good oral bioavailability. [Table 6]

[0134] Oral bioavailability ranged from 36 to 48% (group mean 42 to 47%).

[0135] In general, no significant gender differences were observed in compound exposure or key PK parameters.

[0136] Exposure to the compound increased proportionately.

[0137] Systemic clearance in monkeys was similar to that in rats (but slower than that in dogs).

[0138] Vss was higher than the plasma volume, suggesting possible distribution to tissue compartments.

[0139] The PK properties of the compound of formula I are summarized in the table below. [Table 7]

[0140] The distribution of compounds of formula I in tissues of interest is summarized in the table below. [Table 8]

[0141] N=3 / time point, male Swiss Webster mice, single intravenous dose of 60 mg / kg of compound of formula I

[0142] All references cited herein are incorporated by reference in their entirety.

Claims

1. A method for treating congestive heart failure in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula I. 【Chemistry 5】 thereby treating congestive heart failure.

2. A method for improving cardiac function in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula I. 【Chemistry 6】 thereby improving cardiac function.

3. 3. The method of claim 2, wherein the cardiac function is selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, end systolic volume, end diastolic volume, and cardiac contractility.

4. A method for preventing or reducing a decline in cardiac function in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula I. 【Chemistry 7】 thereby preventing or reducing the decline in cardiac function.

5. 5. The method of claim 4, wherein the cardiac function is selected from the group consisting of ejection fraction, stroke work, stroke volume, cardiac output, relaxation time, end systolic volume, end diastolic volume, and cardiac contractility.

6. A method for reducing cardiac damage in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of formula I. 【Chemistry 8】 thereby reducing cardiac damage.

7. 7. The method of claim 6, wherein the cardiac injury is selected from the group consisting of infarct size, cardiac hypertrophy, and peri-infarct angiogenesis.

8. The method of any one of claims 1 to 7, wherein the subject has or is at risk of having congestive heart failure.

9. 9. The method of any one of claims 1 to 8, wherein the subject has been diagnosed with a disorder selected from the group consisting of myocardial infarction, atherosclerosis, cardiomyopathy, congenital heart disease, and rheumatic fever.

10. The method of any one of claims 1 to 9, wherein said administering comprises periodic administration of the compound over a long-term treatment period.

11. 11. The method of claim 10, wherein the administration frequency is selected from the group including once every 12 hours, once every 24 hours, once every 48 hours, once every 72 hours, once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, and once every week.

12. The method of any one of claims 1 to 11, wherein the method does not produce a concomitant significant increase in circulating hemoglobin concentration in the subject.

13. The method according to any one of claims 1 to 12, wherein the compound of formula I is used in combination with an inhibitor of sodium-glucose cotransporter 2 (SGLT2).

14. 14. The method of claim 13, wherein the SGLT2 inhibitor is selected from the group consisting of canagliflozin, dapagliflozin, and empagliflozin.

Images