Androstane derivatives with activity as pure or nearly pure stimulators of SERCA2a for the treatment of heart failure

Abstract

Compounds and compositions for the activation of SERCA2a are disclosed. + / K + Provided are compounds that only moderately inhibit ATPase, but act as near-pure or pure SERCA2a activators.Generally, the disclosed compounds are derivatives of androstane represented by formula (I).Also disclosed herein are pharmaceutical compositions comprising one or more compounds of formula (I) for use in treating heart failure. TIFF2022551165000145.tif4261

Description

[Technical Field]

[0001] The present invention relates to the field of medicine, in particular to androstane derivatives for use in the treatment of acute heart failure. [Background technology]

[0002] The prevalence of heart failure (HF) is age-dependent, ranging from less than 2% in people under 60 years of age to more than 10% in people 75 years of age and older (Metra M & Teerlink JR, Lancet 2017, 390:1981-1995). Most patients with HF have a history of hypertension, coronary artery disease, cardiomyopathy, valvular disease, or a combination of these disorders (Metra M & Teerlink JR, Lancet 2017, 390:1981-1995). The calculated lifetime risk of developing HF is expected to increase, with individuals with hypertension at higher risk (Lloyd-Jones DM et al., Circulation 2002, 106:3068-3072). Patients with HF have a poor prognosis and higher rates of hospitalization and mortality.

[0003] The clinical symptoms of HF are caused by a dual pathological feature of the heart that results in inotropic abnormalities, resulting in reduced systolic ejection (systolic dysfunction) and impaired compliance that reduces the ability of the ventricles to aspirate blood from the venous system (diastolic dysfunction). This reduces the amount of blood available for systolic contraction (impaired left ventricular (LV) filling). Impaired contraction and relaxation are due to intracellular Ca 2+ Ca stored in the sarcoplasmic reticulum (SR) 2+ Due to a decrease in intracellular Ca uptake 2+ This is the result of an abnormal distribution of Ca in the SR membrane, which is an active membrane transporter (Bers DM et al., Ann NY Acad Sci 2006, 1080:165-177). 2+The SR is driven by the ATPase (SERCA2a). SERCA2a activity is physiologically restricted by its interaction with phospholamban (PLN) (Bers DM., Annu Rev Physiol 2008, 70:23-49; MacLennan DH & Kranias EG, Nat Rev Mol Cell Biol 2003, 4(7): 566-577), and such restriction is normally relieved by PLN phosphorylation by protein kinase A (PKA), a signaling pathway that is significantly suppressed as a result of HF remodeling (Lohse M et al., Circ Res 2003, 93:896-906). SERCA2a function is therefore impaired in failing myocardium (Bers DM et al., Ann NY Acad Sci 2006, 1080:165-177), thus limiting Ca uptake by the SR. 2+ In addition to its effects on muscle cell contraction and relaxation, it is primarily responsible for the abnormal Ca 2+ The distribution also promotes cardiac arrhythmias (Zaza & Rocchetti, Curr Pharm Des 2015, 21:1053-1061) and, in the long term, accelerates myocyte loss by apoptosis (Nakayama H et al., J Clin Invest 2007, 117:2431-44). Decreased SERCA2a function inhibits Ca transport via the less energy-efficient Na-Ca exchanger (NCX). 2+ It also increases the energetic cost of contraction, requiring a compensatory increase in release of SERCA2a (Lipskaya L et al., Expert Opin Biol Ther 2010, 10:29-41). Substantial evidence suggests that normalizing SERCA2a function reduces intracellular Ca release. 2+It has been shown to restore homeostasis and improve cardiac contraction and relaxation in cardiomyocytes and in situ (Byrne MJ et al., Gene Therapy 2008, 15:1550-1557; Sato et al., JBC 2001, 276:9392-99). In summary, restoring SERCA2a function in HF may improve cardiac relaxation, and possibly contractility, while minimizing arrhythmias, myocardial oxygen consumption, and myocyte death (Lipskaya L et al., Expert Opin Biol Ther. 2010, 10:29-41). This highlights the need for "pure" SERCA2a activators. Indeed, SERCA2a activation is essential for Ca upregulation. 2+ Improved sequestration allows Ca 2+ Ca that maintains the wave 2+ -induced-Ca 2+ Ca negative feedback on release 2+ It may increase the intra-SR threshold for wave generation (Fernandez-Tenorio M & Niggli EJ, Mol Cell Cardiol 2018, 119:87-95). Therefore, pure or nearly pure SERCA2a activation may reduce arrhythmogenic risk, and therefore compounds with SERCA2a stimulating properties are of interest.

[0004] In conclusion, novel molecules that can independently enhance SERCA2a function may improve overall cardiac function in HF, providing a strong motivation to explore new compounds with such pharmacodynamic profiles.

[0005] Current long-term treatments for HF aim to prevent "myocardial remodeling" (e.g., beta-blockers, ACE inhibitors, aldosterone antagonists), a chronic maladaptive response to reduced contractility that amplifies the initial injury and underlies disease pathogenesis (Heineke J & Molkentin D, Nat Rev 2006, 7:589-600). While this approach has undisputed merit, it does not target the impaired "contraction" and "relaxation," the dysfunctions that define HF and contribute to its symptoms. Indeed, medications that improve cardiac contractility / relaxation ("inotropic / losorexic agents") remain widely used and essential in patient management, especially in advanced disease stages (Metra M & Teerlink JR, Lancet 2017, 390:1981-1995). These include sympathomimetic amines (dobutamine) and Ca2+, a potent vasodilator. 2+These include the sensitizer levosimendan. Unfortunately, these agents act through mechanisms with potentially harmful effects, including promotion of life-threatening arrhythmias, increased myocardial oxygen consumption, and impaired already inadequate coronary blood flow due to vasodilation-induced reductions in blood pressure (Ashkar H, Makaryus AN StatPearls. Treasure Island (FL): StatPearls Publishing, 2018 Jan-2017 Dec 19 (https: / / www.ncbi.nlm.nih.gov / books / NBK470431 / ); Gong B. et al., J Cardiothorac Vasc Anesth 2015, 29: 1415-25 EDITORIAL). This limits the use of inotropic agents to the late stage of the disease, thereby negating the potential benefit of improving contractility early in the disease course. Furthermore, these drugs do not improve patient prognosis and survival, and their therapeutic use requires careful monitoring (Ashkar H & Makaryus AN, StatPearls. Treasure Island (FL): StatPearls Publishing, 2018 Jan-2017 Dec 19) (Gong B. et al., J Cardiothorac Vasc Anesth 2015, 29: 1415-25 EDITORIAL).

[0006] Among the positive inotropic agents, the cardiac glycoside digoxin inhibits Na + / K +Digoxin is an inhibitor of ATPase enzyme activity and remains one of the most commonly prescribed medications. However, its use has declined over the past several decades due to the difficulty of maintaining digoxin within the beneficial serum concentration range (0.5–0.7 ng / ml) without reaching the threshold level of 0.9 ng / ml, above which an increased risk of death, primarily due to arrhythmias, has been observed (Packer M, Journal of Cardiac Failure 2016, 22:726–730; Packer M, Eur J Heart Failure 2018, 20:851–852).

[0007] Research is also actively underway to develop HF medications with mechanisms of action other than positive inotropy. Among the many most investigated and currently in clinical development are: serelaxin (a recombinant relaxin 2 mediator); ularitide (a recombinant natriuretic peptide); omecamtiv mecarbil (a cardiac myosin activator); BMS986231 (a NO donor); adrenomedullin inhibitor; ANX-042 (a splice variant of NP); and TD1439 (a neprilysin (NEP) inhibitor). However, when evaluated in phase 2-3 clinical trials, none of these new agents met their primary endpoints without safety concerns.

[0008] The clinical course and prognosis of patients with chronic HF (CHF) significantly worsen after the onset of acute HF (AHF) (Solomon SD et al., Circulation 2007, 116:1482-87). AHFS can be defined as the new onset or recurrence of HF symptoms and signs, requiring urgent evaluation and treatment and resulting in unplanned care or hospitalization. Half of patients with AHFS have reduced systolic function (HFrEF), a potential target for future treatment (Braunwald E. Lancet 2015; 385:812-24). Treatment of AHFS in patients with reduced ejection fraction (rEF) focuses on relieving congestion with vasodilators, diuretics, or ultrafiltration, or by increasing cardiac output with positive inotropic agents. Although this treatment strategy reduces the risk of sudden cardiac death, post-discharge event rates remain unacceptably high in patients hospitalized with AHFS. Many undesirable cardiovascular side effects can be caused by available treatments, including myocardial ischemia, cardiac damage, and arrhythmias due to inotropic therapy, particularly in patients with coronary artery disease (CAD) (Abraham WT et al., J Am Coll Cardiol 2005, 46:57-64; Flaherty JD et al., J Am Coll Cardiol. 2009, 53(3):254-63), hypotension, and vasodilator-induced end-organ (renal) hypoperfusion, particularly in hypotensive HF patients. Therefore, the primary goal during hospitalization is to improve cardiac output without causing cardiac and / or renal damage. Furthermore, little focus has been placed on the investigation or treatment of impaired left ventricular (LV) diastolic relaxation, which accounts for HF symptoms in the remaining 50% of patients with HF but preserved EF. Furthermore, AHFS patients with reduced EF also have impaired ventricular relaxation, which contributes to overall impaired cardiac function.Various echocardiographic indices have been developed to measure cardiac relaxation in both animal models and patients with HF (e.g., a decrease in early mitral annular tissue velocity [e'] and a decrease in early mitral inflow [E] deceleration time [DT]), as well as echocardiographic parameters of increased LV filling pressure (e.g., the E / e' ratio). While the response of a single indice is not fully superimposable in some animal models and patients, the overall changes in animal models of impaired ventricular relaxation are certainly translatable to the human condition and are used to study drug effects in AHFS (Shah SA et al., Am Heart J 2009, 157:1035-41).

[0009] In recent years, various therapeutic approaches to improve SERCA2a function have been investigated. These include transgenic overexpression of SERCA2a (Byrne et al., Gene Therapy 2008, 15:1550-1557), inactivation of PLN by expression of dominant-negative mutants (Hoshijima M et al., Nat. Med. 2002, 8: 864-871; Iwanaga Y et al., J Clin Investig 2004, 113: 727-736), AdV-shRNA (Suckau L et al., Circulation 2009, 119: 1241-1252), microRNA (Groβl et al., PLoS One 2014, 9: e92188), or antibodies (Kaye DM et al., J. Am. Coll. Cardiol. 2007, 50:253-260). These approaches suffer from significant problems in construct delivery (e.g., viral vectors) and dose adjustment, which are far from being resolved, as highlighted by the negative results of the largest Phase IIb clinical trial (CUPID2) of SERCA2a gene delivery in HF (Hulot JS, Eur Heart J 2016, 19:1534-1541). A small molecule (pyridone derivative) that inhibits PLN, structurally distinct from istaloxime, has recently been reported (Kaneko M. et al., Eur J Pharmacol 2017, 814:1-7).

[0010] Therefore, the development of small molecule SERCA2a activators would be advantageous for the treatment of HF and still represents a highly promising strategy.

[0011] Istaroxime is a new small molecule drug currently in clinical development for the treatment of AHFS. It is disclosed in EP0825197 and S. De Munari et al. (J. Med. Chem. 2003, 64:3644-3654) and is the compound (3Z,5α)-3-[(2-aminoethoxy)imino]androstane-6,17-dione. Istaroxime activates SERCA2α (Rocchetti M et al., J. Pharmacol Exp Ther. 2005, 313:207-15), while inhibiting Na+. + / K + It has a dual mechanism of action: inhibiting the pump (Micheletti et al., J Pharmacol Exp Ther 2002, 303:592-600). At the same level of inotropy, the proarrhythmic effect of istaloxime is greater than that of pure Na + / K + This is significantly lower than that of the pump inhibitor digoxin (Rocchetti M et al., J Pharmacol Exp Ther. 2005, 313:207-15). 2+ By improving clearance (Alemanni, J Mol Cell Cardiol 2011, 50:910-8), SERCA2a stimulation may improve Na clearance while maintaining its inotropic effect. + / K + It has been suggested that istaloxime may also minimize the proarrhythmic effects of pump blockade (Rocchetti M et al., J Pharmacol Exp Ther. 2005, 313:207-15; Zaza & Rocchetti, Curr Parm Des 2015, 21:1053-1061). This reduction in proarrhythmic effects with istaloxime has been confirmed in clinical studies (Gheorghiade M et al., J Am Coll Cardiol 2008, 51:2276-85).

[0012] In patients with HF, infusion of istaloxime improved both systolic and diastolic function (Horizon study) (Gheorghiade M et al., J Am Coll Cardiol 2008, 51:2276-85; Shah SA et al., Am Heart J 2009, 157:1035-41). Improved systolic function was detected as an increase in contractile tissue velocity (s') and an increase in the slope of end-systolic elastance (ESPVR slope); increased diastolic compliance was manifested by an increase in diastolic tissue velocity (e') and a decrease in end-diastolic elastance (EDPVR slope) (Shah SA et al., Am Heart J 2009, 157:1035-41).

[0013] Although it has an excellent pharmacodynamic profile, istaloxime is not optimal for long-term administration due to its poor gastrointestinal (GI) absorption and high clearance rate. Therefore, it has only been developed for intravenous infusion in hospitalized patients with AHFS, and its administration requires well-trained medical personnel (Dec GW, J Am Coll Cardiol. 2008, 51:2286-88; Shah SA et al., Am Heart J 2009, 157:1035-41).

[0014] Therefore, there is a long-standing need for compounds for use in the treatment of HF that have positive lusitropic effects and that can be administered preferably by the oral route (Butler J et al., Eur J Heart Failure 2018, 20:839-841; Wagner S et al., Circ Res 2015, 116:1956-1970; Hasenfuss G & Teerlink JR., Eur Heart J. 2011, 32(15):1838-45).

[0015] It is possible that improved diastolic function could be achieved with "pure" SERCA2a activators. However, despite intensive research into the discovery of small molecule or gene therapies aimed at selectively activating SERCA2a, promising clinical results have not been achieved so far.

[0016] The present invention fulfills the above needs and overcomes the problems of the prior art. Summary of the Invention

[0017] Certain androstane derivatives have now been found to exhibit pure or nearly pure SERCA2a activation. In other words, the androstane derivatives provided herein significantly activate SERCA2a but do not induce Na+ + / K + They do not inhibit, or only moderately inhibit, the ATPase pump. Generally, these androstane derivatives contain a functional group attached via a carbon linker at carbon-3 (C3) and a functional group at C6 and / or C7. The structure of these pure or nearly pure SERCA2a activators has the general formula (I) shown below: [ka] [In the formula, X is any of carboxylic acids, carboxylic esters, and their bioisosteres (sulfates, sulfonic acids, phosphates, phosphonates, or nitrogen-containing ether cyclic rings such as triazoles and tetrazoles), primary alcohols, ethers, or amine groups (e.g., primary amines, secondary amines, or cyclic amines); n is 1, 2, 3, 4, or 5; The dashed line at C3-C1' represents an optional exocyclic double bond C=C at the C3-C1' position; The dashed line at C2-C3 represents the optional endocyclic double bond C=C; Y at C6 is hydroxyl (OH) in the α- or β-configuration, or hydroxymethyl (CHOH) in the α-configuration; Z at C7 is either -H, or -OH in the α-configuration, or a ketone, and the dashed line represents an optional carbonyl group (C=O) at that position. It has the following structure. The compounds disclosed herein may include enantiomeric and / or diastereomeric mixtures; pharmaceutically acceptable salts, solvates, or hydrates thereof; or metabolites and metabolic precursors thereof.

[0018] In the context of the present invention, metabolites and metabolic precursors refer to compounds of formula (I) that have been converted by metabolic reactions but which substantially retain or increase pharmacological activity.

[0019] Examples of metabolites or metabolic precursors are hydroxylated, carboxylated, sulfonated, glycosylated, methylated or demethylated, acetylated, or covalently attached to glucuronic acid, glycine and other amino acids, glutathione, or oxidized or reduced derivatives of the compounds of formula (I).

[0020] Some compounds of formula (I), particularly the esters, may also be prodrugs of the active form.

[0021] Where the compounds of formula (I) can exhibit tautomerism, the formula is intended to encompass all tautomers and the invention includes within its scope all possible stereoisomers, Z and E isomers, optical isomers, enantiomers and mixtures thereof.

[0022] Also included within the scope of the present invention is pharmaceutically acceptable salt. Pharmaceutically acceptable salt is a salt that retains the biological activity of the base compound, for example, derived from a known pharmacologically acceptable acid, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, fumaric acid, succinic acid, oxalic acid, malic acid, tartaric acid, maleic acid, citric acid, methanesulfonic acid or benzoic acid, and others commonly used in the art (see, for example, Pharmaceutical Salts and Co-crystals, Editors: Johan Wouters, Luc Quere, RSC Publishing, 2011).

[0023] A further object of the present invention are compounds of said general formula (I) for use as medicaments, in particular for the treatment of HF.

[0024] In some embodiments, the compound of claim 1 is (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; 3-[2-(Piperidin-4-yl)ethylidene]butyric acid;(E)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one;(Z)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one;(E)-3-(4-aminobutyl)-6α-hydroxyandrost-2-en-17-one hydroiodide;3-[2-(piperidin-4-yl)ethyl]- 6α-Hydroxyandrost-2-en-17-one hydroiodide;(EZ)-3-(4-aminobutylidene)-6α-hydroxyandrostan-17-one;(E)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;(Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;3β-[ 2-(Piperidin-4-yl)ethyl]-6α-hydroxyandrostan-17-one;Ethyl (6α-hydroxy-17-ketoandrostan-3β-yl)acetate;4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid;4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid;2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl) Butyrate ; Ethyl 6 -(6α-hydroxy-17-oxoandrostan-3-yl) mosquito Proate; 6-(6β-Hydroxy-17-oxoandrostan-3-yl)caproic acid;(EZ)-3-(5-N-methylaminopentylidene)-6α-hydroxymethylandrostan-7,17-dione;(EZ)-3-[2-(pyrrolidin-3yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(EZ)-3-[2-(azetidine- 3 -yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(EZ)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(EZ)-3-(5-N-methylaminopentylidene)-6α-hydroxymethyl-7α-hydroxyandrostan-17-one;3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione;3β-[2-(azetidine- 3 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethylandrostan-7,17-dione; 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethylandrostan-7,17-dione; and 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one.

[0025] A further object of the present invention is pharmaceutical compositions comprising one or more compounds of formula (I), optionally in combination with other therapeutically active ingredients. These pharmaceutical compositions can be formulated for oral administration, intravenous or intramuscular injection, inhalation, intravitreal injection, etc. In certain embodiments, the pharmaceutical compositions disclosed herein are used to treat HF.

[0026] The above and other objects of the present invention will be disclosed in detail by the examples and drawings. [Brief explanation of the drawings]

[0027] [Figure 1] Figure 1 shows the effect of 1 μM CVie216 ​​on sarcoplasmic reticulum (SR) Ca2+ uptake parameters in rat ventricular myocytes isolated from STZ rats (loading protocol). SR-Ca2+ uptake parameters include Ca2+ transient (CaT) amplitude (Panel A); Ca2+-induced Ca2+ release (CICR) gain (Panel B); and the constant of time for Ca2+ decay (τ) (Panel C). The difference between the control (N = 16–18) and CVie216 ​​(N = 20–23) curves was statistically significant in Panels A–C (p < 0.05, two-way ANOVA).

[0028] [Figure 2] Figure 2 shows the effect of 1 μM CVie214 on sarcoplasmic reticulum (SR) Ca2+ uptake parameters in rat ventricular myocytes isolated from STZ rats (loading protocol). SR-Ca2+ uptake parameters include Ca2+ transient (CaT) amplitude (Panel A); Ca2+-induced Ca2+ release (CICR) gain (Panel B); and the time constant of Ca2+ decay (τ) (Panel C). The difference between the control (N = 14) and CVie214 (N = 11) curves was statistically significant in Panel B (p < 0.05) and near significance in Panel C (p = 0.05).

[0029] [Figure 3]Figure 3 shows the effects of CVie216 ​​on short-term variations (STV) of action potentials (APDs) and action potential durations (APDs) in guinea pig ventricular myocytes at various stimulation rates (Hz). Panel A, from left to right, shows the rate-dependence of action potential duration at 90% repolarization (APD90) (left panel), diastolic membrane potential (Ediast) (middle panel), and maximum depolarization rate (dV / dtmax) (right panel) under basal conditions (CTR, filled circles; N > 13) or in the presence of 1 μM CVie216 ​​(open circles; N > 11). Differences measured between the control and CVie216 ​​groups were not statistically significant for all parameters. Panel B shows the linear correlation between STV and mean APD90 for the control (CTR, filled circles) and CVie216 ​​(open circles) groups. Data from all pacing rates were pooled within each group to extend STV assessment to a wide APD range. The solid line is a linear fit of the data points (control slope = 0.013 vs. CVie216 ​​slope = 0.09, NS), indicating that CVie216 ​​did not alter STV sensitivity to APD prolongation.

[0030] [Figure 4]Figure 4 shows the effects of CVie214 on short-term variations (STV) of action potentials (APDs) and action potential durations (APDs) in guinea pig ventricular myocytes at various stimulation rates (Hz). Panel A, from left to right, shows the rate-dependence of action potential duration at 90% repolarization (APD90) (left panel), diastolic membrane potential (Ediast) (middle panel), and maximum depolarization rate (dV / dtmax) (right panel) under basal conditions (CTR, filled circles; N > 17) or in the presence of 1 μM CVie214 (open circles; N > 17). Differences measured between the control and CVie214 groups were not statistically significant for all parameters. Panel B shows the linear correlation between STV and mean APD90 for the control (CTR, filled circles) and CVie214 (open circles) groups. Data from all pacing rates were pooled within each group to extend STV assessment to a wide APD range. The solid line is a linear fit of the data points (control slope = 0.012 vs. CVie214 slope = 0.014, NS), indicating that CVie214 did not alter STV sensitivity to APD prolongation. DETAILED DESCRIPTION OF THE INVENTION

[0031] Disclosed herein are compositions and methods useful for treating heart failure. In particular, provided herein are compositions containing novel androstane derivatives. Furthermore, they activate SERCA2a while inhibiting Na+. + / K + There is one group of novel androstane derivatives described herein that only mildly inhibits the ATPase pump. This group of androstane derivatives is referred to as "nearly pure" SERCA2a stimulators and has the general formula (I) with an amine-containing functional group at the C3 carbon. Another group of novel androstane derivatives described herein is Na + / K +They exhibit potent SERCA2a activation without significant inhibition of the ATPase pump. This group of androstane derivatives is referred to as "pure" SERCA2a stimulators, and is represented by general formula (I) with a carboxylic acid / ester-containing functional group arranged via a spacer at the C3 carbon. In other embodiments, the nearly pure or pure SERCA2a stimulators of general formula (I) may contain an alcohol, sulfate, or phosphate-containing functional group at the C3 carbon. Thus, these compositions have positive lusitropic properties and are capable of increasing Na + / K + They can be used to selectively activate SERCA2a while avoiding the proarrhythmic effects of ATPase pump inhibition. The compositions and methods disclosed herein are described in more detail below.

[0032] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Standard techniques are employed unless otherwise defined. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patents, and other documents mentioned herein are incorporated by reference in their entirety.

[0033] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] The term "about" refers to the variation in the numerical value of a measurement, e.g., volume, time, pressure, concentration, etc., due to the typical error rate of the device used to obtain the measurement. In one embodiment, the term "about" means within 5% of the reported numerical value, and preferably, the term "about" means within 3% of the reported numerical value.

[0035] The term "heart failure" refers to a clinical condition characterized by typical symptoms (e.g., shortness of breath, swollen ankles, and fatigue) that may be accompanied by signs (e.g., elevated jugular venous pressure, lung noise, and peripheral edema) caused by structural and / or functional cardiac abnormalities, resulting in reduced cardiac output and / or elevated intracardiac pressure at rest or during stress.

[0036] The terms "acute heart failure" or "AHF" are used interchangeably herein and generally refer to the rapid onset or worsening of symptoms and / or signs of HF that require immediate treatment and hospitalization. The current definition of "acute heart failure" is fairly nonspecific and may encompass a wide range of conditions, with several phenotypes characterized by different clinical manifestations, etiologies, exacerbating factors, treatment approaches, and prognoses. Furthermore, the majority of patients have a subacute disease course, with progressive worsening of HF signs and symptoms that may begin several days before hospitalization.

[0037] The terms "chronic heart failure" or "CHF" are used interchangeably herein and refer to the current clinical classification of chronic HF based on the presence of signs and symptoms of HF and left ventricular ejection fraction (LVEF), with three recognized categories: "heart failure with reduced ejection fraction" or "HFrEF" (characterized by an LVEF less than about 40%); "heart failure with mildly reduced ejection fraction" or "HFmEF" or "HFmrEF" (characterized by an LVEF of about 40% to about 49%); and "heart failure with preserved ejection fraction" or "HFpEF" (characterized by an LVEF equal to or greater than about 50%). The terms "HFmrEF" and "HFpEF" include two additional criteria: elevated natriuretic peptide levels (BNP > 35 pg / mL and / or NT-proBNP > 125 pg / mL) associated with evidence of structural and / or functional heart disease (left ventricular hypertrophy, and / or left atrial enlargement, and / or evidence of diastolic dysfunction). The effectiveness of HF evidence-based drug therapy has only been confirmed in patients with "HFrEF," whereas no treatment has shown significant improvement in "HFpEF."

[0038] The terms "metabolite" and "metabolic precursor" refer to compounds that have been transformed / modified by metabolic reactions but that substantially retain or exhibit an increase in their pharmacological activity.

[0039] The term "treating" refers to an indication of success in curing or ameliorating a disease or condition. Treatment can include, for example, reducing or alleviating the severity of one or more symptoms of a disease or condition, or it can include reducing the frequency with which an individual, such as a human patient, experiences a symptom of a disease, disorder, disorder, or condition.

[0040] The term "preventing" refers to the prevention of a disease or condition, e.g., acute heart failure, in an individual, such as a human patient. For example, if an individual at risk of developing heart failure is treated with a method of the invention and does not later develop heart failure, the disease has been prevented in that individual.

[0041] The term "treatment or prevention" may be used herein to mean a method of providing some level of cure or improvement for a disease or condition, and contemplates a range of results directed toward that end, but is not limited to complete disease prevention.

[0042] As used herein, the term "pharmaceutically acceptable carrier" refers to a chemical composition that can be combined with an active compound, such as an androstane derivative having general formula (I) or a metabolite thereof, and that, after combination, can be used to administer the compound to a mammal.

[0043] As used herein, the term "pharmaceutically acceptable" salt, solvate, hydrate, or ester refers to a salt, solvate, hydrate, or ester form of an active ingredient that is compatible with the other ingredients of a pharmaceutical composition and is not harmful to the subject to which the composition is administered. The term "pharmaceutically acceptable salt" also refers to a salt form of a compound derived from a pharmacologically acceptable acid that retains the biological activity of the basic compound.

[0044] As used herein, the term "parameter" intended to measure cardiac function refers to any cardiac function that can be observed or measured using appropriate measurement techniques available in the art. Examples of "parameters" of cardiac function include, but are not limited to, calcium transient amplitude (CaT), calcium-induced calcium release (CICR), calcium decay time constant, and velocity-dependent action potential duration at 90% repolarization (APD). 90 ), diastolic membrane potential (E diast ), maximum depolarization rate (dV / dt max ), heart rate, blood pressure, diastolic relaxation, systolic contraction, left ventricular ejection fraction (LVEF), diastolic blood pressure, systolic blood pressure, cardiac output, stroke volume, contraction velocity (s'), early relaxation velocity (e'), late relaxation velocity (a'), left ventricular filling pressure index (E / e'), E wave deceleration time (DT), mitral deceleration index (DT / E), deceleration slope (E / DT), cardiac index, mitral inflow velocity, etc. As will be appreciated by those skilled in the art, measuring one or more "parameters" of cardiac function, in comparison to average normal "parameters," can be used to detect cardiac dysfunction and to determine whether cardiac function has improved during or after treatment.

[0045] The term "substantially pure" in relation to activation or stimulation of SERCA2a refers to the ability to stimulate SERCA2a activity in a statistically significant manner in a cell-free system (SR cardiac microsomes obtained from guinea pig, dog, rat, etc.), while also referring to purified dog kidney Na in a cell-free system. + / K + Only mildly inhibits ATPase (i.e., IC 50 is greater than about 0.5 μM, preferably greater than about 1 μM), androstane derivatives and the like.

[0046] The term "pure" in reference to activation or stimulation of SERCA2a refers to the ability to stimulate SERCA2a activity in a cell-free system (SR cardiac microsomes obtained from guinea pigs, dogs, rats, etc.) in a statistically significant manner and to achieve a statistically significant effect on Na + / K + No significant inhibition of ATPase pumps (i.e., IC 50 is greater than about 100 μM), androstane derivatives and other compounds.

[0047] The terms "therapeutically active" or "active" ingredient or compound refer to a substance that provides a beneficial effect to the individual to whom it is administered. A "therapeutically effective amount" or "therapeutically effective dose" is the amount of a composition or active ingredient sufficient to provide a beneficial effect to the individual to whom it is administered.

[0048] Androstane derivatives with near-pure or pure SERCA2a-stimulating activity The present invention is based on the discovery of androstane derivatives that have near-pure or pure SERCA2a stimulating activity. + / K + Derivatives that show stimulation of SERCA2a with little or no inhibition of the ATPase pump.

[0049] These novel androstane derivatives are functionalized at the C-3 carbon by carbon linkers with various functional groups, such as amine-containing or carboxylic acid / ester-containing functional groups. In addition, these novel androstane derivatives are also functionalized at the C-6 and / or C-7 carbon by hydroxyl, hydroxymethyl, or ketone groups, etc. Preferably, the novel androstane derivatives suitable for use herein are each represented by the general formula (I): [ka] This is shown by:

[0050] wherein X is any of carboxylic acids, carboxylic esters, and their bioisosteres (sulfates, sulfonic acids, phosphates, phosphonates, or nitrogen-containing ether cyclic rings such as triazoles and tetrazoles), primary alcohols, ethers, or amine groups (e.g., primary amines, secondary amines, or cyclic amines);

[0051] The carbon linker at C6 has one or more carbons represented by n, which is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5);

[0052] The dashed lines represent the optional double bond (C=C at C3-C1' or C2-C3) and the C=O at C7;

[0053] The Y group at C6 is hydroxyl (OH) in the α or β configuration, or hydroxymethyl (CHOH) in the α configuration, and

[0054] The Z group at C7 can be either -H, or -OH in the alpha configuration, or a ketone (C=O).

[0055] In certain embodiments, a substantially pure SERCA2a stimulator may be desirable. As such, suitable androstane derivatives for use herein may include those represented by general formula (I) in which X is an amine functional group (e.g., primary amine, secondary amine, or cyclic amine). However, in some embodiments, it may be desirable to select a pure SERCA2a stimulator. As such, suitable androstane derivatives for use may include those represented by general formula (I) in which X is not an amine functional group (e.g., primary amine, secondary amine, or cyclic amine). In a preferred embodiment, the pure SERCA2a stimulator is one represented by general formula (I) in which X has a carboxylic acid or carboxylic acid ester.

[0056] The androstane derivatives disclosed herein preferably contain an oxygen-containing functional group in either Z or Y, or both.

[0057] In this specification, the enantiomeric and / or diastereomeric mixtures of the compounds represented by general formula (I), as well as their pharmaceutically acceptable salts, solvates, and / or hydrates, and their metabolites and / or metabolic precursors are also suitable for use.Metabolites or metabolic precursors include, for example, derivatives of the compounds of formula (I) that are hydroxylated, carboxylated, sulfonated, acetylated, glycosylated, glucuronidated, methylated or demethylated, or covalently bound to glutathione, glycine, or other amino acids, or oxidized or reduced.In addition, some compounds of formula (I), especially esters, can also be prodrugs of active forms. Pharmaceutically acceptable salts include, for example, but are not limited to, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, fumaric acid, succinic acid, oxalic acid, malic acid, tartaric acid, maleic acid, citric acid, methanesulfonic acid, or benzoic acid, and others commonly used in the art (see, e.g., Pharmaceutical Salts and Co-crystals, Editors: Johan Wouters, Luc Quere, RSC Publishing, 2011, the entire contents of which are incorporated herein by reference).

[0058] Where the compounds of formula (I) can exhibit tautomerism, the formula is intended to encompass all tautomers, including, but not limited to, all possible stereoisomers, Z and E isomers, optical isomers, enantiomers and mixtures thereof.

[0059] Specific androstane derivatives suitable for use herein include:

[0060] (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie201) [ka]

[0061] (Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie202) [ka]

[0062] (E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie203) [ka]

[0063] (Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie204) [ka]

[0064] (E)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one (CVie205) [ka]

[0065] (Z)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one (CVie206) [ka]

[0066] (E)-3-(4-aminobutyl)-6α-hydroxyandrost-2-en-17-one hydroiodide (CVie207) [ka]

[0067] 3-[2-(Piperidin-4-yl)ethyl]-6α-hydroxyandrost-2-en-17-one hydroiodide (CVie208) [ka]

[0068] (EZ)-3-(4-aminobutylidene)-6α-hydroxyandrostan-17-one (CVie209) [ka]

[0069] (E)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one (CVie210) [ka]

[0070] (Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one (CVie211) [ka]

[0071] 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxyandrostan-17-one (CVie212) [ka]

[0072] Ethyl (6α-hydroxy-17-ketoandrostan-3β-yl) (CVie213) acetate [ka]

[0073] 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid (CVie214) [ka]

[0074] 4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid (CVie215) [ka]

[0075] 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid (CVie216) [ka]

[0076] ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl) Butyrate (CVie217) [ka]

[0077] Ethyl 6 -(6α-hydroxy-17-oxoandrostan-3-yl) mosquito Proate (CVie218) [ka]

[0078] 6 -(6β-Hydroxy-17-oxoandrostan-3-yl)caproic acid (CVie219) [ka]

[0079] (E,Z)-3-(5-N-methylaminopentylidene]-6α-hydroxymethylandrostane-7,17-dione (CVie401) [ka]

[0080] (E,Z)-3-[2-(pyrrolidin-3yl)ethylidene]-6α-hydroxymethylandrostane-7,17-dione (CVie402) [ka]

[0081] (E,Z)-3-[2-(azetidine- 3 -yl)ethylidene]-6α-hydroxymethylandrostane-7,17-dione (CVie403) [ka]

[0082] (E,Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxymethylandrostane-7,17-dione (CVie405) [ka]

[0083] (E,Z)-3-(5-N-methylaminopentylidene)-6α-hydroxymethyl-7α-hydroxyandrostan-17-one (CVie406) [ka]

[0084] 3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione (CVie407) [ka]

[0085] 3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one (CVie408) [ka]

[0086] 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione (CVie409) [ka]

[0087] 3β-[2-(pyrrolidin-3yl)ethyl]6α-hydroxymethyl-7α-hydroxyandrostan-17-one (CVie410) [ka]

[0088] 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione (CVie411) [ka]

[0089] 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one (CVie412) [ka]

[0090] It is also an object of the present invention to utilize the SERCA2a activating properties of the compounds of formula (I) for the treatment, amelioration, regression, symptomatic relief or suppression, or prevention of diseases associated with reduced SERCA2a activation, such as heart failure (AHF and / or CHF). 2+ Because SERCA2a distribution is involved in the myocardial remodeling process, its correction by SERCA2a stimulation may counteract it, thus preventing the progression of early abnormalities in contractility in overt heart failure.

[0091] As described above, the androstane derivatives disclosed herein act as pure or nearly pure SERCA2a activators. As shown in the following examples, these compounds demonstrate SERCA2a activation. Pure SERCA2a activators, such as CVie201-204 and CVie213-219, exhibit Na + / K + For example, these compounds do not significantly inhibit the Na ATPase of isolated dog kidney. + / K + ATPase with an IC greater than 100 μM 50 On the other hand, almost pure SERCA2a activators such as CVie205-212 and CVie401-412 showed Na + / K + For example, these compounds inhibit the Na+ ATPase of isolated dog kidney. + / K + ATPase with an IC of at least 0.8 μM 50 values, preferably they are measured using isolated canine kidney Na + / K + ATPase with an IC of at least 1 μM 50 Furthermore, the nearly pure SERCA2a activator will have about 6 to about 170 times less Na than istaloxime (Example 3). + / K + Shows ATPase inhibition.

[0092] In some embodiments, the substantially pure and pure SERCA2a activators can be distinguished by their respective functional groups attached to the C3 carbon linker (i.e., X in Formula (I)). In some embodiments, the pure SERCA2a activators are those having a carboxylic acid or carboxylic ester at the C3 carbon linker. In other embodiments, the substantially pure SERCA2a activators are those having an amine functional group (e.g., a primary amine, a secondary amine, or a cyclic amine) at the C3 carbon linker.

[0093] The pure or nearly pure SERCA2a activator compounds provided herein can be used to treat heart failure. + / K + This ability to activate SERCA2a without significantly inhibiting ATPase makes these compounds Na + / K + These compounds can provide lusitropic effect to the heart and improve cardiac function without increasing the risk of arrhythmia or myocardial cell damage associated with ATPase inhibition.Therefore, these compounds can be used as medicines for the treatment of heart failure (acute or chronic) and in methods for treating or preventing heart failure.Therefore, they can be included in pharmaceutical compositions formulated for different administration routes using synthesis and formulation techniques within the scope of those skilled in the art.The pharmaceutical compositions and therapeutic treatment methods that utilize pure or nearly pure SERCA2a activators disclosed herein will be discussed in more detail below.

[0094] Pharmaceutical Composition The compound of formula (I) as a therapeutic agent can be administered alone or as a component of a pharmaceutical preparation (composition).Accordingly, disclosed herein is a pharmaceutical composition comprising any of the compounds of formula (I) disclosed herein or their specific derivatives in admixture with at least one pharmaceutically acceptable vehicle and / or excipient.The pharmaceutical composition can be formulated for administration to an individual parenterally, topically, subcutaneously, intramuscularly, orally, or by topical administration such as aerosol or transdermal administration.In certain embodiments, the route of administration is oral.

[0095] Pharmaceutical compositions can be formulated in any manner and can be administered in a variety of unit dosage forms depending on the disease or disorder and the extent of the disorder, the general condition of each patient, the resulting preferred method of administration, etc. Details of formulation and administration techniques are well explained in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton PA.

[0096] The compounds can be formulated for administration in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavorings and perfuming agents, buffers, preservatives, and antioxidants can also be present in the composition.

[0097] Formulations of the compositions according to the present invention include those suitable for oral, nasal, topical, parenteral (e.g., intramuscular or intravenous injection), rectal, subcutaneous, and / or vaginal administration. The formulations can be conveniently provided in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and / or the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect.

[0098] The pharmaceutical preparations provided herein can be prepared according to any method known in the art for the manufacture of pharmaceuticals. Such preparations can contain sweeteners, flavoring agents, coloring agents, and preservatives. The preparations can be mixed with non-toxic pharmaceutically acceptable excipients suitable for preparation. The preparations can contain one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and can be provided in the form of liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, sustained-release preparations, tablets, pills, gels, patches, implants, etc.

[0099] Pharmaceutical preparations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art, in appropriate dosages. Such carriers allow the pharmaceutical to be formulated in unit dosage form and can be tablets, gel tabs, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for patient ingestion. Pharmaceutical preparations for oral administration can be formulated as solid excipients, optionally milling the resulting mixture and processing the granular mixture to obtain tablets or dragee cores, after adding appropriate additional compounds as needed. Suitable solid excipients include carbohydrate or protein fillers, such as sugars such as lactose, sucrose, mannitol, or sorbitol; starches derived from corn, wheat, rice, potato, or other plants; celluloses such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; gums such as gum arabic and gum tragacanth; and proteins such as gelatin and collagen. Disintegrating or solubilizing agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

[0100] Dragee cores may be provided with a suitable coating, such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solution, and / or a concentrated sugar solution that may contain a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablets or dragee coatings for product identification or to characterize the amount (i.e., dosage) of active compound. The pharmaceutical formulations used to practice the uses and methods provided herein can also be administered orally using, for example, push-fit capsules made of gelatin and sealed soft capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain the active agent mixed with a filler or binder, such as lactose or starch, a lubricant, such as talc or magnesium stearate, and optional stabilizers. In soft capsules, the active agent may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0101] Aqueous suspensions can contain the active agent (e.g., the compositions used to practice the uses and methods provided herein) in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, saccharin, or erythritol or rebaudioside A. The preparations may be adjusted for osmotic pressure.

[0102] Oily suspensions are particularly useful for administering hydrophobic active agents suitable for the uses and methods provided herein. Oily suspensions can be formulated by suspending the active agent in vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, or in mineral oils such as liquid paraffin, or in mixtures thereof. See, for example, U.S. Patent No. 5,716,928, which describes the use of essential oils or essential oil components to increase the bioavailability of orally administered hydrophobic pharmaceutical compounds and reduce inter- and intra-individual variability. See also U.S. Patent No. 5,858,401. Oily suspensions can contain thickening agents such as beeswax, hard paraffin, and cetyl alcohol. Sweeteners such as glycerol, sorbitol, sucrose, erythritol, or rebaudioside A can be added to provide a palatable oral formulation. These formulations can be preserved by adding antioxidants such as ascorbic acid. For examples of injectable oily vehicles, see Minto J., Pharmacol. Exp. Ther. 1997, 281:93-102. The pharmaceutical preparations provided herein can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil or a mineral oil, as described above, or a mixture thereof. Suitable emulsifying agents include naturally occurring gums such as acacia gum and tragacanth gum; naturally occurring phosphatides such as soybean lecithin; esters; or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions can also contain sweeteners and flavorings, such as in the case of syrup and elixir formulations. Such formulations can also contain demulcents, preservatives, or coloring agents.

[0103] According to the present invention, pharmaceutical compounds can also be administered via intranasal, intraocular, and intravaginal routes, including suppositories, insufflation, powder, and aerosol formulations (for examples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol. 1995, 35:1187-1193; Tjwa, Ann. Allergy Asthma Immunol. 1995, 75:107-111, the contents of each of which are incorporated herein by reference in their entirety). Suppository formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at body temperature, thereby melting and releasing the drug in the body. Such materials include cocoa butter and polyethylene glycol.

[0104] In accordance with the present invention, pharmaceutical compounds can be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, cosmetics, powders, and aerosols and delivered by topical routes, transdermally.

[0105] According to the present invention, the pharmaceutical compound of formula (I) can be delivered by inhalation. For example, in an alternative embodiment, the compound of formula (I) for inhalation is prepared for dry dispersion by, for example, spray-drying a solution containing the active ingredient (i.e., the compound of formula (I)), for example, using the methods described in U.S. Patent Nos. 6,509,006; 6,592,904; 7,097,827; and 6,358,530 (the contents of each of which are incorporated herein by reference in their entirety). Dry powder excipients include, for example, low-molecular-weight carbohydrates or polypeptides that are mixed with the compound of formula (I) to aid dispersion. In an alternative embodiment, types of pharmaceutical excipients useful as carriers for dry powder dispersion include stabilizers such as human serum albumin (HSA), which are also useful dispersants, bulking agents such as carbohydrates, amino acids, and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers can be in crystalline or amorphous form, or a mixture of the two. Devices that can be used to deliver powder or aerosol formulations include, for example, those described in US Pat. Nos. 5,605,674 and 7,097,827.

[0106] According to the present invention, pharmaceutical compounds can also be delivered as nanoparticles or microspheres for sustained release in the body.For example, nanoparticles or microspheres can be administered by intradermal or subcutaneous injection, and drug is slowly released subcutaneously; see Rao J., Biomater, Sci. Polym. Ed. 1995,7:623-645;For biodegradable and injectable gel formulations, see Gao, Pharm. Res. 1995,12:857-863;Or for oral administration, see Eyles, J. Pharm. Pharmacol. 1997,49:669-674, the entire contents of each of which are incorporated herein by reference.

[0107] According to the present invention, the pharmaceutical compounds of Formula (I) can be administered parenterally, such as by intramuscular (IM) or intravenous (IV) administration, or into a body cavity or lumen of an organ. These formulations may contain a solution of the active agent dissolved in a pharmaceutically acceptable carrier. Usable vehicles and solvents include water, dextrose-containing water, and Ringer's solution, which is isotonic sodium chloride. Additionally, sterile, fixed oils can be used as solvents or suspending media. For this purpose, any bland, fixed oil, including synthetic mono- or diglycerides, can be used. Furthermore, fatty acids such as oleic acid can also be used in the preparation of injectable solutions. These solutions are sterile and generally free of undesirable matter. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and is selected primarily based on the volume, viscosity, body weight, etc., according to the specific administration method selected and the patient's needs. For intravenous administration, the formulation can be a sterile injectable formulation, such as a sterile injectable aqueous or oily suspension. This suspension can be formulated using appropriate dispersing or wetting agents and suspending agents. The sterile injectable formulation can also be a suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol. Administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into the blood vessels for a predetermined period of time).

[0108] The pharmaceutical compounds and formulations provided herein can be lyophilized. Stable lyophilized formulations containing the compositions provided herein are provided, which can be produced by lyophilizing a solution containing a drug and a bulking agent such as mannitol, trehalose, raffinose, and sucrose, or a mixture thereof, as provided herein. There are many other conventional lyophilizing agents. Among sugars, lactose is the most common. Citric acid, sodium carbonate, EDTA, benzyl alcohol, glycine, sodium chloride, etc. are also used (see, for example, Journal of Excipients and Food Chemistry Vol. 1, Issue 1 (2010) pp 41-54; U.S. Patent Application Publication No. 20040028670).

[0109] Treatment method According to the present invention, the compounds of formula (I) provided herein can be used for prophylactic and / or therapeutic treatment. In therapeutic applications, the pharmaceutical compositions are administered in a therapeutically effective amount to a subject already suffering from a disease or disorder. In other embodiments, the pharmaceutical compositions provided herein are administered in an amount sufficient to treat, prevent, or ameliorate the disease or disorder in an individual in need thereof. The effective dosing schedule and amount, i.e., "dosing regimen," for this application will depend on various factors, including the stage of the disease or disorder, the severity of the disease or disorder, the patient's general health, physical condition, age, etc. The method of administration is also taken into account when calculating a patient's dosing regimen.

[0110] In certain embodiments, the compound of formula (I) is used for treating an individual with heart failure.In preferred embodiments, the individual shows symptoms of acute heart failure or has been diagnosed with acute heart failure.Although the individual can be a non-human animal, in preferred embodiments, the individual is a human patient, for example, a human patient suffering from heart failure.In other embodiments, the compound provided herein is used for stimulating SERCA2a in an individual.

[0111] Generally, the compounds of formula (I) and pharmaceutical compositions described herein can be used to treat heart failure or acute heart failure. The treatment method includes providing or presenting an individual with heart failure or acute heart failure. In some cases, a measuring step is first performed to determine the individual's baseline cardiac function. The measuring step can include measuring one or more parameters of cardiac function, including, but not limited to, heart rate, blood pressure, diastolic relaxation, systolic contraction, left ventricular ejection fraction (LVEF), diastolic blood pressure, systolic blood pressure, cardiac output, stroke volume, deceleration slope (E / DT), contraction velocity (s'), early relaxation velocity (e'), late relaxation velocity (a'), left ventricular filling pressure index (E / e'), E / Ea ratio or E / A ratio, Ea ratio, E wave deceleration time (DT), mitral deceleration index (DT / E), deceleration slope (E / DT), cardiac index, mitral inflow velocity, etc. In individuals with heart failure or cardiac dysfunction, measured parameters may include one or more of: a decrease in heart rate, a decrease in cardiac pressure, a decrease in systolic and / or diastolic blood pressure, a decrease in left ventricular end-diastolic / systolic volume and function (LVEF), an increase in the E / Ea or E / A ratio, a decrease in the Ea ratio, and a decrease in stroke volume. The measuring step may also be used to determine the effectiveness of administration of the pharmaceutical composition (i.e., restoration or partial restoration of cardiac function) and / or to monitor the individual's condition during treatment. Thus, the measuring step may be performed before, during, or after administration of the pharmaceutical composition. As will be appreciated by those skilled in the art, any suitable measurement technique available to the skilled artisan at the time of the measuring step is suitable for use herein, and selecting an appropriate measurement technique corresponding to the parameter of interest is within the purview of those skilled in the art. Suitable measuring devices / techniques include, but are not limited to, blood tests, echocardiography (including tissue Doppler), cardiac catheterization, nuclear stress tests, CAT scans, radionuclide ventriculography scans, stethoscopes, sphygmomanometers, and the like. For example, diastolic relaxation can be measured by echocardiography or PCWP.

[0112] The methods disclosed herein also include administering to an individual a therapeutically effective amount of a compound of general formula (I). In a preferred embodiment, the compound is in a pharmaceutical composition, such as any one of the combinations described above. The compound is administered at a therapeutically effective dose, e.g., about 1 mg / kg to about 20 mg / kg, as disclosed elsewhere herein. In a more preferred embodiment, the route of administration is oral. The measuring step can be performed before, during, or after the administering step. For example, it may be desirable to continuously monitor one or more parameters of cardiac function during and for a period of time thereafter.

[0113] The dosing regimen also takes into account pharmacokinetic parameters well known in the art, i.e., rate of absorption, bioavailability, metabolism, clearance, etc. of the active agent (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; most recently Remington's, supra). The state of the art allows the clinician to determine the dosing regimen for each individual patient, active agent, and disease or condition being treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance for determining the dosage regimen, i.e., dosing schedule and dosage levels, to be administered in practicing the methods provided herein, and are accurate and appropriate.

[0114] The formulation can be administered once or multiple times as needed, depending on the dosage and frequency tolerated by the patient. The formulation should provide a sufficient amount of the active agent to effectively treat, prevent, or ameliorate the illness, disease, or symptoms described herein. For example, exemplary pharmaceutical formulations for oral administration of the compositions used to practice the methods and uses provided herein can be administered in a daily dose of about 1 μg / kg to about 20, 50, 100, or 1000 μg / kg body weight or more per day, or an equivalent amount of a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0115] In alternative embodiments, the effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or hydrate equivalent thereof, administered to an individual in need thereof includes the use of various dosing schedules, for example: A) for the relief of hospitalized patients with AHFS, a compound of Formula (I) can be administered by intravenous infusion at doses ranging from 0.1-0.5 μg / kg to about 10, 50, or 100 μg / kg body weight or more per minute for 12, 24, 48, 72, or more hours; B) for patients who have been rescued from AHFS and discharged from the hospital, the dosing schedule to maintain therapeutic effect can be a daily amount of between 1, 10, 50, 100, or 1000 μg / kg body weight or more per day.

[0116] Compounds of formula (I) useful in practicing the present invention may be administered as a single bolus to deliver a dose of 1 ng / kg to 50 mg / kg body weight, or to deliver an oral or intravenous dose of 1 μg to about 20 mg, or a repeated regimen, or a combination thereof as readily determined by one of skill in the art. In certain embodiments, dosages include at least 0.05 mg / kg, 0.1 mg / kg, or at least 0.2 mg / kg, or at least 0.3 mg / kg, or at least 0.4 mg / kg, or at least 0.5 mg / kg, or at least 0.6 mg / kg, or at least 0.7 mg / kg, or at least 0.8 mg / kg, or at least 0.9 mg / kg, or at least 1 mg / kg, or at least 2 mg / kg, or at least 3 mg / kg, or at least 4 mg / kg, or at least 5 mg / kg, or at least 6 mg / kg, or at least 7 mg / kg, or at least 8 mg / kg, or at least 9 mg / kg, or at least 10 mg / kg, or at least 15 mg / kg, or at least 20 mg / kg, or at least 25 mg / kg, or at least 30 mg / kg, or at least 35 mg / kg, or at least 40 mg / kg, or at least 45 mg / kg, or at least 50 mg / kg, daily or on another suitable regular regimen.

[0117] In one embodiment, the present invention provides a compound of general formula (I) described herein at a therapeutically effective dose of about 0.125 mg / kg to about 10 mg / kg, for example, 0.125 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 1.25 mg / kg, 1.5 mg / kg, 1.75 mg / kg, 2 mg / kg, 2.25 mg / kg, 2.5 mg / kg, 2.75 mg / kg, 3 mg / kg, 3.25 mg / kg, 3.5 mg / kg, 3.75 mg / kg, 4 mg / kg, 4.25 mg / kg. Intravenous or subcutaneous administration of the compound is contemplated at a therapeutically effective dose of between about 0.25 mg / kg and about 5 mg / kg. In another embodiment, the therapeutically effective dose is between about 0.5 mg / kg and about 5 mg / kg. In yet another embodiment, the therapeutically effective dose is from about 0.5 mg / kg to 4 mg / kg, or from about 0.5 mg / kg to about 3 mg / kg.

[0118] In another embodiment, the present invention provides a compound having general formula (I) described herein at a therapeutically effective dose of about 0.25 mg / kg to about 50 mg / kg, e.g., 0.25 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 26 mg / kg, 27 mg / kg, 28 mg / kg, 29 mg / kg, 30 mg / kg, 31 mg / kg, 32 mg / kg, 33 mg / kg, 34 mg / kg, 35 mg / kg, 36 mg / kg, 37 mg / kg, 38 mg / kg, 39 mg / kg, 40 mg / kg, 41 mg / kg, 42 mg / kg, 43 mg / kg, 44 mg / kg, 45 mg / kg, 46 mg / kg, 47 mg / kg, 48 mg / kg, 49 mg / kg, 50 mg / kg, 51 mg / kg, 52 mg / kg, 53 mg / kg, 54 mg / kg, 55 mg / kg, 56 mg / kg, 57 mg / kg, 58 mg / kg, g / kg, 7mg / kg, 7.5mg / kg, 8mg / kg, 8.5mg / kg, 9mg / kg, 9.5mg / kg, 10mg / kg, 10.5mg / kg, 11mg / kg, 11.5mg / kg, 12 mg / kg, 12.5mg / kg, 13mg / kg, 13.5mg / kg, 14mg / kg, 14.5mg / kg, 15mg / kg, 15.5mg / kg, 16mg / kg, 16.5mg / kg, 17mg / kg, 17.5mg / kg, 18mg / kg, 18.5mg / kg, 19mg / kg, 19.5mg / kg, 20mg / kg, 20.5mg / kg, 21mg / kg, 21.5mg / kg, 22mg / kg, 22.5mg / kg, 23mg / kg, 23.5mg / kg, 24mg / kg, 24.5mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30m Intramuscular administration of the androstane derivative at a therapeutically effective dose of about 0.25 mg / kg to about 35 mg / kg is contemplated. In another embodiment, the therapeutically effective dose is about 0.25 mg / kg to about 30 mg / kg. In yet another embodiment, the therapeutically effective dose is about 0.25 mg / kg to about 10 mg / kg. In yet another embodiment, the therapeutically effective dose is from about 0.25 mg / kg to 5 mg / kg.

[0119] In yet another embodiment, the present invention provides a compound having general formula (I) as described herein in a therapeutically effective dose of about 1 μg to about 10 mg, e.g., 1 μg, 1.25 μg, 1.5 μg, 1.75 μg, 2 μg, 2.25 μg, 2.5 μg, 2.75 μg, 3 μg, 3.25 μg, 3.5 μg, 3.75 μg, 4 μg, 4.25 μg, 4.5 μg, 4.75 μg, 5 μg, 5.25 μg, 5.5 μg, 5.75 μg, 6μg, 6.25μg, 6.5μg, 6.75μg, 7μg, 7.25μg, 7.5μg, 7.75μg, 8μg, 8.25μg, 8.5μg, 8.75μg, 9μg, 9.25μg, 9.5μg, 9.75 μg, 10μg, 20μg, 30μg, 40μg, 50μg, 60μg, 70μg, 80μg, 90μg, 100μg, 150μg, 200μg, 250μg, 300μg, 350μg, 400μg, 450 μg, 500μg, 550μg, 600μg, 650μg, 700μg, 750μg, 800μg, 850μg, 900μg, 950μg, 1mg, 1.1mg, 1.2mg, 1.3mg, 1.4mg, 1. 5mg, 1.6mg, 1.7mg, 1.8mg, 1.9mg, 2mg, 2.1mg, 2.2mg, 2.3mg, 2.4mg, 2.5mg, 2.6mg, 2.7mg, 2.8mg, 2.9mg, 3mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, or 10 mg; preferably, the dose is from about 1 μg to about 2000 μg, e.g., from about 1 μg to about 2000 μg, or from about 100 μg to about 1,500 μg, or from about 500 μg to about 1,200 μg, or from about 500 μg to about 1,000 μg. In some embodiments, the therapeutically effective dose of the compound delivered via intravitreal administration is at least about 0.02 mg, e.g., at least about 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, 0.5 mg, 0.55 mg, 0.6 mg, 0.65 mg, 0.7 mg, 0.75 mg, 0.8 mg, 0.85 mg, 0.9 mg, 0.95 mg, or 1 mg.

[0120] In another embodiment, the present invention provides a compound having general formula (I) described herein at a therapeutically effective dose of about 1 mg / kg to about 20 mg / kg, e.g., 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg Oral administration of the compound is contemplated at a therapeutically effective dose of between about 1 mg / kg and about 10 mg / kg, 10.5 mg / kg, 11 mg / kg, 11.5 mg / kg, 12 mg / kg, 12.5 mg / kg, 13 mg / kg, 13.5 mg / kg, 14 mg / kg, 14.5 mg / kg, 15 mg / kg, 15.5 mg / kg, 16 mg / kg, 16.5 mg / kg, 17 mg / kg, 17.5 mg / kg, 18 mg / kg, 18.5 mg / kg, 19 mg / kg, 19.5 mg / kg, or 20 mg / kg. In a preferred embodiment, the compound is administered via oral delivery at a therapeutically effective dose between about 1 mg / kg and about 10 mg / kg. For example, in one particular embodiment, a compound having general formula (I) is orally delivered to a human at a dose of between about 1 and 5 mg / kg. In some embodiments, the oral dose described herein is administered once. In other embodiments, it is administered over the course of one day.

[0121] In another embodiment, the therapeutically effective amount of the compound having general formula (I) described herein is administered according to a dosing schedule, for example, from about 0.1 to about 0.1 μg / kg / min to about 5.0 μg / kg / min, such as 0.1 μg / kg / min, 0.2 μg / kg / min, 0.3 μg / kg / min, 0.4 μg / kg / min, 0.5 μg / kg / min, 0.6 μg / kg / min, 0.7 μg / kg / min, 0.8 μg / kg / min, / kg / min, 0.9μg / kg / min, 1.0μg / kg / min, 1.1μg / kg / min, 1.2μg / kg / min, 1.3μg / kg / min, 1.4μg / kg / min, 1.5μg / kg / min, 1.6μg / kg / min, 1.7μg / kg / min, 1.8μg / kg / min, 1.9μg / kg / min, 2.0μg / kg / min, 2.1μg / kg / min, 2.2μg / kg / min, 2 .3μg / kg / min, 2.4μg / kg / min, 2.5μg / kg / min, 2.6μg / kg / min, 2.7μg / kg / min, 2.8μg / kg / min, 2.9μg / kg / min, 3.0μg / kg / min, 3.1μg / kg / min, 3.2μg / kg / min, 3.3μg / kg / min, 3.4μg / kg / min, 3.5μg / kg / min, 3.6μg / kg / min, 3.7μg / kg / m in, 3.8 μg / kg / min, 3.9 μg / kg / min, 4.0 μg / kg / min, 4.1 μg / kg / min, 4.2 μg / kg / min, 4.3 μg / kg / min, 4.4 μg / kg / min, 4.5 μg / kg / min, 4.6 μg / kg / min, 4.7 μg / kg / min, 4.8 μg / kg / min, 4.9 μg / kg / min, or 5.0 μg / kg / min administered to an individual by infusion. For example, in some embodiments, the compound is administered by infusion at an effective dose of about 0.2 μg / kg / min to about 2.0 μg / kg / min, or about 0.2 μg / kg / min to about 1.5 μg / kg / min, or about 0.25 μg / kg / min to about 1.0 μg / kg / min, or about 0.5 μg / kg / min to about 1.0 μg / kg / min.

[0122] In an alternative embodiment, the effective amount of the compound of formula (I), or a pharmaceutically acceptable salt, solvate, or hydrate equivalent thereof, administered to an individual in need thereof is individualized based on monitoring of pulmonary artery wedge pressure (PCWP), tissue Doppler imaging (TDI) measurements, dyspnea, peripheral and pulmonary venous congestion, urine output, exercise capacity, serum biomarkers such as NT-proBNP, and high-sensitivity cardiac troponin (hs-cTnT).

[0123] In an alternative embodiment, the compound of formula (I), or a pharmaceutically acceptable salt, solvate, or hydrate equivalent thereof, administered to an individual in need thereof is in an amount sufficient to maintain normal exercise tolerance without respiratory distress.

[0124] In alternative embodiments, an effective amount is demonstrated by a decrease in PCWP, orthopnea, paroxysmal nocturnal dyspnea, an increase in exercise tolerance, a decrease in peripheral and pulmonary venous congestion such as lung noise or rales, a decrease in ankle swelling, biomarkers such as urinary output, e.g., NT-proBNP, and high-sensitivity cardiac troponin (hs-cTnT), and the like.

[0125] In alternative embodiments, lower doses of the compound of Formula (I), or its pharmaceutically acceptable salt, solvate, or hydrate equivalent, are used when administered into the bloodstream or IV or IM, e.g., as IV or IM administration (e.g., compared to oral, inhalation, or subcutaneous administration), or when administered into a body cavity or lumen of an organ. Substantially higher doses can be used when administered topically, spray, inhalation, or orally, or by powder, spray, or inhalation. Actual methods for preparing formulations suitable for parenteral or non-parenteral administration will be known or apparent to those skilled in the art and are described in detail in publications such as Remington's (see Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA).

[0126] In certain embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt, solvate, or hydrate equivalent thereof, is given chronically, for example, from the date of diagnosis until the last day of the patient's life, or until the disease is in remission. In alternative embodiments, dose adjustments are required to transition from the treatment phase to a maintenance period through periodic monitoring of certain conventionally known biomarkers or clinical signs of the disease.

[0127] In an alternative embodiment, when evaluating the effectiveness of a treatment, treatment regimen, or specific dosage, or when determining whether treatment should be administered at a controlled dose, individuals, e.g., patients with AHF or CHF, are regularly screened periodically for the presence and extent of organ or tissue involvement or damage, e.g., cardiac (ventricular dilation, third heart sound, cardiac hypertrophy), fatigue, tiredness, decreased exercise tolerance, increased post-exercise recovery time, renal (renal failure, oliguria), pulmonary (orthopnea, paroxysmal nocturnal dyspnea, tachypnea), ankle swelling, elevated jugular venous pressure, etc. A thorough physical examination should be performed at time intervals selected by a specialist in the treatment of AHF or CHF, focusing on cardiovascular disease, particularly cardiac, pulmonary, and peripheral circulatory function. Thus, in an alternative embodiment, treatment with a compound of formula (I), or a pharmaceutically acceptable salt, solvate or hydrate equivalent thereof, is initiated as early as possible, preferably urgently, to prevent rapid progression of symptoms, and continued for several years after the patient is discharged from the hospital, preferably for the rest of the patient's life, or at least for a period consistent with the use of other medications in HF.

[0128] According to the present invention, the uses and methods provided herein can further include co-administration with other drugs or pharmaceuticals. Indeed, the present invention selectively corrects impaired cardiac biochemical function (i.e., SERCA2a activity). This certainly contributes to the alleviation of existing HF clinical symptoms and causes fewer unwanted side effects than available therapies (due to the selectivity described above). However, because CHF and AHF are complex clinical syndromes, the present invention is potentially relevant to existing and future drug classes and / or specific drugs, such as: a) drug classes, e.g., ACE inhibitors, AIRBs, diuretics, Ca2+ 2+ Channel blockers, beta-blockers, digitalis, NO donors, vasodilators, SERCA2a stimulators, neprilysin (NEP) inhibitors, myosin filament activators, recombinant relaxin-2 mediators, recombinant NP protein, activators of soluble guanylate cyclase (sGC), beta-arrestin ligands for angiotensin II receptors; b) specific drugs: hydrochlorothiazide, furosemide, verapamil, diltiazem, carvedilol, metoprolol, hydralazine, eplerenone, spironolactone, lisinopril, ramipril, nitroglycerin, nitrates, digoxin, valsartan, olmesartan, telmisartan, candesartan, losartan, entresto, omecamtib, sacubitril, serelaxin, ularitide, levosimendan, cinaciguat.

[0129] As a therapeutic agent, the compound of the present invention, particularly used for treating HF, can be combined with other therapeutic agents used to treat the same disease.Exemplary other therapeutic agents include diuretics, such as furosemide, bumetanide, and torasemide, metolazone, aldosterone antagonists, such as spironolactone or eplerenone; thiazide diuretics, such as hydrochlorothiazide, metolazone, chlorthalidone, etc.Other drugs include ACE inhibitors, such as lisinopril and ramipril.Angiotensin II receptor blockers (ARBs), such as valsartan, candesartan, losartan, etc., can also be considered.Angiotensin receptor / neprilysin inhibitors (ARNIs), such as sacubitril, can be considered. Other drugs may be selected from beta-blockers, such as carvedilol and metoprolol, or vasodilators, such as hydralazine, optionally combined with isosorbide dinitrate; nitrates, such as nitroglycerin, amlodipine, and felodipine; and non-dihydropyridines, such as diltiazem or verapamil. The compounds of the present invention may also be combined with digoxin, if necessary. Other drugs, such as ivabradine or other anticoagulants, may also be considered. Additionally, other drugs may include Omecamtriv mecarbil.

[0130] The compounds of the present invention can be combined with other therapeutic agents, particularly agents useful for the treatment of cardiovascular disease, more particularly in combination therapy for HF. The combined active ingredients can be administered according to various protocols determined by a physician. According to an embodiment of the present invention, combination therapy can be carried out by administering the compound of formula (I) and additional therapeutic active ingredient(s) simultaneously or at different times. For simultaneous administration, the compound of the present invention and the additional active ingredient(s) can each be formulated into their own pharmaceutical compositions. In this case, the present invention provides a kit containing separate pharmaceutical compositions each containing the compound of the present invention and the additional active ingredient(s), particularly for the treatment of heart failure. In another embodiment, the present invention provides a pharmaceutical unit dosage kit containing the compound of the present invention and the additional active ingredient(s), particularly for the treatment of HF.

[0131] Nanoparticles, nanolipoparticles and liposomes Also provided are nanoparticles, nanolipoparticles, vesicles, and liposomal membranes for delivering pharmaceutically active compounds and compositions, including compounds provided herein, for example, as provided herein (compounds of Formula (I) or pharmaceutically acceptable salts, solvates, or hydrate equivalents thereof), to a subject in need thereof. In alternative embodiments, these compositions are designed to target specific molecules, including biological molecules such as polypeptides, including cell surface polypeptides, for example, to target a desired cell type, e.g., muscle or cardiac cells, endothelial cells, etc.

[0132] Multilamellar liposomes containing the compounds used to practice the methods of the present disclosure are provided, for example, in U.S. Patent Publication No. 20070082042 to Park et al., the contents of which are incorporated herein by reference in their entirety. Multilamellar liposomes with particle sizes of about 200-5000 nm can be prepared using a mixture of oil phase components including squalene, sterols, ceramides, neutral lipids or oils, fatty acids, and lecithin, and can be encapsulated in compositions used to practice the uses and methods provided herein.

[0133] Liposomes can be produced using any method, such as those described in U.S. Pat. No. 4,534,899 and U.S. Patent Publication No. 20070042031, including, for example, a method of producing liposomes by encapsulating an active agent (or combination of active agents) according to the present invention, comprising providing an aqueous solution to a first reservoir; providing an organic lipid solution to a second reservoir, then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution is mixed with the aqueous solution to substantially immediately produce liposomes encapsulating the active agent; and immediately mixing the liposome solution with a buffer to produce a diluted liposome solution.

[0134] In one embodiment, the liposome compositions used to practice the uses and methods provided herein comprise substituted ammonium and / or polyanions, e.g., as described in U.S. Patent Publication No. 20070110798, to target delivery of the compounds used to practice the methods provided herein, the compounds of Formula (I), or pharmaceutically acceptable salts, solvates, or hydrate equivalents thereof, to a desired cell type.

[0135] Nanoparticles comprising the compounds of the invention for use in practicing the uses and methods provided herein in the form of active agent-containing nanoparticles (e.g., secondary nanoparticles) are provided and are described, for example, in U.S. Patent Publication No. 20070077286. In one embodiment, nanoparticles are provided that comprise a lipid-soluble active agent or a lipid-solubilized water-soluble active agent reacted with a divalent or trivalent metal salt for use in practicing the uses and methods provided herein.

[0136] In one embodiment, solid lipid suspensions can be used to formulate and deliver compositions used to practice the uses and methods provided herein to mammalian cells in vivo, in vitro, or ex vivo, as described, for example, in U.S. Patent Publication No. 20050136121.

[0137] The compositions and formulations used to implement the uses and methods provided herein can be delivered by using liposomes or nanoliposomes.The use of liposomes can focus the delivery of active agents to target cells in vivo, especially when the liposome surface carries a ligand specific to the target cell or preferentially directs to a specific organ.See, for example, U.S. Patent Nos. 6,063,400 and 6,007,839;Al-Muhammed, J. Microencapsul. 1996, 13:293-306;Chonn, Curr. Opin. Biotechnol. 1995, 6:698-708;Ostro, Am. J. Hosp. Pharm. 1989, 46:1576-1587.

[0138] Delivery Vehicle In alternative embodiments, any delivery vehicle can be used to implement the uses and methods provided herein, for example, to deliver the compounds provided herein to a subject in need thereof.For example, delivery vehicles comprising polycations, cationic polymers, and / or cationic peptides such as polyethyleneimine derivatives can be used, for example, as described in US Patent No. 20060083737.

[0139] In one embodiment, a dry polypeptide-surfactant complex is used to formulate compositions used to practice the uses and methods provided herein, e.g., as described in U.S. Patent Publication No. 20040151766.

[0140] In one embodiment, the compositions used to carry out the uses and methods provided herein can be applied to cells using a vehicle with a cell membrane-permeable peptide conjugate, as described, for example, in U.S. Patent Nos. 7,306,783 and 6,589,503. In one aspect, the composition to be delivered is conjugated to a cell membrane-permeable peptide. In one embodiment, the composition to be delivered and / or the delivery vehicle is conjugated to a transport-mediating peptide, as described, for example, in U.S. Patent No. 5,846,743, which describes a highly basic transport-mediating peptide that binds to polyphosphoinositides.

[0141] In one embodiment, electropermeabilization is used as a primary or secondary means to deliver compositions to cells using any electroporation system, e.g., as described in U.S. Pat. Nos. 7,109,034, 6,261,815, and 5,874,268.

[0142] Preparation of Compounds of Formula (I) The compounds of the present invention can be synthesized by many methods available to those skilled in the art of organic chemistry.The following describes general and exemplary synthetic schemes for preparing the compounds of the present invention.These schemes are illustrative and are not intended to limit the possible techniques that those skilled in the art can use to prepare the compounds disclosed herein.Various methods for preparing the compounds of the present invention will be apparent to those skilled in the art.In addition, various steps of the synthesis can be carried out in an alternative order to obtain the desired compound or compounds.

[0143] Examples of compounds of the present invention that have been prepared according to the methods described in the general schemes are set forth in the Examples section below.

[0144] The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of organic synthetic chemistry, or variations thereof as will be appreciated by those skilled in the art. The reactions are carried out in a solvent or solvent mixture appropriate to the reagents and materials used and suitable for the transformations being effected. One skilled in the art of organic synthesis will understand that the functional groups present on the molecule should be consistent with the proposed transformations.

[0145] Those skilled in the art can also easily modify the reagents and reaction conditions exemplified in the following schemes to include any combination of the substituents defined above, and can also easily use interchangeable steps for each synthetic process and incorporate isolation and / or purification steps as needed.

[0146] The starting materials and intermediates useful for preparing the compounds of the invention are either commercially available or can be prepared by well-known synthetic procedures.

[0147] The final products obtained by the syntheses described below can be purified using techniques commonly known to those skilled in the art, such as preparative chromatography, thin layer chromatography, HPLC, or crystallization.

[0148] Exemplary processes for synthesizing the compounds of the present invention are described herein.

[0149] In the following preparations, compounds, solvents, reactants, and other optional materials are from commercial sources unless otherwise specified. Generally, compounds of formula (I) can be prepared by a multi-step synthesis starting from dehydroepiandrosterone (prasterone). Dehydroepiandrosterone is commercially available or can be prepared according to well-known methods starting from 4-androstene-3,17-dione (androstenedione).

[0150] Production of 5α-androstane-3β,6α,17β-triol [ka] A suitable intermediate for the synthesis of 6-α-3,17-androstanedione (2) was prepared from dehydroepiandrosterone 1 by hydroboration followed by oxidation, as described by De Munari et al. (J. Med. Chem., 2003, 46(17):3644-54). Briefly, a solution of dehydroepiandrosterone 1 (5 g, 17.5 mmol, 1 equiv.) in THF (85 mL) was stirred under Ar at -20 °C. A 1 M solution of BH3·THF complex in THF was then added to the stirred solution (44 mL, 44 mmol, 2.5 equiv.), and stirring was continued at room temperature for 3 h. HO (85 mL) was carefully added dropwise, followed by the dropwise addition of NaBO3·4HO (5.4 g, 35 mmol, 2 equiv.). After stirring overnight at room temperature, the mixture was filtered. The solid was washed with THF and then discarded. The liquid was saturated with NaCl and extracted with THF (3 × 40 mL). The combined organic extracts were dried with NaCl and NaSO, filtered, and evaporated to dryness. The crude 5α-androstane-3β,6α,17β-triol 2 was crystallized from EtOAc / MeOH (2 / 1, 10 mL / g) to give a white solid (3.8 g, 70%).

[0151] Production of 6α-hydroxyandrostane-3,17-dione [ka] Intermediate 3 was obtained from 2 by selective oxidation at C3 and C17. NBS (3.4 g, 19.5 mmol, 3 equiv.) was added to a stirred solution of 5α-androstane-3β,6α,17β-triol 2 (2 g, 6.5 mmol, 1 equiv.) in dioxane / HO / pyridine (54 / 10 / 1 mL) at 0 °C. After the addition, the mixture was warmed to room temperature and stirred overnight. The orange solution was diluted with water (50 mL) and quenched with NaSO (350 mg). The organic solvent was evaporated under vacuum until a white solid appeared. The solid was filtered and washed with water. After drying at 40 °C, 6α-hydroxyandrostane-3,17-dione 3 was obtained as a white solid (1.3 g, 70%).

[0152] Synthesis of adrostan-3-methylene-17-one [ka] 6-α-3,17-androstanedione 3 was then converted to the exomethane derivative 6 (androstan-3-methylene-17-one) by selective Wittig reaction at the C3 carbonyl followed by cross-metathesis coupling with 5-pentenoic acid. t-BuOK (670 mg, 6 mmol, 4 equiv.) was added to a suspension of methyltriphenylphosphonium bromide (1.66 g, 6 mmol, 4 equiv.) in THF (10 mL) at -5 °C. The solution immediately turned bright orange. After 10 min, 6α-hydroxyandrostan-3,17-dione 3 (450 mg, 1.5 mmol, 1 equiv.) was added while maintaining the temperature below 0 °C. Immediately after the addition, the reaction was quenched by the addition of 1 M aqueous HCl (15 mL) and extracted with EtOAc (3 × 20 mL). The combined organic phases were dried over NaSO and evaporated to dryness. The crude extract was purified by column chromatography (eluent EtOAc:petroleum spirit 4:6) to give 376 mg (83%) of adrostan-3-methylene-17-one 6 as a white foam.

[0153] Direct synthesis of CVie 201 and 202 from precursor 6 via cross-metathesis [ka] Hoveyda-Grubbs second-generation catalyst (12 mg, 0.015 mmol, 0.05 equiv.) was added to a solution of androstane-3-methylene-17-one 6 (100 mg, 0.33 mmol, 1 equiv.) in DCM (1 mL). The solution was then heated to reflux and treated every 20 min with 10 μL of 4-pentenoic acid (330 μL total, 3.3 mmol, 10 equiv.). After the addition was complete, the mixture was refluxed for an additional 2 h. The reaction mixture was concentrated in vacuo and purified by flash chromatography (eluent acetone:petroleum spirit 3:7 + 0.1% HCOH) to give two distinct white solids, (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (4.8 mg, 4%) (CVie201) and (Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (7.2 mg, 6%) (CVie202).

[0154] Alternatively, CVie201 and CVie202 were obtained by varying the Wittig reaction. In the first method (Route A), a polar solvent such as DMSO and a base such as NaH were used to stabilize the betaine intermediate. In the second approach (Route B), an aprotic solvent such as THF was used as a base to stabilize the cyclooxaphosphetane intermediate. Route A produced a mixture of diastereomers (60% Z / syn CVie202; 30% E / anti CVie201), while Route B provided CVie202 derived from the cyclooxaphosphate intermediate. Both methods require the generation of diastereomers 7 and / or 8, as described below.

[0155] Alternative synthesis of CVie201 and 202 via Wittig reaction Route A [ka] 60% NaH in mineral oil (100 mg, 2.56 mmol, 8 equiv.) was carefully added to dry DMSO (1 mL) under an Ar atmosphere. The resulting solution was stirred at 60 °C for 20 min. After cooling at room temperature, (3-carboxypropyl)triphenylphosphonium bromide (550 mg, 1.28 mmol, 4 equiv.) was added. A bright orange color immediately appeared. The solution was stirred for 2 h. 6α-hydroxyandrostane-3,17-dione 3 (100 mg, 0.32 mmol, 1 equiv.) was then added to the mixture. The resulting solution was stirred at room temperature for an additional 4 h. The reaction mixture was diluted with EtOAc (25 mL) and washed with 1 M aqueous HCl (3 × 30 mL). The organic layer, dried over NaSO, was evaporated to dryness to give 25 mg of crude material.

[0156] The crude material was first dissolved in MeOH (1.5 mL), followed by the addition of EDC hydrochloride (115 mg, 0.6 mmol, 2 equiv.) and DMAP (5 mg, 0.03 mmol, 0.1 equiv.). The solution was stirred at room temperature for 3 h and concentrated in vacuo. The crude solid was dissolved in EtOAc (15 mL) and washed with 1 M aqueous HCl (3 × 10 mL). The crude product was purified by flash chromatography on silica gel (acetone:petroleum spirit 3:7) to afford 25 mg of a clear oil (20%) containing a mixture of diastereoisomers 7 and 8.

[0157] Route B [ka] A 1 M solution of LiHMDS in THF (40 mL, 40 mmol, 12 equiv.) was carefully added to a suspension of (3-carboxypropyl)triphenylphosphonium bromide (8.5 g, 20 mmol, 6 equiv.) in dry THF (33 mL) at −40° C. under an Ar atmosphere. The solution was stirred at −40° C. until a bright orange color appeared. 6α-Hydroxyandrostane-3,17-dione 3 (1 g, 3.3 mmol, 1 equiv.) was then added to the solution at −40° C. After stirring overnight at room temperature, the reaction mixture was quenched with 1 M aqueous HCl (300 mL) and extracted with EtOAc (3 × 350 mL). The combined organic layers were dried over NaSO and evaporated to dryness.

[0158] The crude material was dissolved in absolute EtOH (17 mL), and then EDC hydrochloride (1.26 mg, 6.6 mmol, 2 equiv.) and DMAP (50 mg, 0.3 mmol, 0.1 equiv.) were added. The mixture was stirred at room temperature for 3 h. The reaction was diluted with EtOAc (150 mL) and washed with 1 M aqueous HCl (3 × 100 mL). The crude product was purified by flash chromatography on silica gel (acetone:petroleum spirit 3:7) to give 910 mg (72%) of compound 8.

[0159] [ka] Final hydrolysis of the methyl (or ethyl) ester A 1 M aqueous solution of LiOH (150 μL, 2.5 equiv.) was added to a solution of the methyl esters 7 and 8 (25 mg, 0.06 mmol, 1 equiv.) in THF (600 μL) and water (200 μL). After 2 h, the reaction was diluted with water (10 mL) and quenched by adding 1 M HCl until the solution reached pH 1. The aqueous phase was extracted with EtOAc (3 × 15 mL). The combined organic layers were dried over NaSO and evaporated to dryness. The crude product was purified by flash chromatography (AcOEt:petroleum spirit 7:3, 1% HCOOH). Two white solids were obtained, corresponding to the E (7 mg, 31%) and Z (12 mg, 54%) diastereoisomers (CVie201 and CVie202, respectively).

[0160] [ka] Synthetic method for CVie203 and 204: Synthesis of intermediate compound 12 To prepare CVie203 and CVie204, precursor 12 was first generated from 6-α-3,17-androstanedione 3. The carbonyl of 6-α-3,17-androstanedione 3 was protected as a diketal by reaction with ethylene glycol in toluene under an acid catalyst (p-tSA or camphorsulfonic acid), yielding compound 9. Compound 9 was oxidized with PCC or other oxidizing agents to give compound 10, which was reduced with NaBH or KBH to generate protected alcohol 11, which selectively possessed the C6-hydroxyl group in the β-configuration. Final cleavage of the cyclic diketal by acidic treatment in acetone, as described by De Munari et al. (J. Med. Chem., 2003, 46(17):3644-54), gave precursor 12.

[0161] Briefly, a solution of 6α-hydroxyandrostane-3,17-dione (1.5 g, 4.9 mmol, 1 equiv.), ethylene glycol (10.5 mL, 88 mmol, 36 equiv.), and PTSA (561 mg, 2.9 mmol, 0.6 equiv.) in toluene (160 mL) was stirred at reflux for 12 h with a Dean-Stark trap. After cooling to room temperature, the mixture was neutralized with 5% aqueous NaHCO3. The organic layer was separated, washed with HO (2 × 40 mL), dried over Na2SO4, and evaporated to dryness to yield 3,3:17,17-bis(ethylenedioxy)androstan-6α-ol 9 as a white solid (1.9 g, 98%).

[0162] PCC (148 mg, 0.69 mmol, 4 equiv.) was added to a solution of 3,3:17,17-bis(ethylenedioxy)androstan-6α-ol (3 g, 14 mmol, 1 equiv.) 9 and sodium ascorbate (1.2 g, 14 mmol, 4 equiv.) in dry CHCl (87 mL) at 0 °C. The mixture was stirred overnight at room temperature. The mixture was washed with 1 M aqueous HCl (3 × 30 mL) and water (3 × 30 mL). The organic layer was dried over NaSO and evaporated to dryness. The crude product was purified by flash chromatography on a silica gel column (eluent acetone:petroleum spirit 2:8). 3,3:17,17-bis(ethylenedioxy)androstan-6-one 10 was obtained as a white solid (1.53 g, 96%).

[0163] NaBH (144 mg, 3 mmol, 1.2 equiv) was added to a stirred suspension of 3,3:17,17-bis(ethylenedioxy)androstan-6-one 10 (1 g, 2.5 mmol, 1 equiv) in MeOH (13 mL) at 0 °C. After 2 h at 0 °C, HO (40 mL) was added dropwise. The mixture was extracted with EtOAc (3 × 40 mL). The combined organic extracts were dried over NaSO, filtered, and evaporated to dryness to give 3,3:17,17-bis(ethylenedioxy)androstan-6β-ol 11 (915 mg, 92%) as a white solid.

[0164] PTSA (2.26 g, 11.5 mmol, 5 equiv.) was added portionwise over 5 min to a solution of 3,3:17,17-bis(ethylenedioxy)androstan-6β-ol 11 (910 mg, 2.3 mmol, 1 equiv.) in acetone (46 mL). After stirring at room temperature for 1 h, the solution was quenched by adding 5% aqueous NaHCO until pH 7. After stirring for 5 min, a white solid appeared. The volatiles were removed in vacuo. The suspension was extracted with CHCl (3 × 30 mL), and the combined organic extracts were washed with brine (40 mL), dried over NaSO, filtered, and evaporated. The resulting solid was stirred with n-hexane / EtOAc 8 / 2 (10 mL) for 45 min and then collected by filtration. The solid was dried at 45 °C for 3 h. 568 mg (81%) of a white solid (i.e., 6β-hydroxyandrostane-3,17-dione 12) was obtained.

[0165] 12 Final Conversions to CVie 203 and 204 [ka] CVie203 and CVie204 were obtained from precursor 12 via Wittig reaction using the same methodology as described above for CVie201 and CVie202. The configuration of the C3-C1′ double bond was identified in the two isomers by NOESY experiments.

[0166] Briefly, 60% NaH in mineral oil (100 mg, 2.56 mmol, 8 equiv.) was carefully added to dry DMSO (1 mL) under an Ar atmosphere. The resulting solution was stirred at 60 °C for 20 min. After cooling to room temperature, (3-carboxypropyl)triphenylphosphonium bromide (550 mg, 1.28 mmol, 4 equiv.) was added. A bright orange color immediately appeared. The solution was stirred for 2 h. Then, 6β-hydroxyandrostane-3,17-dione 12 (100 mg, 0.32 mmol, 1 equiv.) was added to the mixture. The resulting solution was stirred at room temperature for an additional 4 h. The reaction mixture was diluted with EtOAc (25 mL) and washed with 1 M aqueous HCl (3 × 30 mL). The organic layer was dried over NaSO and evaporated to dryness to give 25 mg of crude material.

[0167] The crude material was then dissolved in MeOH (1.5 mL). EDC hydrochloride (115 mg, 0.6 mmol, 2 equiv.) and DMAP (5 mg, 0.03 mmol, 0.1 equiv.) were added. The solution was stirred at room temperature for 3 h. It was concentrated in vacuo. The crude solid was dissolved in EtOAc (15 mL) and washed with 1 M aqueous HCl (3 × 10 mL). The crude product was purified by flash chromatography on silica gel (acetone:petroleum spirit 3:7) to give a mixture of diastereoisomers 13 and 14 in 17% and 30% yields, respectively.

[0168] The reaction mixture was concentrated in vacuo and purified by flash chromatography (eluent acetone:petroleum spirit 3:7 + 0.1% HCOH) to give two distinct white solids, (E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie203) and (Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie204).

[0169] Hydrogenation and ester hydrolysis to produce CVie214, CVie215, and CVie217 [ka] Compound CVie217 was prepared from the mixture of diastereomers 7 and 8 described above. Briefly, hydrogenation of the C3-C1' double bond of the diastereomers was carried out using a Pd-C catalyst in EtOAc. The resulting compound was CVie217. The configuration of the stereocenter formed at C3 was identified by NOESY experiments. Compound CVie217 was then hydrolyzed with 1 M LiOH or NaOH in THF to produce CVie214. Similarly, diastereomers 13 and 14 were hydrogenated with a Pd-C catalyst in EtOAc to produce the ester compound 19, which was then hydrolyzed with 1 M LiOH or NaOH in THF to produce CVie215.

[0170] Wittig reaction followed by C=C hydrogenation and ester hydrolysis to prepare CVie213 and CVie216 [ka] Compound 6-α-3,17-androstanedione 3 was also used as the starting point for the synthesis of CVie213 and CVie216 ​​via the Horner-Emmons reaction. First, triethylphosphonoacetate (6.5 mL, 33 mmol, 5 equiv.) was carefully added to a suspension of 60% NaH in mineral oil (1.3 g, 33 mmol, 5 equiv.) in DMF (200 mL) at 0 °C under an Ar atmosphere. The resulting solution was warmed to room temperature and stirred for 20 min. Next, 6-α-3,17-androstanedione 3 (2 g, 6.5 mmol, 1 equiv.) was added at 0 °C. After stirring overnight at room temperature, the reaction was quenched by careful addition of HO (100 mL) and extracted with EtO (3 × 150 mL). The combined organic layers were dried over NaSO and evaporated in vacuo. The crude material was purified by flash chromatography on a silica gel column (acetone:petroleum spirit 3:7) to yield 2.1 g (86%) of a mixture of two diastereoisomers (compound 21) as a clear oil.

[0171] Under an Ar atmosphere, 10% Pd-C (700 mg) was added to a solution of diastereoisomeric compound 21 (2 g, 5.3 mmol, 1 equiv.) in degassed EtOAc (200 mL). After three vacuum / hydrogen cycles, the reaction was stirred overnight at room temperature under an H atmosphere. After removing the hydrogen by vacuum / Ar cycles, the reaction mixture was filtered through CELITE®. The filtered solution was evaporated to dryness. CVie213 product was obtained without purification in an amount of 1.8 g (90%). CVie216 ​​was further produced by hydrolysis of CVie213 with 1 M LiOH or NaOH in THF.

[0172] Wittig reaction followed by C=C hydrogenation and ester hydrolysis to prepare CVie218 and CVie219 [ka] Similarly, 6-α-3,17-androstanedione 3 was reacted with an appropriate triphenylphosphonium salt (e.g., 5-carboxytriphenylphosphonium bromide, LiHMDS, THF, followed by EtOH (or MeOH)) to generate compound 24. Using a Pd-C catalyst in the presence of hydrogen, CVie218, which contained a C6 chain at the C-3 position, was generated. Hydrolysis of CVie218 with 1 M LiOH or NaOH in THF generated CVie219.

[0173] Synthesis of derivatives bearing primary amine groups from precursor 6 by metathesis reaction with Boc-protected amines followed by Boc deprotection [ka] For the synthesis of derivatives bearing a primary amine group as the X substituent of formula (I), a cross-metathesis reaction was carried out on precursor 6 using the same experimental conditions as described above for the synthesis of CVie201 and CVie202.

[0174] Briefly, Hoveyda-Grubbs second-generation catalyst was added to a solution of androstan-3-methylene-17-one 6 in DCM. Androstan-3-methylene-17-one 6 was then mixed with an appropriate Boc-protected amine (e.g., tert-butylpent-4-en-1-yl-carbamate or N-Boc-4-pentyn-1-amine) containing the exo-methylene group to generate diastereoisomer 25 (25% yield). Compound 25 (50 mg, 0.1 mmol, 1 equiv.) was treated with 500 μL of a 1:1 mixture of TFA / DCM trifluoroacetic acid in DCM and stirred at room temperature to directly cleave the Boc group. After stirring at room temperature for 1 min, the reaction was diluted with EtOAc (50 mL) and washed with saturated aqueous NaHCO (3 × 30 mL). The organic phase was dried over Na2SO4, filtered, and evaporated to dryness to yield (EZ)-3-(4-aminobutylidene]-6α-hydroxyandrostan-17-one (CVie209) as a white solid (28 mg, 75%).

[0175] Alternatively, compound 25 was reacted with iodotrimethylsilane in an alcohol solvent (e.g., MeOH) to undergo Boc cleavage accompanied by migration of the exocyclic double bond, yielding CVie205, which possesses an endocyclic double bond between C2 and C3. Briefly, 1 M TMSI in DCM (100 μL, 0.1 mmol, 1 equiv.) was added to a solution of diastereoisomer 25 (50 mg, 0.1 mmol, 1 equiv.) at room temperature. After stirring at the same temperature for 2 h, the solvent was removed in vacuo. Methanol (2 mL) was added to the residue and allowed to stand at room temperature for 1 h. After removing the solvent in vacuo, CVie207 was obtained without further purification.

[0176] Synthesis of cyclic amine derivatives with exocyclic unsaturation: CVie205, CVie206, CVie210, and CVie211 [ka] Cyclic amine derivatives were synthesized by the sodium hydride (NaH)-DMSO Wittig reaction as described above for CVie203 and CVie204 using the appropriate N-protected phosphonium salt, e.g., N-Boc-4-(2-triphenylphosphoniumethyl)azetidine iodide to give compounds 26 and 27, or N-Boc-3-(2-triphenylphosphoniumethyl)piperidine iodide to give compounds 28 and 29. After purification of the diastereoisomeric mixture, the N-Boc group was cleaved by acidic hydrolysis with TFA to give CVie205, CVie206, CVie210, and CVie211.

[0177] Synthesis of CVie208 with endocyclic unsaturation (C=C double bond migration during Boc deprotection) [ka] Additionally, compounds 28 and 29 were treated with TMSI to generate CVie208, as described above for the synthesis of CVie207.

[0178] Hydrogenation with TFA and Boc cleavage to generate CVie212 [ka] Alternatively, compound 30 was synthesized by catalytic hydrogenation (H 2 , Pd—C, EtOAc) of the double bond of compounds 28 and 29, followed by Boc cleavage with TFA in DCM to generate CVie212.

[0179] The synthesis of compounds containing 6α-hydroxymethylandrostane-7,17-dione was achieved starting from the general intermediate 37. Compound 37 itself was synthesized from 4-androstene-3,17-dione 31 by protection of the two ketone moieties with a cyclic acetal followed by simultaneous double bond migration (32), oxidation of the allylic position with sodium dichromate (33), formation of a silyl enol ether (35), hydroxymethylation with MeAl and formaldehyde (36), and final cleavage of the acetal under acidic conditions. The synthesis is described in detail in the following paragraphs.

[0180] Synthesis of Compound 32: (20S,7R)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolane]-12-ene [ka] A mixture of androst-4-ene-3,17-dione 31 (400.0 g, 1.4 mol) and PTSA·HO (13.3 g, 70.0 mmol) in ethylene glycol (8.0 L) was stirred at 100 °C until the reaction was complete. Approximately 5.0 L of glycol was distilled under vacuum to a boiling point of approximately 80–85 °C. The mixture was cooled to room temperature. The pH of the mixture was adjusted to ∼9. The mixture was then poured into ice water. The mixture was filtered, and the solid was washed with water, collected, and treated with acetone to give crude compound 32 (469.0 g, 89%) as a yellow solid.

[0181] Synthesis of Compound 33: (20S,7R)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolane]-12-en-14-one [ka] A mixture of compound 32 (440.0 g, 1.2 mol), HOSU (541.2 g, 4.7 mol), and NaCrO·HO (527.5 g, 1.8 mol) in acetone (8.0 L) was vigorously stirred at 50 °C for 2 days. After cooling to room temperature, the mixture was quenched with aqueous NaSO and stirred for 20 min. The mixture was poured into ice water. The resulting mixture was stirred for 20 min and then filtered. The solid filtrate was washed with water, collected, and dried in vacuo to give crude compound 33 (390.0 g, 85%) as a yellow solid.

[0182] Synthesis of Compound 34: (7S,20S)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolan]-14-one [ka] A mixture of compound 33 (50.0 g, 128.9 mmol) in EtOAc (1250 mL) was added to Pd / C (16.0 g). The mixture was then stirred under H at room temperature overnight. TLC showed that the reaction was complete. The mixture was filtered, concentrated, and purified by flash chromatography (PE / EA=2 / 1) to give compound 34 (25.0 g, 50.0%) as a white solid.

[0183] Synthesis of Compound 35: 1-((20S,7R)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecane-14',2''-1,3-dioxolane]-13-en-14-yloxy)-1,1-dimethyl-1-silaethane [ka] A mixture of compound 34 (20.0 g, 51.3 mmol) in dry THF (100.0 mL) was stirred at −78°C, followed by the dropwise addition of 1.5 M LDA (205.2 mL, 307.8 mmol) in toluene. After stirring at the same temperature for 1 h, Me3SiCl (50.0 mL, 400.1 mmol) was added dropwise. After stirring at −70°C for 3 h, the temperature was raised to −30°C, and triethylamine (33.5 g, 331.5 mmol) was added. After stirring at the same temperature for 1 h, the mixture was warmed to room temperature, and water (200.0 mL) and EtOAc (100.0 mL) were added. The separated aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by flash chromatography (PE / EA=2 / 1) to give compound 35 (14.3 g, 60.3%) as a white solid.

[0184] Synthesis of Compound 36: (13S,20S,7R)-13-(hydroxymethyl)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolan]-14-one [ka] A mixture of 2,6-diphenylphenol (10.0 g, 27.6 mmol) in dry DCM (450.0 mL) was added dropwise to a solution of MeAl in toluene (41.4 mL, 82.9 mmol) while cooling in an ice / water bath so that the temperature did not exceed room temperature. After stirring at room temperature for 1 hour, the solution was cooled to 0 °C, and a solution of trioxane (24.8 g, 276.0 mmol) in dry DCM (100.0 mL) was added dropwise. The pale yellow solution was stirred at 0 °C for an additional 1 hour, and then the temperature was cooled to -78 °C. A solution of compound 35 (10.0 g, 27.6 mmol) in dry DCM (125 mL) was added. After stirring at -78 °C for 1 hour, the temperature was raised to -20 °C, and the reaction mixture was stirred at that temperature overnight. 5% aqueous NaHCO (85.0 mL) was added at room temperature. The jelly mixture was filtered through a CELITE® pad and washed thoroughly with DCM. The separated organic layer was washed with water and evaporated. Approximately 1 M TBAF in THF (24.0 mL) was added to the residue, and the solution was stirred at room temperature for 1.5 hours. The solution was washed with water, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by flash chromatography to give compound 36 (6.5 g, 71.4%) as a yellow solid.

[0185] Synthesis of Compound 37: (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyldecahydro-1H-cyclopenta[a]phenanthrene-3,7,17(2H,4H,8H)-trione [ka] A mixture of compound 36 (8.0 g, 19.0 mmol) in acetone (100.0 mL) was added to 10% aqueous HCl (50.0 mL). The mixture was then heated to 70° C. for 1 hour. TLC showed the reaction was complete. The mixture was quenched with 5% aqueous NaOH and extracted with DCM (50.0 mL × 2). The combined organic phases were washed with brine (50.0 mL), dried over NaSO, filtered, concentrated, and purified by flash chromatography (DCM / EA = 4 / 1) to give the crude product, which was treated with ether to give the pure product 37 (3.3 g, 52.4%) as a white solid.

[0186] Synthesis of Compound 38: (13S,14S,20S,7R)-13-(hydroxymethyl)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolan]-14-ol [ka] NaBH4 (4.0 g, 104.8 mmol) was slowly added to a mixture of compound 36 (22.0 g, 52.4 mmol) in MeOH (1000 mL) at 0 °C. The mixture was then stirred at room temperature for 1 hour. TLC showed the reaction was complete. The mixture was quenched with 5% aqueous NaH2PO4 (220 mL) and extracted with DCM (300 mL × 3). The combined organic phases were washed with brine (200 mL), dried over Na2SO4, filtered, concentrated, and purified by flash chromatography (DCM / EA = 4 / 1) to give the crude product, which was treated with ether to give compound 38 (7.5 g, 34.1%) as a white solid.

[0187] Synthesis of Compound 39: (8S,9S,15S,2R)-9-hydroxy-8-(hydroxymethyl)-2,15-dimethyltetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecane-5,14-dione [ka] To a mixture of compound 38 (5.7 g, 13.5 mmol) in acetone (70 mL) was added 10% aqueous HCl (35 mL). The mixture was then heated to 70 °C for 1 h. TLC showed the reaction was complete. The mixture was quenched with 5% aqueous NaOH and extracted with DCM (50 mL × 2). The combined organic phases were washed with brine (50 mL), dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography (DCM / EA = 4 / 1) to give the crude product, which was treated with ether to give the pure product 39 (1.8 g, 40.0%) as a white solid.

[0188] Synthesis of Compound 40: tert-butyl-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)azetidine-1-carboxylate [ka] To a solution of the phosphonium salt (2.57 g, 4.5 mmol) in THF (25 mL) was added a solution of n-BuLi in THF (2.5 M, 3.6 mL, 9.0 mmol) at −78° C. The mixture was stirred at 30° C. for 1 hour. Compound 37 (500 mg, 1.5 mmol) was then added to the mixture at −20° C., followed by warming to 30° C. for 2 hours. The mixture was quenched with saturated NH4Cl (25 mL) and extracted with EtOAc (25 mL × 3). The combined organic layers were concentrated, and the residue was purified by silica gel column chromatography (hexane / EtOAc = 1 / 1) to give the crude compound. The compound was purified by reverse-phase column chromatography to give pure compound 40 (60 mg, 8%) as a white solid.

[0189] Synthesis of Compound 41: tert-butyl-3-(2-((3S,6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)azetidine-1-carboxylate [ka] To a solution of compound 40 (60 mg, 0.12 mmol) in EA (3 mL) was added Pd / C (60 mg). The mixture was then stirred under H at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give compound 41 (52 mg, 86%) as a white solid.

[0190] CVie407: Synthesis of (3S,6S,10R,13S)-3-(2-(azetidin-3-yl)ethyl)-6-(hydroxymethyl)-10,13-dimethyldodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 41 (52 mg, 0.10 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 hour. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL x 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound Cvie407 (13 mg, 32%) as a white solid.

[0191] Synthesis of Compound 42: tert-butyl-3-((E)-2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)azetidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 0.7 mL, 1.80 mmol) was added to a solution of the compound phosphonium salt (514 mg, 0.90 mmol) in THF (5 mL) at -78 °C. The mixture was stirred at 40 °C for 1 hour. Compound 39 (100 mg, 0.30 mmol) was then added to the mixture at 0 °C, and the mixture was then warmed to 40 °C overnight. The reaction was repeated 9 times. The mixture was quenched with saturated NH4Cl (80 mL) and extracted with EtOAc (100 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound 42 (20 mg, 1%) as a yellow solid.

[0192] CVie403: Synthesis of (6S,10R,13S)-3-(2-(azetidin-3-yl)ethylidene)-6-(hydroxymethyl)-10,13-dimethyldodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 40 (130 mg, 0.26 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 h. The mixture was basified to pH 8-9 with saturated NaHCO3. The mixture was extracted with DCM (30 mL × 3). The combined organic layers were concentrated, and the residue was purified by preparative HPLC to give compound CVie403 (13 mg, 13% yield) as a yellow solid.

[0193] Synthesis of Compound 43: 3-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)azetidine-1-carboxylate [ka] A mixture of compound 42 (20 mg, 0.04 mmol), Pd / C (10%, 20 mg), and Pd(OH) (20%, 20 mg) in EtOAc (2 mL) was stirred under H (balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 43 (20 mg, 100%) as a brown solid.

[0194] CVie408: (6S,7S,10R,13S)-3-(2-(azetidin-3-yl)ethyl)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyltetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one Synthesis of [ka] A mixture of compound 43 (20 mg, 0.04 mmol) in TFA / DCM (1:1, 2 mL) was stirred at 0 °C for 30 min. The mixture was diluted with saturated NaHCO and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie408 (6.4 mg, 40%) as a yellow solid.

[0195] Synthesis of Compound 44: tert-butyl-3-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)pyrrolidine-1-carboxylate [ka] A solution of n-BuLi in THF (1.5 M, 1.57 mL, 3.94 mmol) was added to a solution of the phosphonium salt of the compound (1.5 g, 2.62 mmol) in THF (15 mL) at -78 °C. The reaction mixture was stirred at 35 °C for 1 hour. Then, a solution of compound 37 (350 mg, 1.05 mmol) was added to the mixture at -20 °C and warmed to room temperature for 2 hours. The mixture was quenched with saturated NH4Cl (25 mL) and extracted with EtOAc (25 mL x 3). The combined organic layers were concentrated, and the residue was purified by flash chromatography (hexane:EA = 1:1) to obtain the crude compound. The compound was then purified by reverse-phase column chromatography to obtain pure compound 44 (53 mg, 10%) as a white solid.

[0196] CVie402: Synthesis of (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(pyrrolidin-3-yl)ethylidene)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 44 (89 mg, 0.173 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 h. The mixture was diluted with saturated NaHCO3 and adjusted to pH = 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound CVie402 (38 mg, 53%) as a white solid.

[0197] Synthesis of Compound 45: tert-butyl-3-(2-((3S,6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)pyrrolidine-1-carboxylate [ka] A solution of compound 44 (53 mg, 0.103 mmol) in EA (3 mL) was added to Pd / C (60 mg). The mixture was then stirred under H at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give compound 45 (50 mg, 94%) as a white solid.

[0198] CVie409: Synthesis of (3S,6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(pyrrolidin-3-yl)ethyl)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 45 (50 mg, 0.09 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 hour. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL x 3). The combined organic layers were concentrated, and the residue was purified by preparative HPLC to give compound CVie409 (12 mg, 32%) as a white solid.

[0199] Synthesis of Compound 46: tert-butyl-3-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)pyrrolidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 0.7 mL, 1.80 mmol) was added to a solution of the compound phosphonium salt (527 mg, 0.90 mmol) in THF (5 mL) at -78 °C. The reaction mixture was stirred at 35 °C for 1 hour. Compound 39 (100 mg, 0.30 mmol) was then added to the mixture at 0 °C, and the mixture was then warmed to 35 °C overnight. The reaction was repeated four times. The mixture was quenched with saturated NH Cl (80 mL) and extracted with EtOAc (100 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound 46 (26 mg, 3%) as a white solid.

[0200] Synthesis of Compound 47: tert-butyl-3-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)pyrrolidine-1-carboxylate [ka] A mixture of compound 46 (26 mg, 0.05 mmol), Pd / C (10%, 30 mg), and Pd(OH) (20%, 30 mg) in EtOAc (3 mL) was stirred under H (balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 47 (26 mg, 100%) as a yellow solid.

[0201] CVie410: Synthesis of (6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(pyrrolidin-3-yl)ethyl)tetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one [ka] A solution of compound 47 (26 mg, 0.05 mmol) in TFA / DCM (1:2, 2 mL) was stirred at 0 °C for 1 h. The mixture was diluted with saturated NaHCO and adjusted to pH 8-9. The mixture was extracted with DCM (20 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie410 (9 mg, 43%) as a yellow solid.

[0202] Synthesis of Compound 48: tert-butyl-4-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)piperidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 2.90 mL, 7.20 mmol) was added to a mixture of compound phosphonium salt (2.16 g, 3.60 mmol) in THF (16 mL) at -78 °C. The reaction mixture was stirred at 30 °C for 1 hour. Compound 37 (400 mg, 1.20 mmol) was then added to the mixture at -20 °C. The mixture was stirred at -20 °C for 30 minutes and then warmed to 30 °C for 2 hours. The mixture was quenched with saturated NH Cl (15 mL) and extracted with EtOAc (30 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound 48 (28 mg, 4%) as a yellow solid.

[0203] CVie405: Synthesis of (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(piperidin-4-yl)ethylidene)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 46 (80 mg, 0.152 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 30 minutes. The mixture was basified with saturated NaHCO to pH = 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie405 (30 mg, 46%) as a yellow solid.

[0204] Synthesis of Compound 49: tert-butyl-4-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)piperidine-1-carboxylate [ka] A mixture of compound 48 (28 mg, 0.05 mmol) and Pd / C (10%, 50 mg) in EtOAc (2 mL) was stirred under H (in a balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 49 (28 mg, 100%) as a yellow solid.

[0205] CVie411: Synthesis of (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(piperidin-4-yl)ethyl)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 49 (28 mg, 0.05 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 30 minutes. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie411 (10 mg, 43%) as a yellow solid.

[0206] Synthesis of Compound 50: tert-butyl-4-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)piperidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 0.70 mL, 1.80 mmol) was added to a solution of phosphonium salt (540 mg, 0.90 mmol) in THF (5 mL) at −78° C. The reaction mixture was stirred at 40° C. for 1 h. Compound 39 (100 mg, 0.30 mmol) was then added to the mixture at 0° C., and then warmed to 40° C. for 2 h. The reaction was repeated five times. The mixture was quenched with saturated NH4Cl (80 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were concentrated, and the residue was purified by preparative HPLC to give crude compound 50 (35 mg, 4%) as a white solid.

[0207] Synthesis of Compound 51: tert-butyl-4-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)piperidine-1-carboxylate [ka] A mixture of compound 50 (35 mg, 0.07 mmol), Pd / C (10%, 40 mg), and Pd(OH) (20%, 40 mg) in EtOAc (2 mL) was stirred under H (in a balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 51 (35 mg, 100%) as a brown solid.

[0208] CVie412: Synthesis of (6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(piperidin-4-yl)ethyl)tetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one [ka] A solution of compound 51 (35 mg, 0.07 mmol) in TFA / DCM (1:2, 2 mL) was stirred at room temperature for 30 minutes. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie412 (13 mg, 46%) as a yellow solid.

[0209] Synthesis of Compound 52: tert-butyl ((E)-5-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthren-3(2H,4H,10H)-ylidene)pentyl)(methyl)carbamate [ka] To a mixture of N-Boc-N-methyl-5-triphenylphosphonium penteneamine iodide (4.26 g, 7.23 mmol) in THF (50 mL) was added n-BuLi (3.18 mL, 7.95 mmol) dropwise at −78° C. The mixture was stirred at 0° C. for 20 minutes. The mixture was then cooled to −30° C. Compound 37 (800 mg, 2.41 mmol) was then added to the reaction mixture. The mixture was stirred at room temperature overnight. The reaction mixture was quenched with HO and concentrated. The residue was purified by silica gel column chromatography (PE / EtOAc=1 / 2) and then by preparative HPLC to yield compound 52 (36 mg, 200 mg) as a colorless oil.

[0210] CVie401: Synthesis of (6S,10R,13S,E)-6-(hydroxymethyl)-10,13-dimethyl-3-(5-(methylamino)pentylidene)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A mixture of compound 52 (60 mg, 0.116 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature overnight. The mixture was then concentrated, diluted with EtOAc, washed with saturated Na2CO3, dried over Na2SO4, filtered, and concentrated to give compound CVie401 (38 mg, 79%) as a yellow oil.

[0211] Synthesis of Compound 53: tert-butyl (5-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxododecahydro-1H-cyclopenta[a]phenanthren-3(2H,4H,10H)-ylidene)pentyl)(methyl)carbamate [ka] To a mixture of N-Boc-N-methyl-5-triphenylphosphonium penteneamine iodide (4.39 g, 7.45 mmol) in THF (45 mL) was added dropwise a solution of nBuLi in THF (4.46 mL, 2.5 N, 11.16 mmol) at −78° C. The mixture was then stirred at 0° C. for 20 minutes. The mixture was cooled to −50° C., and compound 39 (830 mg, 2.48 mmol) was added. The mixture was stirred at room temperature overnight. The mixture was quenched with HO, concentrated, and purified by column chromatography (PE / EtOAc=1 / 1), followed by preparative HPLC to give compound 53 (80 mg, 300 mg) as a white solid.

[0212] CVie406: Synthesis of (6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3-(5-(methylamino)pentylidene)tetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one [ka] A solution of compound 53 (80 mg, 0.155 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 10 minutes. The mixture was basified with saturated NaHCO to pH 8-9. The mixture was extracted with DCM (25 mL × 2). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie406 (60 mg, 94%) as a yellow solid.

[0213] The present invention can be described in one or more of the following aspects or combinations thereof.

[0214] Aspect 1: Formula (I) [ka] [In the formula, X is any of carboxylic acids, carboxylic esters, and their bioisosteres (sulfates, sulfonic acids, phosphates, phosphonates, or nitrogen-containing ether cyclic rings such as triazoles and tetrazoles), primary alcohols, ethers, or amine groups (e.g., primary amines, secondary amines, or cyclic amines); n is 1, 2, 3, 4, or 5; The dashed line at C3-C1' represents an optional exocyclic double bond C=C at the C3-C1' position; The dashed line at C2-C3 represents the optional endocyclic double bond C=C; Y at C6 is hydroxyl (OH) in the α- or β-configuration, or hydroxymethyl (CH2OH) in the α-configuration; Z at C7 is either -H, or -OH in the alpha configuration, or a ketone, and the dashed line represents an optional carbonyl group (C=O) at that position. or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0215] Embodiment 2: The compound of embodiment 1, wherein X is selected from the group consisting of a carboxylic acid, a carboxylic acid ester, a primary amine, a secondary amine, and a cyclic amine.

[0216] Embodiment 3: The compound of embodiment 1, wherein X is a carboxylic acid or a carboxylic acid ester.

[0217] Embodiment 4: The compound of embodiment 1, wherein X is not a primary amine, a secondary amine, or a cyclic amine.

[0218] Aspect 5: The following: (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(E)-3-[2-(azetidic acid) (Z)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one;(E)-3-(4-aminobutyl)-6α-hydroxyandrost-2-en-17-one hydroiodide;3-[2-(piperidin-4-yl)ethyl]-6α-hydroxyandrost 3-[2-(Piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;(E)-3-[2-(Piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;(Z)-3-[2-(Piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;3β-[2-(Piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid; 4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid; 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl) Butyrate ; Ethyl 6 -(6α-hydroxy-17-oxoandrostan-3-yl) mosquito Proate; 6 -(6β-Hydroxy-17-oxoandrostan-3-yl)caproic acid;(E,Z)-3-(5-N-methylaminopentylidene)-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-[2-(pyrrolidin-3yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-[2-(azetidine-3 -yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-(5-N-methylaminopentylidene)-6α-hydroxymethyl-7α-hydroxyandrostan-17-one;3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione;3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethylandrostan-7,17-dione; 3β-[2-(pyrrolidin-3yl)ethyl]6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethylandrostan-7,17-dione; and 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one, 2. The compound according to embodiment 1, selected from the group consisting of:

[0219] Aspect 6: The following: (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;Ethyl (6α-hydroxy-17-ketoandrostan-3β-yl)acetate;4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid;4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid;2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl) Butyrate ; Ethyl 6-(6α-hydroxy-17-oxoandrostan-3-yl) mosquito Proate; and 6 -(6β-hydroxy-17-oxoandrostan-3-yl)caproic acid, 2. The compound according to embodiment 1, selected from the group consisting of:

[0220] Embodiment 7: The compound of embodiment 1, wherein the compound is selected from the group consisting of 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid, and 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid.

[0221] Aspect 8: The compound of any one of aspects 1-7, wherein the pharmaceutically acceptable salt is selected from chloride, bromide, sulfate, phosphate, nitrate, fumarate, succinate, oxalate, malate, tartrate, maleate, citrate, methanesulfonate, and benzoate.

[0222] Embodiment 9: A pharmaceutical composition for use in a method for treating heart failure, comprising a therapeutically effective amount of one or more of the compounds according to any one of embodiments 1-8 in combination with at least one pharmaceutically acceptable vehicle and / or excipient.

[0223] Embodiment 10: A pharmaceutical composition according to embodiment 9, which is formulated for enteral administration, parenteral administration, or inhalation.

[0224] Embodiment 11: The pharmaceutical composition of embodiment 10, which is formulated for oral administration.

[0225] Embodiment 12: The pharmaceutical composition of embodiment 11, wherein the composition is administered at a dose of about 1 mg / kg to about 20 mg / kg, optionally at a dose of about 1 mg / kg to about 10 mg / kg.

[0226] Embodiment 13: The pharmaceutical composition of embodiment 9, which is formulated for intravenous injection.

[0227] Embodiment 14: The pharmaceutical composition of embodiment 13, wherein the composition is administered at a dose of about 0.125 mg / kg to about 10 mg / kg, optionally at a dose of about 0.25 mg / kg to about 5 mg / kg.

[0228] Embodiment 15: The pharmaceutical composition of embodiment 9, which is formulated for intramuscular injection.

[0229] Embodiment 16: The pharmaceutical composition of embodiment 15, wherein the composition is administered at a dose of about 0.25 mg / kg to about 50 mg / kg, optionally at a dose of about 0.25 mg / kg to about 35 mg / kg.

[0230] Embodiment 17: A pharmaceutical composition according to any one of embodiments 9 to 16, which is administered at least once daily.

[0231] Embodiment 18: A pharmaceutical composition according to any one of embodiments 9 to 17, further comprising one or more additional therapeutically active ingredients.

[0232] Aspect 19: The one or more additional therapeutically active ingredients are an ACE inhibitor, an AIRB, a diuretic, a Ca 2+ 20. The pharmaceutical composition of embodiment 18, wherein the therapeutic agent is selected from the group consisting of a channel blocker, a beta-blocker, a digitalis, an NO donor, a vasodilator, a SERCA2a stimulator, a neprilysin (NEP) inhibitor, a myosin filament activator, a recombinant relaxin-2 mediator, a recombinant NP protein, an activator of soluble guanylate cyclase (sGC), and a beta-arrestin ligand for the angiotensin II receptor.

[0233] Aspect 20: The pharmaceutical composition of aspect 19, wherein the diuretic is selected from the group consisting of furosemide, bumetanide, torasemide, metolazone, aldosterone antagonists, thiazide diuretics.

[0234] Embodiment 21: The pharmaceutical composition according to embodiment 19, wherein the ACE inhibitor is lisinopril or ramipril.

[0235] Embodiment 22: The pharmaceutical composition of embodiment 18, wherein the one or more additional therapeutically active ingredients are selected from the group consisting of valsartan, candesartan, olmesartan, telmisartan, losartan, sacubitril, carvedilol, omecamtib, and metoprolol.

[0236] Aspect 23: A compound according to any one of aspects 1 to 8 for use as a medicament.

[0237] Embodiment 24: A compound according to embodiment 23 for use in the treatment of heart failure.

[0238] Embodiment 25: A compound according to embodiment 24 for use in the treatment of acute heart failure.

[0239] Embodiment 26: A compound according to embodiment 24 for use in the treatment of chronic heart failure.

[0240] Embodiment 27: A method of treating an individual having heart failure, the method comprising: (1) providing an individual having heart failure; (2) administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier, and (ii) a substantially pure SERCA2a stimulator, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and (3) measuring one or more parameters of cardiac function; wherein administering the pharmaceutical composition results in improvement of cardiac function.

[0241] Aspect 28: A substantially pure SERCA2a stimulator is represented by the general formula (I): [ka] [In the formula, X is selected from the group consisting of carboxylic acids, carboxylic esters, and bioisosteres thereof (e.g., sulfates, sulfonic acids, phosphates, phosphonates, and nitrogen-containing ether cyclic rings such as triazoles and tetrazoles), primary alcohols, ethers, or amine groups (e.g., primary amines, secondary amines, or cyclic amines); Y is hydroxyl (OH) in the α-configuration, hydroxyl (OH) in the β-configuration, or hydroxymethyl (CHOH) in the α-configuration; Z is selected from the group consisting of hydrogen (H), hydroxyl (OH) in the α-configuration, and ketone (O); n is 1 to 5; and The dotted line represents an optional double bond (C=C). 28. The method of embodiment 27, wherein

[0242] Embodiment 29: The method of embodiment 28, wherein X is selected from the group consisting of a carboxylic acid, a carboxylic ester, a primary amine, a secondary amine, and a cyclic amine.

[0243] Embodiment 30: The method of embodiment 28, wherein X is a carboxylic acid or a carboxylic acid ester.

[0244] Embodiment 31 The method of embodiment 28, wherein X is not a primary amine, a secondary amine, and a cyclic amine.

[0245] Aspect 32: A substantially pure SERCA2a stimulator, comprising: (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(E)-3-[2-(azetidic acid) (Z)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one;(E)-3-(4-aminobutyl)-6α-hydroxyandrost-2-en-17-one hydroiodide;3-[2-(piperidin-4-yl)ethyl]-6α-hydroxyandrost -2-en-17-one hydroiodide;(E,Z)-3-(4-aminobutylidene)-6α-hydroxyandrostan-17-one;(E)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;(Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one;3β-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid; 4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid; 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl) Butyrate ; Ethyl 6 -(6α-hydroxy-17-oxoandrostan-3-yl) mosquito Proate; 6 -(6β-Hydroxy-17-oxoandrostan-3-yl)caproic acid;(E,Z)-3-(5-N-methylaminopentylidene)-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-[2-(pyrrolidin-3yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-[2-(azetidine-3 -yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxymethylandrostan-7,17-dione;(E,Z)-3-(5-N-methylaminopentylidene)-6α-hydroxymethyl-7α-hydroxyandrostan-17-one;3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione;3β-[2-(azetidine- 3 -yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethylandrostan-7,17-dione; 3β-[2-(pyrrolidin-3yl)ethyl]6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethylandrostan-7,17-dione; and 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one, 29. The method of embodiment 28, wherein the compound is selected from the group consisting of:

[0246] Aspect 33: A substantially pure SERCA2a stimulator, comprising: (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;(Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid;Ethyl (6α-hydroxy-17-ketoandrostan-3β-yl)acetate;4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid;4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid;2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl) Butyrate ; Ethyl 6-(6α-hydroxy-17-oxoandrostan-3-yl) mosquito Proate; and 6 -(6β-hydroxy-17-oxoandrostan-3-yl)caproic acid, 29. The method of embodiment 28, wherein the compound is selected from the group consisting of:

[0247] Embodiment 34: The method of embodiment 28, wherein the substantially pure SERCA2a stimulator is selected from the group consisting of 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid, and 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid.

[0248] Embodiment 35: The method of any one of embodiments 28 to 34, wherein the pharmaceutically acceptable salt is selected from chloride, bromide, sulfate, phosphate, nitrate, fumarate, succinate, oxalate, malate, tartrate, maleate, citrate, methanesulfonate, and benzoate.

[0249] Embodiment 36: The method according to any one of embodiments 28 to 35, wherein the pharmaceutical composition is administered orally.

[0250] Embodiment 37: The method of embodiment 36, wherein the pharmaceutical composition is administered at a dose of about 1 mg / kg to about 20 mg / kg, optionally at a dose of about 1 mg / kg to about 10 mg / kg.

[0251] Embodiment 38: A method according to any one of embodiments 28 to 35, wherein the pharmaceutical composition is administered intravenously.

[0252] Embodiment 39: The method of embodiment 38, wherein the pharmaceutical composition is administered at a dose of about 0.125 mg / kg to about 10 mg / kg, optionally at a dose of about 0.25 mg / kg to about 5 mg / kg.

[0253] Embodiment 40: A method according to any one of embodiments 28 to 35, wherein the pharmaceutical composition is administered intramuscularly.

[0254] Embodiment 41: The method of embodiment 40, wherein the pharmaceutical composition is administered at a dose of about 0.25 mg / kg to about 50 mg / kg, optionally at a dose of about 0.25 mg / kg to about 35 mg / kg.

[0255] Embodiment 42: The method according to any one of embodiments 28 to 41, wherein the pharmaceutical composition comprises one or more additional therapeutically active ingredients.

[0256] Aspect 43: The one or more additional therapeutically active ingredients are an ACE inhibitor, an AIRB, a diuretic, a Ca 2+ 43. The method of embodiment 42, wherein the therapeutic agent is selected from the group consisting of a channel blocker, a beta-blocker, a digitalis, an NO donor, a vasodilator, a SERCA2a stimulator, a neprilysin (NEP) inhibitor, a myosin filament activator, a recombinant relaxin-2 mediator, a recombinant NP protein, an activator of soluble guanylate cyclase (sGC), and a beta-arrestin ligand for the angiotensin II receptor.

[0257] Embodiment 44: The method of embodiment 43, wherein the diuretic is selected from the group consisting of furosemide, bumetanide, torasemide, metolazone, aldosterone antagonists, and thiazide diuretics.

[0258] Embodiment 45: A method according to embodiment 43, wherein the ACE inhibitor is lisinopril or ramipril.

[0259] Embodiment 46: The method of embodiment 42, wherein the one or more additional therapeutically active ingredients are selected from the group consisting of valsartan, candesartan, olmesartan, telmisartan, losartan, sacubitril, carvedilol, omecamtib, and metoprolol.

[0260] Embodiment 47: The method according to any one of embodiments 28 to 46, wherein the individual is a human.

[0261] Aspect 48: The cardiac function of a subject is characterized in that one or more parameters of cardiac function are Ca 2+ Transient (CaT) amplitude, Ca 2+Inducible Ca 2+ release (CICR), velocity dependence of action potential duration at 90% repolarization (APD 90 ), diastolic membrane potential (E diast ), maximum depolarization rate (dV / dt max 48. The method of any one of aspects 28 to 47, wherein the measured value is selected from the group consisting of: heart rate, cardiac pressure, systolic blood pressure, diastolic blood pressure, LVEF, E / e′ ratio, E / Ea ratio, E / A ratio, and stroke volume.

[0262] Embodiment 49: The method according to any one of embodiments 28 to 48, wherein the measuring step is carried out before, during and / or after the administering step. [Example]

[0263] The present invention is further illustrated by the following examples.

[0264] Example 1: Preparation of compounds of formula (I) In the following examples, compounds, solvents, reactants, and other optional materials are from commercial sources unless otherwise specified. Generally, compounds of formula (I) were prepared by a multi-step synthesis starting from dehydroepiandrosterone (prasterone). Dehydroepiandrosterone is commercially available or can be prepared according to well-known methods starting from 4-androstene-3,17-dione (androstenedione).

[0265] Production of 5α-androstane-3β,6α,17β-triol [ka] A suitable intermediate for the synthesis of 6-α-3,17-androstanedione (2) was prepared from dehydroepiandrosterone 1 by hydroboration followed by oxidation, as described by De Munari et al. (J. Med. Chem., 2003, 46(17):3644-54), the entire contents of which are incorporated herein by reference. Briefly, a solution of dehydroepiandrosterone 1 (5 g, 17.5 mmol, 1 equiv.) in THF (85 mL) was stirred under Ar at -20 °C. A 1 M solution of BH3·THF complex in THF was then added to the stirred solution (44 mL, 44 mmol, 2.5 equiv.), and stirring was continued at room temperature for 3 h. HO (85 mL) was carefully added dropwise, followed by the dropwise addition of NaBO3·4HO (5.4 g, 35 mmol, 2 equiv.). After stirring overnight at room temperature, the mixture was filtered. The solid was washed with THF and then discarded. The liquid was saturated with NaCl and extracted with THF (3 × 40 mL). The combined organic extracts were dried with NaCl and NaSO, filtered, and evaporated to dryness. The crude 5α-androstane-3β,6α,17β-triol 2 was crystallized from EtOAc / MeOH (2 / 1, 10 mL / g) to give a white solid (3.8 g, 70%).

[0266] Spectroscopic data of 5α-androstane-3β,6α,17β-triol 2

[0267] 1 H NMR (DMSO-d6) δ 4.44 (m, 1H, OH), 4.42 (m, 1H, OH), 4.24 (d, 1H, OH), 3.42 (dt, 1H, 16-Ha), 3.26 (m, 1H, 3-H), 3.12 (m, 1H, 6-H), 0.72 (s, 3H, CH3), 0.60 (s, 3H, CH3).mp 232-234 ℃.

[0268] Production of 6α-hydroxyandrostane-3,17-dione [ka] Intermediate 3 was obtained from 2 by selective oxidation at C3 and C17. NBS (3.4 g, 19.5 mmol, 3 equiv.) was added to a stirred solution of 5α-androstane-3β,6α,17β-triol 2 (2 g, 6.5 mmol, 1 equiv.) in dioxane / HO / pyridine (54 / 10 / 1 mL) at 0 °C. After the addition, the mixture was warmed to room temperature and stirred overnight. The orange solution was diluted with water (50 mL) and quenched with NaSO (350 mg). The organic solvent was evaporated under vacuum until a white solid appeared. The solid was filtered and washed with water. After drying at 40 °C, 6α-hydroxyandrostane-3,17-dione 3 was obtained as a white solid (1.3 g, 70%).

[0269] Spectroscopic data of 6α-hydroxyandrostane-3,17-dione 3

[0270] 1 H NMR (acetone-d6) δ 3.61 (d, 1H, OH), 3.48 (m, 1H, 6-H), 1.11 (s, 3H, CH3), 0.86 (s, 3H, CH3). mp 204-206 ℃ lit. 206-207 (Hammerschmidt & Spiteller, 1973)

[0271] Synthesis of adrostan-3-methylene-17-one [ka] 6-α-3,17-androstanedione 3 was then converted to the exomethane derivative 6 (androstan-3-methylene-17-one) by selective Wittig reaction at the C3 carbonyl followed by cross-metathesis coupling with 5-pentenoic acid. t-BuOK (670 mg, 6 mmol, 4 equiv.) was added to a suspension of methyltriphenylphosphonium bromide (1.66 g, 6 mmol, 4 equiv.) in THF (10 mL) at -5 °C. The solution immediately turned bright orange. After 10 min, 6α-hydroxyandrostan-3,17-dione 3 (450 mg, 1.5 mmol, 1 equiv.) was added while maintaining the temperature below 0 °C. Immediately after the addition, the reaction was quenched by the addition of 1 M aqueous HCl (15 mL) and extracted with EtOAc (3 × 20 mL). The combined organic phases were dried over NaSO and evaporated to dryness. The crude extract was purified by column chromatography (eluent EtOAc:petroleum spirit 4:6) to give 376 mg (83%) of adrostan-3-methylene-17-one 6 as a white foam.

[0272] Spectroscopic data of adrostan-3-methylene-17-one 6

[0273] 1 H NMR (400 MHz, Chloroform-d) δ 4.63 (dt, 2H, 3α-CH2), 3.47 (td, 1H, 6-H), 2.55 (ddd, 1H, 16Ha), 2.44 (ddd, 1H, 16-Hb), 0.89 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.80 - 0.70 (m, 1H, 5-H).

[0274] 13 C NMR (101 MHz, Chloroform-d) δ 220.79 (17-C), 148.22 (3-C), 107.40 (3-CH2), 69.85 (6-C), 13.79 (CH3), 12.91 (CH3).

[0275] Direct synthesis of CVie 201 and 202 from precursor 6 via cross-metathesis [ka] Hoveyda-Grubbs second-generation catalyst (12 mg, 0.015 mmol, 0.05 equiv.) was added to a solution of androstane-3-methylene-17-one 6 (100 mg, 0.33 mmol, 1 equiv.) in DCM (1 mL). The solution was then heated to reflux and treated every 20 min with 10 μL of 4-pentenoic acid (330 μL total, 3.3 mmol, 10 equiv.). After the addition was complete, the mixture was refluxed for an additional 2 h. The reaction mixture was concentrated in vacuo and purified by flash chromatography (eluent acetone:petroleum spirit 3:7 + 0.1% HCOH) to give two distinct white solids, (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (4.8 mg, 4%) (CVie201) and (Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (7.2 mg, 6%) (CVie202).

[0276] Spectroscopic data from CVie201

[0277] 1 H NMR (Chloroform-d) δ 5.10 (t, J = 7.3 Hz, 1H, 3α-H), 3.50 (td, J = 10.3, 9.8, 6.0 Hz, 1H, 6-H), 2.91 (d, 1H, 16-Ha), 0.89 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.73 (m, 1H, 5-H).

[0278] 13 C NMR (101 MHz, Chloroform-d) δ 221.30 (17-C), 178.72 (CO2), 139.71 (3-C), 119.89(3α-C), 69.95(6-C), 54.03 (5-C), 24.03 (16-C), 13.93(CH3), 13.06(CH3).

[0279] MS (ESI) C 23 H 33 O4 - [M - ] Calculated value: 373.2. Detected value: 373.4

[0280] Spectroscopic data of CVie202

[0281] 1 H NMR (400 MHz, Chloroform-d) δ 5.10 (t, 1H, 3α -H), 3.50 (td, 1H, 6-H), 2.91 (d, 1H, 16-Ha), 0.89 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.73 (t, 1H, 5-H).

[0282] 13 C NMR (101 MHz, Chloroform-d) δ 220.86 (17-C), 177.66 (CO2), 139.84 (C-3), 119.47 (Ca), 70.08 (C-6), 13.78(CH3), 12.91 (CH3).

[0283] Alternatively, CVie201 and CVie202 were obtained by varying the Wittig reaction. In the first method (Route A), a polar solvent such as DMSO and a base such as NaH were used to stabilize the betaine intermediate. In the second approach (Route B), an aprotic solvent such as THF was used as a base to stabilize the cyclooxaphosphetane intermediate. Route A produced a mixture of diastereomers (60% Z / syn CVie202; 30% E / anti CVie201), while Route B provided CVie202 derived from the cyclooxaphosphate intermediate. Both methods require the generation of diastereomers 7 and / or 8, as described below.

[0284] Alternative synthesis of CVie201 and 202 via Wittig reaction Route A [ka] 60% NaH in mineral oil (100 mg, 2.56 mmol, 8 equiv.) was carefully added to dry DMSO (1 mL) under an Ar atmosphere. The resulting solution was stirred at 60 °C for 20 min. After cooling at room temperature, (3-carboxypropyl)triphenylphosphonium bromide (550 mg, 1.28 mmol, 4 equiv.) was added. A bright orange color immediately appeared. The solution was stirred for 2 h. 6α-hydroxyandrostane-3,17-dione 3 (100 mg, 0.32 mmol, 1 equiv.) was then added to the mixture. The resulting solution was stirred at room temperature for an additional 4 h. The reaction mixture was diluted with EtOAc (25 mL) and washed with 1 M aqueous HCl (3 × 30 mL). The organic layer, dried over NaSO, was evaporated to dryness to give 25 mg of crude material.

[0285] The crude material was first dissolved in MeOH (1.5 mL), followed by the addition of EDC hydrochloride (115 mg, 0.6 mmol, 2 equiv.) and DMAP (5 mg, 0.03 mmol, 0.1 equiv.). The solution was stirred at room temperature for 3 h and concentrated in vacuo. The crude solid was dissolved in EtOAc (15 mL) and washed with 1 M aqueous HCl (3 × 10 mL). The crude product was purified by flash chromatography on silica gel (acetone:petroleum spirit 3:7) to afford 25 mg of a clear oil (20%) containing a mixture of diastereoisomers 7 and 8.

[0286] Spectroscopic data for the two diastereoisomers 7 and 8

[0287] 1 H NMR (Chloroform-d) δ 5.16-4.96 (m, 1H, 3α-H), 3.66 (s, 3H, CH3O), 3.47 (m, 1H, 6-OH), 0.89 (s, 3H,CH3), 0.86 (s, 3H, CH3), 0.74 (m, 1H, 5-H).

[0288] Route B [ka] A 1 M solution of LiHMDS in THF (40 mL, 40 mmol, 12 equiv.) was carefully added to a suspension of (3-carboxypropyl)triphenylphosphonium bromide (8.5 g, 20 mmol, 6 equiv.) in dry THF (33 mL) at −40° C. under an Ar atmosphere. The solution was stirred at −40° C. until a bright orange color appeared. 6α-Hydroxyandrostane-3,17-dione 3 (1 g, 3.3 mmol, 1 equiv.) was then added to the solution at −40° C. After stirring overnight at room temperature, the reaction mixture was quenched with 1 M aqueous HCl (300 mL) and extracted with EtOAc (3 × 350 mL). The combined organic layers were dried over NaSO and evaporated to dryness.

[0289] The crude material was dissolved in absolute EtOH (17 mL), and then EDC hydrochloride (1.26 mg, 6.6 mmol, 2 equiv.) and DMAP (50 mg, 0.3 mmol, 0.1 equiv.) were added. The mixture was stirred at room temperature for 3 h. The reaction was diluted with EtOAc (150 mL) and washed with 1 M aqueous HCl (3 × 100 mL). The crude product was purified by flash chromatography on silica gel (acetone:petroleum spirit 3:7) to give 910 mg (72%) of compound 8.

[0290] Spectroscopic data of compound 8

[0291] 1 H NMR (Chloroform-d) δ 5.07 (t, 1H, 3α-H), 4.10 (q, 2H, OCH2), 3.47 (td, 1H, 6-H), 2.91 (d, 1H), 0.88 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.71 (m, 1H, 5-H).

[0292] 13C NMR (101 MHz, Chloroform-d) δ 173.67 (17-C), 139.60 (3α-C), 119.94(3-C), 69.98 (6-C), 60.41 (OCH2), 51.33 (5-C), 40.37 (CH3), 13.92 (CH3), 13.08 (CH3).

[0293] [ka] Final hydrolysis of the methyl (or ethyl) ester A 1 M aqueous solution of LiOH (150 μL, 2.5 equiv.) was added to a solution of the methyl esters 7 and 8 (25 mg, 0.06 mmol, 1 equiv.) in THF (600 μL) and water (200 μL). After 2 h, the reaction was diluted with water (10 mL) and quenched by adding 1 M HCl until the solution reached pH 1. The aqueous phase was extracted with EtOAc (3 × 15 mL). The combined organic layers were dried over NaSO and evaporated to dryness. The crude product was purified by flash chromatography (AcOEt:petroleum spirit 7:3, 1% HCOOH). Two white solids were obtained, corresponding to the E (7 mg, 31%) and Z (12 mg, 54%) diastereoisomers (CVie201 and CVie202, respectively).

[0294] 1 H NMR (Chloroform-d) δ 5.12 (bt, 1H, 3α-H), 3.51-3.42 (m, 1H, 6-H), 0.90 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.79-0.69 (m, 1H, 5-H).

[0295] [ka] CVie Synthetic method of 203 and 204: Synthesis of intermediate compound 12 To prepare CVie203 and CVie204, precursor 12 was first generated from 6-α-3,17-androstanedione 3. The carbonyl of 6-α-3,17-androstanedione 3 was protected as a diketal by reaction with ethylene glycol in toluene under an acid catalyst (p-tSA or camphorsulfonic acid), yielding compound 9. Compound 9 was oxidized with PCC or other oxidizing agents to give compound 10, which was reduced with NaBH or KBH to generate protected alcohol 11, which selectively possessed the C6-hydroxyl group in the β-configuration. Final cleavage of the cyclic diketal by acidic treatment in acetone, as described by De Munari et al. (J. Med. Chem., 2003, 46(17):3644-54), gave precursor 12.

[0296] Briefly, a solution of 6α-hydroxyandrostane-3,17-dione (1.5 g, 4.9 mmol, 1 equiv.), ethylene glycol (10.5 mL, 88 mmol, 36 equiv.), and PTSA (561 mg, 2.9 mmol, 0.6 equiv.) in toluene (160 mL) was stirred at reflux for 12 h with a Dean-Stark trap. After cooling to room temperature, the mixture was neutralized with 5% aqueous NaHCO3. The organic layer was separated, washed with HO (2 × 40 mL), dried over Na2SO4, and evaporated to dryness to yield 3,3:17,17-bis(ethylenedioxy)androstan-6α-ol 9 as a white solid (1.9 g, 98%).

[0297] Spectroscopic data for 3,3:17,17-bis(ethylenedioxy)androstan-6α-ol 9

[0298] 1 H NMR (DMSO-d6) δ4.25 (d, 1H, OH), 3.88-3.70 (m, 8H, OCH2), 3.11 (m, 1H, 6-H), 0.74 (s, 3H, CH3), 0.73 (s, 3H, CH3).

[0299] PCC (148 mg, 0.69 mmol, 4 equiv.) was added to a solution of 3,3:17,17-bis(ethylenedioxy)androstan-6α-ol (3 g, 14 mmol, 1 equiv.) 9 and sodium ascorbate (1.2 g, 14 mmol, 4 equiv.) in dry CHCl (87 mL) at 0 °C. The mixture was stirred overnight at room temperature. The mixture was washed with 1 M aqueous HCl (3 × 30 mL) and water (3 × 30 mL). The organic layer was dried over NaSO and evaporated to dryness. The crude product was purified by flash chromatography on a silica gel column (eluent acetone:petroleum spirit 2:8). 3,3:17,17-bis(ethylenedioxy)androstan-6-one 10 was obtained as a white solid (1.53 g, 96%).

[0300] Spectroscopic data for 3,3:17,17-bis(ethylenedioxy)androstan-6-one 10

[0301] 1 H NMR (Acetone-d6) δ 3.97-3.76 (m, 8H, CH2O), 2.19 (dd, 1H, 16-Ha), 0.84 (s, 3H, CH3), 0.75 (s, 3H, CH3).

[0302] NaBH (144 mg, 3 mmol, 1.2 equiv) was added to a stirred suspension of 3,3:17,17-bis(ethylenedioxy)androstan-6-one 10 (1 g, 2.5 mmol, 1 equiv) in MeOH (13 mL) at 0 °C. After 2 h at 0 °C, HO (40 mL) was added dropwise. The mixture was extracted with EtOAc (3 × 40 mL). The combined organic extracts were dried over NaSO, filtered, and evaporated to dryness to give 3,3:17,17-bis(ethylenedioxy)androstan-6β-ol 11 (915 mg, 92%) as a white solid.

[0303] Spectroscopic data for 3,3:17,17-bis(ethylenedioxy)androstan-6β-ol 11

[0304] 1H NMR (acetone-d6) δ 3.95-3.75 (m, 8H, OCH2), 3.70 (m, 1H, 6-H), 3.33 (d, 1H, 6-OH), 1.05 (s, 3H, CH3), 0.84 (s, 3H, CH3).

[0305] PTSA (2.26 g, 11.5 mmol, 5 equiv.) was added portionwise over 5 min to a solution of 3,3:17,17-bis(ethylenedioxy)androstan-6β-ol 11 (910 mg, 2.3 mmol, 1 equiv.) in acetone (46 mL). After stirring at room temperature for 1 h, the solution was quenched by adding 5% aqueous NaHCO until pH 7. After stirring for 5 min, a white solid appeared. The volatiles were removed in vacuo. The suspension was extracted with CHCl (3 × 30 mL), and the combined organic extracts were washed with brine (40 mL), dried over NaSO, filtered, and evaporated. The resulting solid was stirred with n-hexane / EtOAc 8 / 2 (10 mL) for 45 min and then collected by filtration. The solid was dried at 45 °C for 3 h. 568 mg (81%) of a white solid (i.e., 6β-hydroxyandrostane-3,17-dione 12) was obtained.

[0306] Spectroscopic data for 6β-hydroxyandrostane-3,17-dione 12

[0307] 1 H NMR (DMSO-d6) δ 4.47 (d, 1H, OH), 3.57 (m, 1H, 6-H), 1.13 (s, 3H, CH3), 0.81 (s, 3H, CH3).

[0308] 12 Final Conversions to CVie 203 and 204 [ka] CVie203 and CVie204 were obtained from precursor 12 via Wittig reaction using the same methodology as described above for CVie201 and CVie202. The configuration of the C3-C1′ double bond was identified in the two isomers by NOESY experiments.

[0309] Briefly, 60% NaH in mineral oil (100 mg, 2.56 mmol, 8 equiv.) was carefully added to dry DMSO (1 mL) under an Ar atmosphere. The resulting solution was stirred at 60 °C for 20 min. After cooling to room temperature, (3-carboxypropyl)triphenylphosphonium bromide (550 mg, 1.28 mmol, 4 equiv.) was added. A bright orange color immediately appeared. The solution was stirred for 2 h. Then, 6β-hydroxyandrostane-3,17-dione 12 (100 mg, 0.32 mmol, 1 equiv.) was added to the mixture. The resulting solution was stirred at room temperature for an additional 4 h. The reaction mixture was diluted with EtOAc (25 mL) and washed with 1 M aqueous HCl (3 × 30 mL). The organic layer was dried over NaSO and evaporated to dryness to give 25 mg of crude material.

[0310] The crude material was then dissolved in MeOH (1.5 mL). EDC hydrochloride (115 mg, 0.6 mmol, 2 equiv.) and DMAP (5 mg, 0.03 mmol, 0.1 equiv.) were added. The solution was stirred at room temperature for 3 h. It was concentrated in vacuo. The crude solid was dissolved in EtOAc (15 mL) and washed with 1 M aqueous HCl (3 × 10 mL). The crude product was purified by flash chromatography on silica gel (acetone:petroleum spirit 3:7) to give a mixture of diastereoisomers 13 and 14 in 17% and 30% yields, respectively.

[0311] Spectroscopic data of compound 13

[0312] 11H NMR (400 MHz, Chloroform-d) δ 5.07 (bs, 1H, 3α-H), 3.85 (d, 1H, 6-H), 3.66 (s, 3H, OCH3), 2.54 - 2.39 (m, 2H, 3γ-H), 1.10 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.80 - 0.69 (m, 1H, 5-H).

[0313] 13 13C NMR (101 MHz, Chloroform-d) δ 219.74 (17-C), 167.24 (CO2), 140.38 (3-C), 119.60 (3α-C), 71.52 (6-C), 54.45 (5-C), 51.22 (OCH3), 14.09 (CH3), 13.86 (CH3).

[0314] Spectral data of Compound 14

[0315] 1H NMR (400 MHz, Chloroform-d) δ 5.07 (s, 1H, 3α-H), 3.89 (d, 1H, 6-H), 3.66 (s, 3H, OCH3), 2.46 (dd, 1H, 16-Ha), 1.10 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.79 - 0.64 (m, 1H, 5-H).

[0316] 13C NMR (101 MHz, Chloroform-d) δ 221.21 (17-C), 176.10 (CO2), 140.33 (3-C), 119.39 (3α-C), 71.75 (6-C), 54.49 (5-C), 51.20 (OCH3) 15.24 (CH3), 13.87 (CH3).

[0317] The reaction mixture was concentrated in vacuo and purified by flash chromatography (eluent acetone:petroleum spirit 3:7 + 0.1% HCOH) to give two distinct white solids, (E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie203) and (Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid (CVie204).

[0318] Spectroscopic data for compound CVie203

[0319] 1 H NMR (400 MHz, Acetone-d6) δ 4.84 (bt, 1H, 3α-H), 3.55 (d, 1H, 6-H), 0.90 (s, 3H, CH3), 0.61 (s, 3H, CH3), 0.51 (ddd, 1H, 5-H).

[0320] 13 C NMR (101 MHz, acetone-d6) δ 218.86 (17-C), 173.42(CO2), 140.74 (3-C), 119.29(3α-C), 70.18 (6-C), 14.74 (CH3), 13.18 (CH3).

[0321] Spectroscopic data for compound CVie204

[0322] 1 H NMR (400 MHz, Chloroform-d) δ 5.07 (s, 1H, 3α-H), 3.88 (s, 1H, 6-H), 1.08 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.72 (s, 1H, 5-H).

[0323] 13 C NMR (101 MHz, acetone) δ 216.39 (17-C), 173.80 (CO2), 135.48(3-C), 113.80 (3α-C), 66.52 (6-C), 10.07 (CH3), 8.70 (CH3).

[0324] Hydrogenation and ester hydrolysis to produce CVie214, CVie215, and CVie217 [ka] Compound CVie217 was prepared from the mixture of diastereomers 7 and 8 described above. Briefly, hydrogenation of the C3-C1' double bond of the diastereomers was carried out using a Pd-C catalyst in EtOAc. The resulting compound was CVie217. The configuration of the stereocenter formed at C3 was identified by NOESY experiments. Compound CVie217 was then hydrolyzed with 1 M LiOH or NaOH in THF to produce CVie214. Similarly, diastereomers 13 and 14 were hydrogenated with a Pd-C catalyst in EtOAc to produce the ester compound 19, which was then hydrolyzed with 1 M LiOH or NaOH in THF to produce CVie215.

[0325] Spectroscopic data of compound 19

[0326] 1 H NMR (400 MHz, Chloroform-d) δ 3.83 (bs, 1H, 6-H), 3.66 (bs, 3H, OCH3), 2.44 (dd, 1H, 16-Ha), 2.28 (t, 2H, 3γ-H), 0.99 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.79 - 0.70 (m, 1H, 5-H).

[0327] 13 C NMR (101 MHz, Chloroform-d) δ 221.43 (17-C), 174.25 (CO2), 71.96 (6-C), 51.25 (OCH3), 15.74 (CH3), 13.86(CH3).

[0328] Spectroscopic data for compound CVie214

[0329] 1 H NMR (400 MHz, Chloroform-d) δ 3.43 (bt, 1H, 6-H), 2.44 (dd, 1H, 16Ha), 2.31 (t, 2H, 3γ-H), 0.84 (s, 3H, CH3), 0.77 (s, 3H, CH3).

[0330] 13 C NMR (101 MHz, Chloroform-d) δ 221.35(17-C), 179.11(CO2), 69.90(6-C), 13.78(CH3), 13.37(CH3) .

[0331] Internal CVie215 core

[0332] 1 H NMR (400 MHz, Chloroform-d) δ 3.85 (s, 1H, 6-H), 2.45 (dd, 1H, 16-Ha), 2.34 (t, 2H, 3γ-H), 1.00 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.74 (d, 1H, 5-H).

[0333] 13 C NMR (101 MHz, Chloroform-d) δ 221.50(17-C), 179.04(CO2H), 72.03(6-C), 15.76(CH3), 13.87(CH3).

[0334] The default CVie217 interface

[0335] 1 H NMR (400 MHz, Chloroform-d) δ 4.09 (q, 2H, CH2O), 3.40 (td, 1H, 6-H), 2.49 - 2.36 (dd, 1H, 16-Ha), 2.24 (t, 2H, 3δ-H), 0.83 (s, 3H, CH3), 0.76 (s, 3H, CH3).

[0336] 13 C NMR (101 MHz, Chloroform-d) δ 220.91 (17-C), 173.77 (CO2), 69.63 (6-C), 60.14 (CH2O), 14.23 (CH3), 13.76 (CH3), 13.36 (CH3).

[0337] Wittig reaction followed by C=C hydrogenation and ester hydrolysis to prepare CVie213 and CVie216 [ka] Compound 6-α-3,17-androstanedione 3 was also used as the starting point for the synthesis of CVie213 and CVie216 ​​via the Horner-Emmons reaction. First, triethylphosphonoacetate (6.5 mL, 33 mmol, 5 equiv.) was carefully added to a suspension of 60% NaH in mineral oil (1.3 g, 33 mmol, 5 equiv.) in DMF (200 mL) at 0 °C under an Ar atmosphere. The resulting solution was warmed to room temperature and stirred for 20 min. Next, 6-α-3,17-androstanedione 3 (2 g, 6.5 mmol, 1 equiv.) was added at 0 °C. After stirring overnight at room temperature, the reaction was quenched by careful addition of HO (100 mL) and extracted with EtO (3 × 150 mL). The combined organic layers were dried over NaSO and evaporated in vacuo. The crude material was purified by flash chromatography on a silica gel column (acetone:petroleum spirit 3:7) to yield 2.1 g (86%) of a mixture of two diastereoisomers (compound 21) as a clear oil.

[0338] Spectroscopic data for diastereoisomeric compound 21

[0339] 1H NMR (Chloroform-d) δ 5.60 (d,Hz, 1H, 3α-H), 4.08 (q, 2H, CH2O), 3.45 (dq,1H, 6-H), 2.41 (dd, 1H, 16-Ha), 0.90 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.77-0.66 (m, 1H, 5-H).

[0340] 13 C NMR (101 MHz, Chloroform-d) δ 220.85 (17-C), 166.81 (CO2), 161.87 (3-C), 113.75 (3α-C), 69.41 (6-C), 13.76 (CH3), 13.00 (CH3).

[0341] Under an Ar atmosphere, 10% Pd-C (700 mg) was added to a solution of diastereoisomeric compound 21 (2 g, 5.3 mmol, 1 equiv.) in degassed EtOAc (200 mL). After three vacuum / hydrogen cycles, the reaction was stirred overnight at room temperature under an H atmosphere. After removing the hydrogen by vacuum / Ar cycles, the reaction mixture was filtered through CELITE®. The filtered solution was evaporated to dryness. CVie213 product was obtained without purification in an amount of 1.8 g (90%). CVie216 ​​was further produced by hydrolysis of CVie213 with 1 M LiOH or NaOH in THF.

[0342] Spectroscopic data for compound CVie213

[0343] 1 H NMR (Chloroform-d) δ 4.12 (q, 2H, OCH2), 3.38 (td, 1H, 6-H), 2.42 (dd, 1H, 16-Ha), 2.27 (t, 2H, 3α-CH2), 0.82 (s, 3H, CH3), 0.76 (s, 3H, CH3).

[0344] 13C NMR (101 MHz, Chloroform-d) δ 221.03 (17-C), 172.93 (CO2), 69.43 (6-C), 13.76 (CH3), 13.32(CH3).

[0345] Spectroscopic data for compound CVie216

[0346] 1 H NMR (400 MHz, Chloroform-d) δ 3.43 (td, 1H, 6-H), 2.45 (dd, 1H, 16-Ha), 2.28 (t, 3α-H), 0.85 (s, 3H, CH3), 0.80 (s, 3H, CH3).

[0347] 13 C NMR (101 MHz, acetone) δ 220.44 (17-C), 174.27 (CO2), 68.57 (6-C), 12.99 (CH3), 12.59 (CH3).

[0348] Wittig reaction followed by C=C hydrogenation and ester hydrolysis to prepare CVie218 and CVie219 [ka] Similarly, 6-α-3,17-androstanedione 3 was reacted with an appropriate triphenylphosphonium salt (e.g., 5-carboxytriphenylphosphonium bromide, LiHMDS, THF, followed by EtOH (or MeOH)) to generate compound 24. Using a Pd-C catalyst in the presence of hydrogen, CVie218, which contained a C6 chain at the C-3 position, was generated. Hydrolysis of CVie218 with 1 M LiOH or NaOH in THF generated CVie219.

[0349] Spectroscopic data for compound CVie218

[0350] 1H NMR (400 MHz, Chloroform-d) δ 4.15 - 4.05 (m, 2H, OCH2), 3.40 (td, 1H, 6-H), 2.43 (dd, 1H, 16-Ha), 2.26 (td, 2H, 3ε-H), 0.84 (s, 3H, CH3), 0.76 (s, 3H, CH3).

[0351] 13 C NMR (101 MHz, Chloroform-d) δ 220.91 (17-C), 173.87 (CO2), 69.81 (6-C), 60.14 (CH2O), 14.24 (CH3), 13.79 (CH3), 13.39 (CH3).

[0352] Spectroscopic data for compound CVie219

[0353] 1 H NMR (400 MHz, Chloroform-d) δ 3.47 - 3.39 (bt, 1H, 6-H), 2.44 (dd, 1H, 17-Ha), 2.33 (t, 2H, 3ε-H), 0.85 (s, 3H, CH3), 0.77 (s, 3H, CH3).

[0354] 13 C NMR (101 MHz, Chloroform-d) δ 221.06 (17-C), 178.93 (CO2), 69.91 (6-C), 13.80 (CH3), 13.39 (CH3).

[0355] Synthesis of derivatives bearing primary amine groups from precursor 6 by metathesis reaction with Boc-protected amines followed by Boc deprotection [ka] For the synthesis of derivatives bearing a primary amine group as the X substituent of formula (I), a cross-metathesis reaction was carried out on precursor 6 using the same experimental conditions as described above for the synthesis of CVie201 and CVie202.

[0356] Briefly, Hoveyda-Grubbs second-generation catalyst was added to a solution of androstan-3-methylene-17-one 6 in DCM. Androstan-3-methylene-17-one 6 was then mixed with an appropriate Boc-protected amine (e.g., tert-butylpent-4-en-1-yl-carbamate or N-Boc-4-pentyn-1-amine) containing the exo-methylene group to generate diastereoisomer 25 (25% yield). Compound 25 (50 mg, 0.1 mmol, 1 equiv.) was treated with 500 μL of a 1:1 mixture of TFA / DCM trifluoroacetic acid in DCM and stirred at room temperature to directly cleave the Boc group. After stirring at room temperature for 1 min, the reaction was diluted with EtOAc (50 mL) and washed with saturated aqueous NaHCO (3 × 30 mL). The organic phase was dried over Na2SO4, filtered, and evaporated to dryness to yield (EZ)-3-(4-aminobutylidene]-6α-hydroxyandrostan-17-one (CVie209) as a white solid (28 mg, 75%).

[0357] Spectroscopic data for diastereoisomeric mixture 25

[0358] 1H NMR (400 MHz, Chloroform-d) δ 5.18 - 5.03 (m, 1H, 3α -H), 3.46 (td, 1H, 6-H), 1.44 (s, 9H, t-Bu), 0.90 (d, J = 1.8 Hz, 3H, CH3), 0.86 (s, 3H, CH3), 0.74 (m, 1H, 5-H).

[0359] Spectroscopic data of CVie209

[0360] 1H NMR (400 MHz, CD3OD) δ 5.13 (d, 1H, 3α-H), 3.40 (tt, 1H, 6-H), 3.35 - 3.28 (m, 2H, 3γ-H), 0.96 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.83 - 0.70 (m, 1H, 5-H).

[0361] 13 C NMR (101 MHz, CD3OD) δ 224.41 (17-C), 143.00 (3A-C), 142.68 (3αB-C), 124.31 (3αA-C), 124.02 (3αB-C), 72.75 (C-6), 16.70 (CH3), 15.78 (CH3).

[0362] Alternatively, compound 25 was reacted with iodotrimethylsilane in an alcohol solvent (e.g., MeOH) to undergo Boc cleavage accompanied by migration of the exocyclic double bond, yielding CVie205, which possesses an endocyclic double bond between C2 and C3. Briefly, 1 M TMSI in DCM (100 μL, 0.1 mmol, 1 equiv.) was added to a solution of diastereoisomer 25 (50 mg, 0.1 mmol, 1 equiv.) at room temperature. After stirring at the same temperature for 2 h, the solvent was removed in vacuo. Methanol (2 mL) was added to the residue and allowed to stand at room temperature for 1 h. After removing the solvent in vacuo, CVie207 was obtained without further purification.

[0363] Spectroscopic data of CVie207

[0364] 1 H NMR (400 MHz, CD3OD) δ 5.36 (d, 1H, H-2), 3.44 (td, 1H, 6-H), 2.94 (t, 2H, 3γ-H), 2.45 (dd, 1H, 16-Ha), 0.88 (s, 3H, CH3), 0.79 (s, 3H, CH3).

[0365] 13C NMR (101 MHz, CD3OD) δ 222.35 (17-C), 134.89 (3-C), 119.69 (2-C), 70.31 (6-H), 12.75 (CH3), 12.07 (CH3).

[0366] Synthesis of cyclic amine derivatives with exocyclic unsaturation: CVie205, CVie206, CVie210, and CVie211 [ka] Cyclic amine derivatives were synthesized by the sodium hydride (NaH)-DMSO Wittig reaction as described above for CVie203 and CVie204 using the appropriate N-protected phosphonium salt, e.g., N-Boc-4-(2-triphenylphosphoniumethyl)azetidine iodide to give compounds 26 and 27, or N-Boc-3-(2-triphenylphosphoniumethyl)piperidine iodide to give compounds 28 and 29. After purification of the diastereoisomeric mixture, the N-Boc group was cleaved by acidic hydrolysis with TFA to give CVie205, CVie206, CVie210, and CVie211.

[0367] Synthesis of CVie208 with endocyclic unsaturation (C=C double bond migration during Boc deprotection) [ka] Additionally, compounds 28 and 29 were treated with TMSI to generate CVie208, as described above for the synthesis of CVie207.

[0368] Hydrogenation with TFA and Boc cleavage to generate CVie212 [ka] Alternatively, compound 30 was synthesized by catalytic hydrogenation (H 2 , Pd—C, EtOAc) of the double bond of compounds 28 and 29, followed by Boc cleavage with TFA in DCM to generate CVie212.

[0369] The synthesis of compounds containing 6α-hydroxymethylandrostane-7,17-dione was achieved starting from the general intermediate 37. Compound 37 itself was synthesized from 4-androstene-3,17-dione 31 by protection of the two ketone moieties with a cyclic acetal followed by simultaneous double bond migration (32), oxidation of the allylic position with sodium dichromate (33), formation of a silyl enol ether (35), hydroxymethylation with MeAl and formaldehyde (36), and final cleavage of the acetal under acidic conditions. The synthesis is described in detail in the following paragraphs.

[0370] Synthesis of Compound 32: (20S,7R)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolane]-12-ene [ka] A mixture of androst-4-ene-3,17-dione 31 (400.0 g, 1.4 mol) and PTSA·HO (13.3 g, 70.0 mmol) in ethylene glycol (8.0 L) was stirred at 100 °C until the reaction was complete. Approximately 5.0 L of glycol was distilled under vacuum to a boiling point of approximately 80–85 °C. The mixture was cooled to room temperature. The pH of the mixture was adjusted to ∼9. The mixture was then poured into ice water. The mixture was filtered, and the solid was washed with water, collected, and treated with acetone to give crude compound 32 (469.0 g, 89%) as a yellow solid.

[0371] Synthesis of Compound 33: (20S,7R)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolane]-12-en-14-one [ka] A mixture of compound 32 (440.0 g, 1.2 mol), HOSU (541.2 g, 4.7 mol), and NaCrO·HO (527.5 g, 1.8 mol) in acetone (8.0 L) was vigorously stirred at 50 °C for 2 days. After cooling to room temperature, the mixture was quenched with aqueous NaSO and stirred for 20 min. The mixture was poured into ice water. The resulting mixture was stirred for 20 min and then filtered. The solid filtrate was washed with water, collected, and dried in vacuo to give crude compound 33 (390.0 g, 85%) as a yellow solid.

[0372] Synthesis of Compound 34: (7S,20S)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolan]-14-one [ka] A mixture of compound 33 (50.0 g, 128.9 mmol) in EtOAc (1250 mL) was added to Pd / C (16.0 g). The mixture was then stirred under H at room temperature overnight. TLC showed that the reaction was complete. The mixture was filtered, concentrated, and purified by flash chromatography (PE / EA=2 / 1) to give compound 34 (25.0 g, 50.0%) as a white solid.

[0373] Spectroscopic data for (7S,20S)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolane]-14-one 34

[0374] 1 H NMR (400 MHz, DMSO-d6): δ 3.85-3.75 (m, 8H), 2.44-2.35 (m, 2H), 2.08-2.03 (m, 1H), 1.87-1.79 (m, 2H), 1.70-1.49 (m, 8H), 1.41-1.28 (m, 4H), 1.17-1.10 (m, 2H), 1.03 (s, 3H), 1.00-0.97 (m, 1H), 0.76 (s, 3H).

[0375] Synthesis of Compound 35: 1-((20S,7R)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecane-14',2''-1,3-dioxolane]-13-en-14-yloxy)-1,1-dimethyl-1-silaethane [ka] A mixture of compound 34 (20.0 g, 51.3 mmol) in dry THF (100.0 mL) was stirred at −78°C, followed by the dropwise addition of 1.5 M LDA (205.2 mL, 307.8 mmol) in toluene. After stirring at the same temperature for 1 h, Me3SiCl (50.0 mL, 400.1 mmol) was added dropwise. After stirring at −70°C for 3 h, the temperature was raised to −30°C, and triethylamine (33.5 g, 331.5 mmol) was added. After stirring at the same temperature for 1 h, the mixture was warmed to room temperature, and water (200.0 mL) and EtOAc (100.0 mL) were added. The separated aqueous phase was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by flash chromatography (PE / EA=2 / 1) to give compound 35 (14.3 g, 60.3%) as a white solid.

[0376] Synthesis of Compound 36: (13S,20S,7R)-13-(hydroxymethyl)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolan]-14-one [ka] A mixture of 2,6-diphenylphenol (10.0 g, 27.6 mmol) in dry DCM (450.0 mL) was added dropwise to a solution of MeAl in toluene (41.4 mL, 82.9 mmol) while cooling in an ice / water bath so that the temperature did not exceed room temperature. After stirring at room temperature for 1 hour, the solution was cooled to 0 °C, and a solution of trioxane (24.8 g, 276.0 mmol) in dry DCM (100.0 mL) was added dropwise. The pale yellow solution was stirred at 0 °C for an additional 1 hour, and then the temperature was cooled to -78 °C. A solution of compound 35 (10.0 g, 27.6 mmol) in dry DCM (125 mL) was added. After stirring at -78 °C for 1 hour, the temperature was raised to -20 °C, and the reaction mixture was stirred at that temperature overnight. 5% aqueous NaHCO (85.0 mL) was added at room temperature. The jelly mixture was filtered through a CELITE® pad and washed thoroughly with DCM. The separated organic layer was washed with water and evaporated. Approximately 1 M TBAF in THF (24.0 mL) was added to the residue, and the solution was stirred at room temperature for 1.5 hours. The solution was washed with water, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by flash chromatography to give compound 36 (6.5 g, 71.4%) as a yellow solid.

[0377] Synthesis of Compound 37: (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyldecahydro-1H-cyclopenta[a]phenanthrene-3,7,17(2H,4H,8H)-trione [ka] A mixture of compound 36 (8.0 g, 19.0 mmol) in acetone (100.0 mL) was added to 10% aqueous HCl (50.0 mL). The mixture was then heated to 70° C. for 1 hour. TLC showed the reaction was complete. The mixture was quenched with 5% aqueous NaOH and extracted with DCM (50.0 mL × 2). The combined organic phases were washed with brine (50.0 mL), dried over NaSO, filtered, concentrated, and purified by flash chromatography (DCM / EA = 4 / 1) to give the crude product, which was treated with ether to give the pure product 37 (3.3 g, 52.4%) as a white solid.

[0378] Spectroscopic data for (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyldecahydro-1H-cyclopenta[a]phenanthrene-3,7,17(2H,4H,8H)-trione 37

[0379] 1 H NMR (400 MHz, DMSO-d6): δ 4.14 (t, 1H), 3.63-3.59 (m, 1H), 3.50-3.47 (m, 1H), 2.74-2.69 (m, 1H), 2.42-2.27 (m, 5H), 2.15-1.93 (m, 3H), 1.87-1.79 (m, 1H), 1.68-1.59 (m, 3H), 1.54-1.44 (m, 2H), 1.32-1.06 (m, 7H), 0.81 (s, 3H).

[0380] LCMS [Mobile phase: 55% water (0.05% FA) and 45% CHCN (0.05% FA) in 6.0 min to 55% water (0.05% FA) and 45% CHCN (0.05% FA) in 0.5 min, finally at these conditions for 0.5 min], purity >90%, Rt = 2.514 min; MS calculated: 332.2; MS found: 333.2 [M+1] + .

[0381] Synthesis of Compound 38: (13S,14S,20S,7R)-13-(hydroxymethyl)-7,20-dimethyldispiro[1,3-dioxolane-2,5'-tetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecan-14',2''-1,3-dioxolan]-14-ol [ka] NaBH4 (4.0 g, 104.8 mmol) was slowly added to a mixture of compound 36 (22.0 g, 52.4 mmol) in MeOH (1000 mL) at 0 °C. The mixture was then stirred at room temperature for 1 hour. TLC showed the reaction was complete. The mixture was quenched with 5% aqueous NaH2PO4 (220 mL) and extracted with DCM (300 mL × 3). The combined organic phases were washed with brine (200 mL), dried over Na2SO4, filtered, concentrated, and purified by flash chromatography (DCM / EA = 4 / 1) to give the crude product, which was treated with ether to give compound 38 (7.5 g, 34.1%) as a white solid.

[0382] Synthesis of Compound 39: (8S,9S,15S,2R)-9-hydroxy-8-(hydroxymethyl)-2,15-dimethyltetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecane-5,14-dione [ka] To a mixture of compound 38 (5.7 g, 13.5 mmol) in acetone (70 mL) was added 10% aqueous HCl (35 mL). The mixture was then heated to 70 °C for 1 h. TLC showed the reaction was complete. The mixture was quenched with 5% aqueous NaOH and extracted with DCM (50 mL × 2). The combined organic phases were washed with brine (50 mL), dried over NaSO, filtered, and concentrated. The residue was purified by flash chromatography (DCM / EA = 4 / 1) to give the crude product, which was treated with ether to give the pure product 39 (1.8 g, 40.0%) as a white solid.

[0383] Spectroscopic data for (8S,9S,15S,2R)-9-hydroxy-8-(hydroxymethyl)-2,15-dimethyltetracyclo[8.7.0.0<2,7>.0<11,15>]heptadecane-5,14-dione 39

[0384] 1 H NMR (400 MHz, DMSO-d6): δ4.37 (brs, 1H), 4.27 (d, J = 4.8 Hz, 1H), 3.87-3.86 (m, 1H), 3.44-3.42 (m, 2H), 2.45-1.87 (m, 10H), 1.63-1.23 (m, 9H), 0.99 (s, 3H), 0.81 (s, 3H).

[0385] LCMS [Mobile phase: 55% water (0.05% FA) and 45% CHCN (0.05% FA) in 6.0 min to 55% water (0.05% FA) and 45% CHCN (0.05% FA), and finally these conditions for 0.5 min], purity >90%, Rt = 2.515 min; MS calculated: 334.2; MS found: 352.2 [M+18]. + .

[0386] Synthesis of Compound 40: tert-butyl-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)azetidine-1-carboxylate [ka] To a solution of the phosphonium salt (2.57 g, 4.5 mmol) in THF (25 mL) was added a solution of n-BuLi in THF (2.5 M, 3.6 mL, 9.0 mmol) at −78° C. The mixture was stirred at 30° C. for 1 hour. Compound 37 (500 mg, 1.5 mmol) was then added to the mixture at −20° C., followed by warming to 30° C. for 2 hours. The mixture was quenched with saturated NH4Cl (25 mL) and extracted with EtOAc (25 mL × 3). The combined organic layers were concentrated, and the residue was purified by silica gel column chromatography (hexane / EtOAc = 1 / 1) to give the crude compound. The compound was purified by reverse-phase column chromatography to give pure compound 40 (60 mg, 8%) as a white solid.

[0387] Data for compound 40:

[0388] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikwa Diamonsil plus; mobile phase: B(ACN):A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 1.820 min; MS calculated: 499, MS found: 400 [M+H-Boc] + .

[0389] Synthesis of Compound 41: tert-butyl-3-(2-((3S,6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)azetidine-1-carboxylate [ka] To a solution of compound 40 (60 mg, 0.12 mmol) in EA (3 mL) was added Pd / C (60 mg). The mixture was then stirred under H at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give compound 41 (52 mg, 86%) as a white solid.

[0390] LCMS column: C18; Column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; Mobile phase: B(ACN):A (0.02%NH4Ac+5%ACN); Gradient (B%): 3 min-5-95-POS; Flow rate: 1.5 mL / min, Stop time: 3 min. Rt = 1.911 min; MS calculated: 501, MS found: 402 [M+H-Boc] + .

[0391] CVie407: Synthesis of (3S,6S,10R,13S)-3-(2-(azetidin-3-yl)ethyl)-6-(hydroxymethyl)-10,13-dimethyldodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 41 (52 mg, 0.10 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 hour. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL x 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound Cvie407 (13 mg, 32%) as a white solid.

[0392] Spectroscopic data of CVie407

[0393] 1 H NMR (CD3OD, 400 MHz):δ 3.86-3.82 (m, 2H), 3.71-3.67 (m, 1H), 3.54-3.47 (m, 2H), 2.78-2.67 (m, 2H), 2.56-2.39 (m, 3H), 2.15-2.06 (m, 1H), 1.85-1.50 (m, 12H), 1.21-1.14 (m, 9H), 1.07-1.01 (m, 2H), 0.88 (s, 3H).

[0394] LCMS column: C18; Column size: 4.6*50 mm; Mobile phase: B (ACN) : A (0.02% NH4Ac); Gradient (B%): 6.5 min-5-95-POS; Rt = 3.114 min; MS calculated: 401, MS found: 402 [M+H] + .

[0395] Synthesis of Compound 42: tert-butyl-3-((E)-2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)azetidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 0.7 mL, 1.80 mmol) was added to a solution of the compound phosphonium salt (514 mg, 0.90 mmol) in THF (5 mL) at -78 °C. The mixture was stirred at 40 °C for 1 hour. Compound 39 (100 mg, 0.30 mmol) was then added to the mixture at 0 °C, and the mixture was then warmed to 40 °C overnight. The reaction was repeated 9 times. The mixture was quenched with saturated NH4Cl (80 mL) and extracted with EtOAc (100 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound 42 (20 mg, 1%) as a yellow solid.

[0396] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikwa Diamonsil plus; mobile phase: B (ACN):A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 1.945 min; MS calculated: 501, MS found: 402 [M+H-Boc] + .

[0397] CVie403: Synthesis of (6S,10R,13S)-3-(2-(azetidin-3-yl)ethylidene)-6-(hydroxymethyl)-10,13-dimethyldodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 40 (130 mg, 0.26 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 h. The mixture was basified to pH 8-9 with saturated NaHCO3. The mixture was extracted with DCM (30 mL × 3). The combined organic layers were concentrated, and the residue was purified by preparative HPLC to give compound CVie403 (13 mg, 13% yield) as a yellow solid.

[0398] Spectroscopic data of CVie403

[0399] 1 H NMR (CD3OD, 400 MHz):δ 5.00-4.96 (m, 1H), 3.92-3.81 (m, 3H), 3.66-3.62 (m, 1H), 3.54-3.52 (m, 2H), 2.77-2.73 (m, 1H), 2.67-2.61 (m, 2H), 2.49-2.43 (m, 2H), 2.38-2.29 (m, 2H), 2.23-2.18 (m, 2H), 2.06-1.96 (m, 2H), 1.83-1.76 (m, 2H), 1.71-1.61 (m, 3H), 1.51-1.35 (m, 3H), 1.18 (s, 3H), 1.12-1.05 (m, 2H), 0.98-0.90 (m, 1H), 0.80 (s, 3H).

[0400] LCMS column: Rt = 3.964 min; MS calculated: 399, MS found: 400 [M+H]+.

[0401] Synthesis of Compound 43: 3-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)azetidine-1-carboxylate [ka] A mixture of compound 42 (20 mg, 0.04 mmol), Pd / C (10%, 20 mg), and Pd(OH) (20%, 20 mg) in EtOAc (2 mL) was stirred under H (balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 43 (20 mg, 100%) as a brown solid.

[0402] LCMS column: C18; Column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; Mobile phase: B(ACN):A (0.02%NH4Ac+5%ACN); Gradient (B%): 3 min-5-95-POS; Flow rate: 1.5 mL / min, Stop time: 3 min. Rt = 1.978 min; MS calculated: 503, MS found: 404 [M+H-Boc] + .

[0403] CVie408: (6S,7S,10R,13S)-3-(2-(azetidin-3-yl)ethyl)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyltetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one Synthesis of [ka] A mixture of compound 43 (20 mg, 0.04 mmol) in TFA / DCM (1:1, 2 mL) was stirred at 0 °C for 30 min. The mixture was diluted with saturated NaHCO and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie408 (6.4 mg, 40%) as a yellow solid.

[0404] Spectroscopic data of CVie408

[0405] 1 H NMR (CD3OD, 400 MHz): δ 4.08 (s, 1H), 3.84 (t, J = 8.4 Hz, 1H), 3.77-3.68 (m, 2H), 3.52-3.48 (m, 2H), 2.81-2.73 (m, 1H), 2.52-2.44 (m, 1H), 2.18-2.06 (m, 2H), 1.86-1.71 (m, 4H), 1.69-1.59 (m, 6H), 1.52-1.43 (m, 2H), 1.39-1.30 (m, 4H), 1.24-1.15 (m, 4H), 1.12-1.04 (m, 1H), 0.95-0.92 (m, 1H), 0.90(s, 3H), 0.87 (s, 3H).

[0406] LCMS column: C18; Column size: 4.6*50 mm; Mobile phase: B (ACN): A (0.02% NH4Ac); Gradient (B%): 6.5 min-5-95-POS; Rt = 3.078 min; MS calculated: 403, MS found: 404 [M+H] + .

[0407] Synthesis of Compound 44: tert-butyl-3-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)pyrrolidine-1-carboxylate [ka] A solution of n-BuLi in THF (1.5 M, 1.57 mL, 3.94 mmol) was added to a solution of the phosphonium salt of the compound (1.5 g, 2.62 mmol) in THF (15 mL) at -78 °C. The reaction mixture was stirred at 35 °C for 1 hour. Then, a solution of compound 37 (350 mg, 1.05 mmol) was added to the mixture at -20 °C and warmed to room temperature for 2 hours. The mixture was quenched with saturated NH4Cl (25 mL) and extracted with EtOAc (25 mL x 3). The combined organic layers were concentrated, and the residue was purified by flash chromatography (hexane:EA = 1:1) to obtain the crude compound. The compound was then purified by reverse-phase column chromatography to obtain pure compound 44 (53 mg, 10%) as a white solid.

[0408] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B (ACN) : A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 2.017 min; MS calculated: 513, MS found: 414 [M+H-Boc] + .

[0409] CVie402: Synthesis of (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(pyrrolidin-3-yl)ethylidene)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 44 (89 mg, 0.173 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 h. The mixture was diluted with saturated NaHCO3 and adjusted to pH = 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound CVie402 (38 mg, 53%) as a white solid.

[0410] Spectroscopic data of CVie402

[0411] 1 H NMR (CD3OD, 400 MHz): δ 5.08-5.05 (m, 1H), 3.88-3.82 (m, 1H), 3.63-3.59 (m, 1H), 3.12-3.02 (m, 2H), 2.99-2.92 (m, 1H), 2.66-2.55 (m, 3H), 2.48-2.42 (m, 2H), 2.37-2.30 (m, 1H), 2.23-2.19 (m, 1H), 2.15-2.04 (m, 2H), 2.03-1.91 (m, 4H), 1.83-1.78 (m, 2H), 1.75-1.61 (m, 3H), 1.51-1.34 (m, 4H), 1.19-1.17 (m, 3H), 1.12-1.03 (m, 2H), 0.97-0.90 (m, 1H), 0.80 (s, 3H).

[0412] LCMS column: Rt = 3.060 min; MS calculated: 413, MS found: 414 [M+H]+.

[0413] Synthesis of Compound 45: tert-butyl-3-(2-((3S,6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)pyrrolidine-1-carboxylate [ka] A solution of compound 44 (53 mg, 0.103 mmol) in EA (3 mL) was added to Pd / C (60 mg). The mixture was then stirred under H at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give compound 45 (50 mg, 94%) as a white solid.

[0414] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B (ACN) : A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 1.984 min; MS calculated: 515, MS found: 416 [M+H-Boc] + .

[0415] CVie409: Synthesis of (3S,6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(pyrrolidin-3-yl)ethyl)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 45 (50 mg, 0.09 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 1 hour. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL x 3). The combined organic layers were concentrated, and the residue was purified by preparative HPLC to give compound CVie409 (12 mg, 32%) as a white solid.

[0416] Spectroscopic data of CVie409

[0417] 1H NMR (CD3OD, 400 MHz):δ 3.87-3.82 (m, 1H), 3.70-3.64 (m, 1H), 3.28-3.24 (m, 1H), 3.21-3.16 (m, 1H), 3.11-3.04 (m, 1H), 2.72-2.62 (m, 2H), 2.57-2.51 (m, 1H), 2.47-2.39 (m, 2H), 2.17-2.05 (m, 3H), 1.85-1.70 (m, 5H), 1.66-1.49 (m, 8H), 1.38-1.34 (m, 2H), 1.25-1.19 (m, 6H), 1.14-1.10 (m, 2H), 0.88 (s, 3H).

[0418] LCMS column: C18; Column size: 4.6*50 mm; Mobile phase: B (ACN) : A (0.02% NH4Ac); Gradient (B%): 6.5 min-5-95-POS; Rt = 3.180 min; MS calculated: 415, MS found: 416 [M+H] +

[0419] Synthesis of Compound 46: tert-butyl-3-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)pyrrolidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 0.7 mL, 1.80 mmol) was added to a solution of the compound phosphonium salt (527 mg, 0.90 mmol) in THF (5 mL) at -78 °C. The reaction mixture was stirred at 35 °C for 1 hour. Compound 39 (100 mg, 0.30 mmol) was then added to the mixture at 0 °C, and the mixture was then warmed to 35 °C overnight. The reaction was repeated four times. The mixture was quenched with saturated NH Cl (80 mL) and extracted with EtOAc (100 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound 46 (26 mg, 3%) as a white solid.

[0420] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B (ACN) : A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 2,000 min; MS calculated: 515, MS found: 416 [M+H-Boc] + .

[0421] Synthesis of Compound 47: tert-butyl-3-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)pyrrolidine-1-carboxylate [ka] A mixture of compound 46 (26 mg, 0.05 mmol), Pd / C (10%, 30 mg), and Pd(OH) (20%, 30 mg) in EtOAc (3 mL) was stirred under H (balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 47 (26 mg, 100%) as a yellow solid.

[0422] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B (ACN) : A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 2.059 min; MS calculated: 517, MS found: 418 [M+H-Boc] + .

[0423] CVie410: Synthesis of (6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(pyrrolidin-3-yl)ethyl)tetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one [ka] A solution of compound 47 (26 mg, 0.05 mmol) in TFA / DCM (1:2, 2 mL) was stirred at 0 °C for 1 h. The mixture was diluted with saturated NaHCO and adjusted to pH 8-9. The mixture was extracted with DCM (20 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie410 (9 mg, 43%) as a yellow solid.

[0424] Spectroscopic data of CVie410

[0425] 1H NMR (CD3OD, 400 MHz): δ 3.95 (s, 1H), 3.64-3.57 (m, 2H), 3.12-3.07 (m, 1H), 3.04-2.96 (m, 1H), 2.93-2.89 (m, 1H), 2.48-2.44 (m, 1H), 2.38-2.32 (m, 1H), 2.05-1.93 (m, 4H), 1.69-1.57 (m, 5H), 1.55-1.46 (m, 4H), 1.37-1.33 (m, 4H), 1.25-1.18 (m, 5H), 1.08-0.90 (m, 3H), 0.82-0.80 (m, 1H), 0.77 (s, 3H), 0.75 (s, 3H).

[0426] LCMS column: C18; column size: 4.6*50 mm; mobile phase: B (ACN): A (0.02% NH4Ac); gradient (B%): 6.5 min-5-95-POS; Rt = 3.139 min; MS calculated: 417, MS found: 418 [M+H] + .

[0427] Synthesis of Compound 48: tert-butyl-4-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)piperidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 2.90 mL, 7.20 mmol) was added to a mixture of compound phosphonium salt (2.16 g, 3.60 mmol) in THF (16 mL) at -78 °C. The reaction mixture was stirred at 30 °C for 1 hour. Compound 37 (400 mg, 1.20 mmol) was then added to the mixture at -20 °C. The mixture was stirred at -20 °C for 30 minutes and then warmed to 30 °C for 2 hours. The mixture was quenched with saturated NH Cl (15 mL) and extracted with EtOAc (30 mL × 3). The combined organic layer was concentrated, and the residue was purified by preparative HPLC to give compound 48 (28 mg, 4%) as a yellow solid.

[0428] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B (ACN):A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-30-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 2.013 min; MS calculated: 527, MS found: 428 [M+H-Boc] + .

[0429] CVie405: Synthesis of (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(piperidin-4-yl)ethylidene)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 46 (80 mg, 0.152 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 30 minutes. The mixture was basified with saturated NaHCO to pH = 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie405 (30 mg, 46%) as a yellow solid.

[0430] Spectroscopic data of CVie405

[0431] 1 H NMR (CD3OD, 400 MHz): δ 5.14 (t, J = 7.2 Hz, 1H), 3.88-3.80 (m, 1H), 3.74-3.66 (m, 1H), 3.09-3.06 (m, 2H), 2.67-2.49 (m, 6H), 2.45-2.38 (m, 1H), 2.33-2.26 (m, 1H), 2.19-2.04 (m, 2H), 2.01-1.83(m, 3H), 1.80-1.69 (m, 6H), 1.61-1.47 (m, 2H), 1.45-1.34 (m, 2H), 1.30-1.25 (m, 5H), 1.19-1.08(m, 4H), 1.04-0.90 (m, 1H), 0.88 (s, 3H).

[0432] LCMS column: Rt = 3.219 min; MS calculated: 427, MS found: 428 [M+H]+.

[0433] Synthesis of Compound 49: tert-butyl-4-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)piperidine-1-carboxylate [ka] A mixture of compound 48 (28 mg, 0.05 mmol) and Pd / C (10%, 50 mg) in EtOAc (2 mL) was stirred under H (in a balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 49 (28 mg, 100%) as a yellow solid.

[0434] LCMS column: C18; Column dimensions: 4.6*30 mm 5 μm; Dikwa Diamonsil plus; Mobile phase: B(ACN):A (0.02%NH4Ac+5%ACN); Gradient (B%): 3 min-5-95-POS; Flow rate: 1.5 mL / min, Stop time: 3 min. Rt = 2.109 min; MS calculated: 529, MS found: 430 [M+H-Boc] + .

[0435] CVie411: Synthesis of (6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(piperidin-4-yl)ethyl)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A solution of compound 49 (28 mg, 0.05 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 30 minutes. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL x 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie411 (10 mg, 43%) as a yellow solid.

[0436] Spectroscopic data of CVie411

[0437] 1 H NMR (CD3OD, 400 MHz): δ 3.85-3.81 (m, 1H), 3.71-3.66 (m, 1H), 3.07-3.04 (m, 2H), 2.69 (t, J = 11.2 Hz, 1H), 2.63-2.51 (m, 3H), 2.48-2.39 (m, 2H), 2.15-2.05 (m, 1H), 1.84-1.72 (m, 7H), 1.63-1.46 (m, 4H), 1.37-1.33 (m, 6H), 1.28-1.14 (m, 7H), 1.09-0.98 (m, 3H), 0.88 (s, 3H).

[0438] LCMS column: C18; Column size: 4.6*50 mm; Mobile phase: B (ACN): A (0.02% NH4Ac); Gradient (B%): 6.5 min-5-95-POS; Rt = 4.188 min; MS calculated: 429, MS found: 430 [M+H] + .

[0439] Synthesis of Compound 50: tert-butyl-4-(2-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthrene-3(2H,4H,10H)-ylidene)ethyl)piperidine-1-carboxylate [ka] A solution of n-BuLi in THF (2.5 M, 0.70 mL, 1.80 mmol) was added to a solution of phosphonium salt (540 mg, 0.90 mmol) in THF (5 mL) at −78° C. The reaction mixture was stirred at 40° C. for 1 h. Compound 39 (100 mg, 0.30 mmol) was then added to the mixture at 0° C., and then warmed to 40° C. for 2 h. The reaction was repeated five times. The mixture was quenched with saturated NH4Cl (80 mL) and extracted with EtOAc (100 mL × 3). The combined organic layers were concentrated, and the residue was purified by preparative HPLC to give crude compound 50 (35 mg, 4%) as a white solid.

[0440] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B(ACN):A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 2.104 min; MS calculated: 529, MS found: 430 [M+H-Boc] + .

[0441] Synthesis of Compound 51: tert-butyl-4-(2-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl)piperidine-1-carboxylate [ka] A mixture of compound 50 (35 mg, 0.07 mmol), Pd / C (10%, 40 mg), and Pd(OH) (20%, 40 mg) in EtOAc (2 mL) was stirred under H (in a balloon) at room temperature overnight. The mixture was filtered, and the filtrate was concentrated to give crude compound 51 (35 mg, 100%) as a brown solid.

[0442] LCMS column: C18; column dimensions: 4.6*30 mm 5 μm; Dikma Diamonsil plus; mobile phase: B(ACN):A (0.02%NH4Ac+5%ACN); gradient (B%): 3 min-5-95-POS; flow rate: 1.5 mL / min, stop time: 3 min. Rt = 2.126 min; MS calculated: 531, MS found: 432 [M+H-Boc] + .

[0443] CVie412: Synthesis of (6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3-(2-(piperidin-4-yl)ethyl)tetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one [ka] A solution of compound 51 (35 mg, 0.07 mmol) in TFA / DCM (1:2, 2 mL) was stirred at room temperature for 30 minutes. The mixture was diluted with saturated NaHCO3 and adjusted to pH 8-9. The mixture was extracted with DCM (25 mL × 3). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie412 (13 mg, 46%) as a yellow solid.

[0444] Spectroscopic data of CVie412

[0445] 1 H NMR (CD3OD, 400 MHz): δ 4.04 (s, 1H), 3.72-3.61 (m, 2H), 3.09-3.06 (m, 2H), 2.63 (t, J = 11.6 Hz, 2H), 2.48-2.41 (m, 1H), 2.13-2.03 (m, 2H), 1.75-1.66 (m, 5H), 1.63-1.55 (m, 5H), 1.45-1.35 (m, 3H), 1.31-1.17 (m, 10H), 1.10-1.03 (m, 2H), 0.89 (s, 1H), 0.87 (s, 3H), 0.84 (s, 3H).

[0446] LCMS column: C18; Column size: 4.6*50 mm; Mobile phase: B (ACN) : A (0.02% NH4Ac); Gradient (B%): 6.5 min-5-95-POS; Rt = 3.203 min; MS calculated: 431, MS found: 432 [M+H] + .

[0447] Synthesis of Compound 52: tert-butyl ((E)-5-((6S,10R,13S)-6-(hydroxymethyl)-10,13-dimethyl-7,17-dioxododecahydro-1H-cyclopenta[a]phenanthren-3(2H,4H,10H)-ylidene)pentyl)(methyl)carbamate [ka] To a mixture of N-Boc-N-methyl-5-triphenylphosphonium penteneamine iodide (4.26 g, 7.23 mmol) in THF (50 mL) was added n-BuLi (3.18 mL, 7.95 mmol) dropwise at −78° C. The mixture was stirred at 0° C. for 20 minutes. The mixture was then cooled to −30° C. Compound 37 (800 mg, 2.41 mmol) was then added to the reaction mixture. The mixture was stirred at room temperature overnight. The reaction mixture was quenched with HO and concentrated. The residue was purified by silica gel column chromatography (PE / EtOAc=1 / 2) and then by preparative HPLC to yield compound 52 (36 mg, 200 mg) as a colorless oil.

[0448] Spectroscopic data of compound 52

[0449] 1 H NMR (CD3OD, 400 MHz): 5.16-5.12 (m, 1H), 3.89-3.85 (m, 1H), 3.77-3.73 (m, 1H), 3.22-3.19 (m, 2H), 2.83 (s, 3H), 2.75-2.60 (m, 2H), 2.60-2.50 (m, 2H), 2.46-2.41 (m, 1H), 2.33-2.27 (m, 1H), 2.15-2.02 (m, 4H), 1.90-1.79 (m, 2H), 1.77-1.71 (m, 3H), 1.60-1.52 (m, 4H), 1.50 (s, 9H), 1.45-1.42 (m, 1H), 1.34-1.29 (m, 2H), 1.26 (s, 3H), 1.24-1.21 (m, 1H), 1.19-1.05 (m, 2H), 1.05-0.99 (m, 1H).

[0450] CVie401: Synthesis of (6S,10R,13S,E)-6-(hydroxymethyl)-10,13-dimethyl-3-(5-(methylamino)pentylidene)dodecahydro-1H-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione [ka] A mixture of compound 52 (60 mg, 0.116 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature overnight. The mixture was then concentrated, diluted with EtOAc, washed with saturated Na2CO3, dried over Na2SO4, filtered, and concentrated to give compound CVie401 (38 mg, 79%) as a yellow oil.

[0451] Spectroscopic data of CVie401

[0452] 1 H NMR (CD3OD, 400 MHz): 5.36 (s, 1H), 3.89-3.86 (m, 1H), 3.71-3.67 (m, 1H), 2.80-2.77 (m, 2H), 2.73-2.67 (m, 1H), 2.55-2.39 (m, 6H), 2.15-2.01 (m, 4H),1.92-1.85 (m, 1H),1.77-1.69 (m, 4H), 1.65-1.45 (m, 7H), 1.38-1.25 (m, 6H), 1.19 (s, 3H), 0.89 (s, 4H).

[0453] LCMS: Rt = 2.128 min, [M+1] + = 416.

[0454] Synthesis of Compound 53: tert-butyl (5-((6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-17-oxododecahydro-1H-cyclopenta[a]phenanthren-3(2H,4H,10H)-ylidene)pentyl)(methyl)carbamate [ka] To a mixture of N-Boc-N-methyl-5-triphenylphosphonium penteneamine iodide (4.39 g, 7.45 mmol) in THF (45 mL) was added dropwise a solution of nBuLi in THF (4.46 mL, 2.5 N, 11.16 mmol) at −78° C. The mixture was then stirred at 0° C. for 20 minutes. The mixture was cooled to −50° C., and compound 39 (830 mg, 2.48 mmol) was added. The mixture was stirred at room temperature overnight. The mixture was quenched with HO, concentrated, and purified by column chromatography (PE / EtOAc=1 / 1), followed by preparative HPLC to give compound 53 (80 mg, 300 mg) as a white solid.

[0455] Spectroscopic data of compound 53

[0456] 1 H NMR (CD3OD, 400 MHz): 5.10-5.08 (m, 1H), 4.05 (s, 1H), 3.78-3.72 (m, 1H), 3.23-3.20 (m, 2H), 2.83 (s, 3H), 2.56-2.53 (m, 1H), 2.48-2.41 (m, 1H), 2.12-2.10 (m, 1H), 2.07-2.00 (m, 5H), 1.85-1.81 (m, 1H), 1.74-1.51 (m, 10H), 1.49 (s, 9H), 1.39-1.30 (m, 4H), 1.21-1.18 (m, 1H), 1.08-1.04 (m, 1H), 0.96 (s, 3H), 0.88 (s, 3H).

[0457] CVie406: Synthesis of (6S,7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3-(5-(methylamino)pentylidene)tetradecahydro-1H-cyclopenta[a]phenanthren-17(2H)-one [ka] A solution of compound 53 (80 mg, 0.155 mmol) in TFA / DCM (1 mL / 2 mL) was stirred at room temperature for 10 minutes. The mixture was basified with saturated NaHCO to pH 8-9. The mixture was extracted with DCM (25 mL × 2). The combined organic layers were dried over NaSO, filtered, and concentrated. The residue was purified by preparative HPLC to give compound CVie406 (60 mg, 94%) as a yellow solid.

[0458] LCMS column: Rt = 0.370 min; MS calculated: 417, MS found: 418 [M+H] + .

[0459] Example 2. General Procedures for Measuring Biological Activity Examples of animal management The study complied with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996) and with animal care guidelines approved by the participating institutions.

[0460] Measurements in isolated left ventricular cardiomyocytes The compounds inhibited (i) SR-Ca in freshly isolated myocytes from rat and guinea pig ventricles by retrograde coronary perfusion with the enzyme solution. 2+ It was characterized for (ii) its uptake function and its effect on action potentials (APs) (Rocchetti M et al., J Pharmacol Exper Therap 2005, 313(1):207-215).

[0461] statistical analysis Whole animal experiments: Data are reported as mean ± SD. Statistical analysis was performed by Student's t-test (paired t-test).

[0462] Isolated myocyte experiments: Data are reported as mean ± SE. Curves containing multiple means were compared by two-way ANOVA for repeated measurements, and drug-induced changes in overall curve steepness were defined according to the significance of the "group by factor X" interaction. 2+ In some cells for which CaT data are reported, τ is poorly defined due to the poor single-exponential fit of the decay. decay is not estimated, and sample sizes (N) are reported in the corresponding figures. The dependence of STV on mean APD was quantified by linear regression. P<0.05 was considered statistically significant for all comparisons.

[0463] Example 3. In vitro screening of compounds of formula (I) Dog kidney sodium + / K + Inhibition of ATPase activity As described herein, the compounds of the present invention are pure or nearly pure SERCA2a stimulators. + / K + These compounds show little or no inhibition of the enzymatic activity of ATPase in dogs. + / K + Its inhibitory effect on ATPase enzyme was tested.

[0464] Renal Na + / K +Purification of ATPase was performed according to the method described by Jorgensen (Methods Enzymol. 1988, 156:29-43). Kidneys were removed from 1- to 3-year-old male beagle dogs (WuXi AppTec, Suzhou Co., Ltd., 1318 Wuzhong Ave., Wuzhong District, Suzhou, 215104, PR China) under pentobarbital anesthesia (import permit 0009171-09 / 04 / 2015-DGSAF-COD_UO-P, 2015, from the Italian Ministry of Health). The kidneys were sliced, and the outer medulla was dissected and suspended in a sucrose-histidine solution (1 g / 10 ml) containing 250 mM sucrose, 30 mM histidine, and 5 mM EDTA, pH 7.2. The tissue was homogenized using an UltraTurrax homogenizer. The sample was centrifuged at 6,000 g for 15 minutes. The supernatant was then decanted and centrifuged at 48,000 g for 30 minutes. The pellet was suspended in sucrose-histidine buffer and incubated for 20 minutes with a solution of sodium dodecyl sulfate (SDS) dissolved in a pH 7.5 gradient buffer containing 25 mM imidazole and 1 mM EDTA. The sample was layered on a discontinuous sucrose gradient (10, 15, and 29.4%) and centrifuged at 60,000 g for 115 minutes. The pellet was suspended in the gradient buffer.

[0465] Na + / K + ATPase activity was measured as described in the literature (Ferrandi M. et al., Hypertension 1996, 28:1018-25). 32 from P-ATP 32The assay was carried out in vitro by measuring the release of 0.3 μg of purified dog kidney enzyme. Increasing concentrations of standard ouabain or test compounds were incubated with 0.3 μg of purified dog kidney enzyme for 10 minutes at 37°C in a final volume of 120 μl of medium containing 140 mM NaCl, 3 mM MgCl, 50 mM Hepes-Tris, 3 mM ATP, pH 7.5. The medium was then incubated with 10 mM KCl and 20 nCi of ATP. 32 10 μl of a solution containing P-ATP (3-10 Ci / mmol, Perkin Elmer) was added. The reaction was continued for 15 min at 37°C and then stopped by acidification with 20% v / v ice-cold perchloric acid. The cells were then centrifuged over activated charcoal (Norit A, Serva). 32 P was separated and the radioactivity was counted. Inhibitory activity was expressed as a percentage of control samples performed in the absence of ouabain or test compound. + / K + The concentration of compound that causes 50% inhibition of ATPase activity (IC 50 ) was calculated using a multi-parameter nonlinear regression optimization program (Kaleidagraph™, Sinergy Software).

[0466] Compounds CVie201, CVie202, CVie203, CVie204, CVie213, CVie214, CVie215, CVie216, CVie217, CVie218, and CVie219 were prepared from purified NaCl as shown in Table 1. + / K + Does not inhibit ATPase enzyme activity and has IC 50 Compounds CVie205, CVie206, CVie207, CVie208, CVie209, CVie210, CVie211, CVie212, CVie401, CVie402, CVie403, CVie404, CVie405, CVie406, CVie407, CVie408, CVie409, CVie410, CVie411, and CVie412 were Na + / K + ATPase was only mildly inhibited (IC50 ranged from 0.8 to 24 μM) (Table 1).

[0467] The compounds were tested using the reference drug digoxin (IC 50 , 0.18 μM) and istaloxime (IC 50 , 0.14 μM) (Table 1). Table 1. Na in dog kidney + / K + ATPase inhibition [Table 1]

[0468] SERCA2a ATPase activity in SR microsomes from normal guinea pig hearts The compounds disclosed herein were also tested for their ability to stimulate SERCA2a activity in SR microsomes derived from normal guinea pig heart tissue at concentrations ranging from 1 to 200 nM. Cardiac SERCA2a microsomes were prepared using 2-month-old guinea pigs (Envigo, Udine, Italy, 350-450 g). Guinea pigs were sacrificed under pentobarbital anesthesia. The left ventricle (LV) was rapidly dissected and immediately frozen in liquid nitrogen. LV tissue was processed according to the method described by Nediani C. et al. (J. Biol. Chem. 1996, 271:19066-7). The tissue was suspended in 4 volumes of buffer containing 10 mM NaHCO3, pH 7, 1 mM PMSF, 10 μg / ml aprotinin and leupeptin, and homogenized using an Ultra Turrax homogenizer. The sample was centrifuged at 12,000 g for 15 minutes. The resulting supernatant was filtered and centrifuged at 100,000 g for 30 minutes. The pellet was suspended in 0.6 M KCl, 30 mM histidine, pH 7, and centrifuged at 100,000 g for 30 minutes to extract contractile proteins. The final pellet was reconstituted in 0.3 M sucrose, 30 mM histidine, pH 7, and stored in aliquots at -80°C until use.

[0469] SERCA2a activity was measured in the absence and presence of various Ca2+ concentrations in the presence of test compounds as described previously (Micheletti R. et al., Am J Card 2007, 99:24A-32A). 2+ At concentrations (100-4000nM), 32 In vitro P-ATP hydrolysis was measured. Increasing concentrations of each compound (ranging from 1 to 200 nM) were preincubated with 2 μg of SERCA2a-enriched microsomes in 80 μl of a solution containing 100 mM KCl, 5 mM MgCl2, 1 μM A23187, and 20 mM Tris, pH 7.5, for 5 min at 4°C. Then, 50 nCi of 32 20 μl of 5 mM Tris-ATP containing P-ATP (3–10 Ci / mmol, Perkin Elmer) was added. ATP hydrolysis was continued for 15 min at 37°C, and the reaction was stopped by acidification with 100 μl of 20% v / v ice-cold perchloric acid. The reaction was then centrifuged over activated charcoal (Norit A, SERVA). 32 P was isolated and radioactivity was measured. SERCA2a-dependent activity was determined as the fraction of total hydrolytic activity inhibited by 10 μM cyclopiazonic acid (Seidler NW et al., J Biol Chem. 1989, 264:17816-23).

[0470] Dose-response curves were fitted using a sigmoidal equation to measure activity at maximum velocity (Vmax), and Ca 2+ The Kd for Ca was calculated (Synergy Software KaleidaGraph 3.6). The effect of the compound on normal guinea pig specimens was calculated as the Kd-Ca of control samples run in the absence of compound. 2+ % decrease in (Ca 2+ The effect was expressed as a function of the affinity for Ca (meaning an increase in affinity for Ca) (Table 2). 2+These results suggest that acetaminophen increases SERCA2a activity at concentrations below 2000mg / mL (Rocchetti M et al., J Pharmacol Exp Ther. 2005, 313:207-15; Rocchetti M et al., J Pharmacol Exp Ther. 2008, 326:957-65; Ferrandi M et al., Br J Pharmacol 2013, 169:1849-1861). Data are means ± SD, n = number of experiments, * indicates p<0.05.

[0471] At nanomolar concentrations, the test compounds inhibited Ca in microsomes from guinea pig heart specimens. 2+ Dose-response curve of SERCA2a-Kd-Ca 2+ These results indicate that the compound reduces Ca in the physiological range. 2+ We demonstrated that iodine increased SERCA2a activity at concentrations suggesting a lusitropic effect. Istaroxime was used as a comparison agent, demonstrating its ability to stimulate SERCA2a (Table 2). In contrast, digoxin failed to stimulate SERCA2a activity (Ferrandi M et al., Br J Pharmacol 2013, 169:1849-61; Rocchetti M et al., J Pharmacol Exp Ther 2005, 313:207-215). Table 2. SERCA2a-Kd-Ca in SR microsomes from normal guinea pig hearts. 2+ Effect of test compounds on [Table 2-1] [Table 2-2]

[0472] Example 4. Studies on CVie214 and CVie216 ​​in isolated ventricular myocytes SR-Ca in rat ventricular myocytes 2+ Import function To test the efficacy of compounds in a model of diastolic dysfunction, male Sprague Dawley rats (150–175 g) were made diabetic by a single tail vein injection of streptozotocin (STZ, 50 mg / kg, Sigma-Aldrich). STZ was freshly prepared in 0.1 M sodium citrate buffer, pH 4.5. Fasting blood glucose was measured after 1 week, and rats with values ​​>300 mg / dl were considered diabetic. SR-Ca 2+ Drug effects on uptake function were assessed in isolated left ventricular myocytes 9 weeks after STZ injection. Myocytes were incubated for at least 30 minutes in the presence of the specified drug to ensure its membrane permeation. Statistical analysis was performed using a "group comparison" model.

[0473] SR-Ca 2+ The effect of the drug on the uptake rate excludes the contribution of the Na / Ca exchanger (NCX) and is consistent with low levels of SR-Ca. 2+ To assess the uptake rate starting from the loading, a specially designed SR "loading protocol" was used. Under voltage clamp conditions, intracellular Ca 2+ The concentration was measured dynamically by epifluorescence (Fluo4-AM). The time-dependent component was mainly I CaL The membrane current, which reflects the Ca concentration in the SR, was simultaneously recorded. The SR loading protocol involves emptying the SR with a short caffeine pulse, followed by the release of sarcolemmal Ca. 2+ Channel (I CaL ) via Ca 2+ The NCX was constructed by gradually replenishing the SR with a voltage step that activated Na influx from the intracellular and extracellular solutions. + The methodology is consistent with published methods, with minor modifications (Rocchetti M et al., J Pharmacol Exper Therap 2005, 313:207-215).

[0474] SR-Ca 2+ The effect of drugs on uptake was assessed by several parameters: 1) Ca 2+2) the rate of Ca transient (CaT) amplitude; and 2+ induced Ca 2+ 3) the rate at which CICR gain increases during the loading protocol (which reflects the rate of SR replenishment and system gain); and 4) the rate at which cytosolic Ca release (CICR) increases during each pulse. 2+ Decay time constant (τ decay ) (This is the net Ca passing through the SR membrane. 2+ The transport rate (by SERCA2a) is reflected (τ decay The decrease in SR-Ca 2+ The analysis was carried out taking into account the uptake of

[0475] The specificity of the "loading protocol" in detecting SERCA2a activation was confirmed by Na + / K + This is supported by the observation that we did not detect any effect of digoxin, an inotropic agent that blocks the ATPase pump but has no SERCA2a stimulating effect (Rocchetti M et al., J Pharmacol Exp Ther 2005, 313:207-215; Alemanni M et al., JMCC 2011, 50:910-918).

[0476] CVie216 ​​(1 μM) inhibits Ca during SR reloading. 2+ The rate of increase in CaT transients (Figure 1A) was increased, which was correlated with an increase in CICR gain (Figure 1B) and τ decay was associated with a decrease in (Figure 1C).

[0477] CVie214 (1 μM) altered CaT parameters during the SR loading protocol in a similar manner to CVie216 ​​(Figure 2B). CICR gain (Figure 2B) and τ decay (Figure 2C) was affected by the drug, as expected from the enhancement of SERCA2a. CVie214 did not significantly increase the rate of CaT increase during SR reloading (Figure 2A). However, the increase in CICR gain was not conclusive, suggesting that this does not negate its effect on SERCA2a, but rather coincides with the increase in I. CaLThis suggests that this may reflect inhibition.

[0478] The results in Figures 1 and 2 show that CVie216 ​​and CVie214 inhibit Ca2+ production by SR. 2+ Under the applied experimental conditions, SR-Ca uptake was significantly increased. 2+ Uptake was entirely supported by SERCA2a, and therefore the results are consistent with activation of SERCA2a by the two agents.

[0479] Action potential measurement The effects of Cvie216 ​​and Cvie214 on action potential parameters were assessed at a concentration of 1 μM to modulate SERCA2a in guinea pig myocytes. Action potential (AP) curves provide a first estimate of the integrated function of membrane ion channels, and their alterations may reveal ancillary actions that could result in adverse compound effects. To increase the sensitivity of AP curves as reporters, we also tested the effect on the rate dependence of AP parameters, providing a multiparametric (more rigorous) approach. To ensure that AP curves recapitulate human APs, APs were recorded in guinea pig ventricular myocytes. Myocytes were incubated in the presence of the drugs for at least 30 min to ensure no effects even after long exposure times. Statistical analysis was performed using a "group comparison" model.

[0480] APs were recorded in guinea pig ventricular myocytes in normal Tyrode's solution at 36.5°C. The following parameters were measured: diastolic membrane potential (E diast ), maximum depolarization rate (dV / dt max ), and action potential duration (APD at 90% of repolarization) were measured at four stimulation rates (0.5, 1, 2, and 4 Hz). Short-term APD variation (STV) during steady-state pacing, an index of repolarization stability, was measured using the APD n fAPD n+1 It was measured as the sum of the absolute orthogonal deviations of the plot (Poincaré plot) from the same line (Altomare C et al., Circulation A&E 2015, 8:1265-1275).

[0481] At 1 μM, CVie216 ​​(FIG. 3A) and CVie214 (FIG. 4A) significantly increased the action potential duration (APD) of 90 ), diastolic membrane potential (E diast ), maximum depolarization rate (dV / dt max ), and did not affect the velocity dependence of the respective AP parameters.

[0482] Short-term APD variation (STV) is a marker of electrical instability and correlates with arrhythmogenic risk. STV is a function of mean APD; therefore, STV was measured at multiple pacing rates (0.5, 1, 2, and 4 Hz) to extend its assessment to a wide APD range. STV and its dependence on mean APD were not significantly affected by both CVie216 ​​(Figure 3B) and CVie214 (Figure 4B).

[0483] Taken together, the multiparametric approach used for action potential analysis means that there are no unwanted drug effects on cardiac electrical activity. Thus, according to this analysis, Cvie216 ​​and Cvie214 selectively exert SERCA2a modulation (positive lusitropic drugs), i.e., they do not affect electrical activity and the membrane currents involved.

[0484] Example 5. In vivo testing of CVie214 and CVie216 Bioavailability in rats Bioavailability in rats was measured by Sundia MediTech Service, China. Specifically, the bioavailability of CVie214-salt and CVie216-salt was measured in rats after intravenous injection (iv) of 1 mg / kg and oral administration (os) of 10 mg / kg. Plasma concentrations of test compounds CVie214-salt and CVie216-salt were measured at intervals from 0 to 24 hours and detected by LC-MS. F values ​​(%) were calculated to be 41.5% and 16.9% for CVie214-salt and CVie216-salt, respectively.

[0485] Acute toxicity in mice The acute toxicity of test compounds CVie214-salt and CVie216-salt was measured in mice (Albino Swiss CD-1, 30 g body weight). Increasing doses of the compounds were administered orally or intravenously to identify the dose causing 50% mortality. Death occurred within 30 minutes of administration, with survival after 24 hours.

[0486] Table 3 reports the acute toxicity results of CVie214-salt and CVie216-salt. For comparison, the acute toxicity of the reference compounds digoxin and istaloxime is also shown. For digoxin, data are taken from the literature (www.lookchem.com, Reference for intravenous digoxin: Afifi AM, Ammar EM. Pharmacological Research Communications. Vol. 6, Pg. 417, 1974; Reference for oral digoxin: Archives Internationales de Pharmacodynamie et de Therapie. Vol. 153, Pg. 436, 1965) (Table 3). Table 3. Acute toxicity (LD) of CVie214-salt and CVie216-salt in mice 50 ) [Table 3]

[0487] Hemodynamics in streptozotocin-induced diabetic rats (echocardiography, 2M-Doppler, tissue Doppler) CVie214 and CVie216 ​​were tested in a diabetic rat model. Briefly, rats were injected with streptozotocin (STZ). Seven to nine weeks after STZ injection, rats underwent transthoracic echocardiography and Doppler evaluation under urethane anesthesia. Two-dimensionally guided M-mode recordings were used to obtain short-axis measurements of left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and diastolic thickness of the posterior wall (PW) and septal wall (SW) according to the American Society of Echocardiography guidelines (Lang RM et al., Eur J Echocardiography 2006, 7:79-108). Left ventricular fractional shortening was calculated as FS = (LVEDD - LVESD) / LVEDD. Relative wall thickness was calculated as PWTd + IVSTd / LVEDD. Mitral inflow was measured at the mitral valve tip by pulsed Doppler on apical four-chamber tomography to obtain early and late filling velocities (E, A) and deceleration time of early filling velocities (DT). Deceleration gradient was calculated as the E / DT ratio. Mitral deceleration index was calculated as the DT / E ratio. Tissue Doppler imaging (TDI) was assessed on apical four-chamber tomography to record septal mitral annular motion, i.e., peak myocardial systolic velocity (s') and early and late diastolic velocity (e' and a').

[0488] After baseline hemodynamic measurements, rats were administered digoxin, CVie214, and CVie216 ​​and compared with controls. Digoxin, used as the reference drug, was infused intravenously at 0.11 mg / kg / min for 15 minutes, with echocardiographic parameters measured 1 hour later. CVie214-salt and CVie216-salt were infused intravenously at 0.2 mg / kg / min into STZ-diabetic rats, with echocardiographic parameters measured 15 and 30 minutes later.

[0489] Tables 4-6 show the hemodynamic parameters of digoxin, CVie214-salt, and CVie216-salt in STZ-diabetic rats. Data shown in Tables 4-6 are means ± SD, and values ​​marked with an asterisk are statistically significant at at least p<0.05.

[0490] The data indicate that the streptozotocin-induced diabetic rat model is characterized by diastolic dysfunction compared with healthy control rats (control, n = 18 rats; STZ, n = 20 rats) (Table 4). In particular, STZ rats showed increases in DT and DT / E and decreases in E, DT / E, e', and HR. CVie214-salt and CVie216-salt improved STZ-impaired diastolic function compared with controls (Table 4), demonstrating significant decreases in DT and DT / E and increases in E / DT and e' associated with improvements in SV and CO (Tables 5-6). E / e' significantly decreased 30 min after CVie216-salt infusion (Table 6). Only CVie214 modestly but significantly increased s and HR at 15 min (Table 5). Digoxin, used as a reference compound, improved diastolic function, decreased DT, DT / E, and increased E / DT, e', and systolic function (FS, s'), but did not affect global cardiac function such as SV and CO (Table 4). Table 4. Hemodynamic parameters in control and STZ-diabetic rats and the effect of IV infusion of digoxin in STZ rats [Table 4] FS (%): Left ventricular shortening fraction, systolic function. E (m / s): Early filling velocity of mitral inflow. A (m / s): Late filling velocity of mitral inflow. E / A: Index of LV function. DT (ms): Deceleration time of E wave. DT / E (s2 / m): Mitral decay index. E / DT (m / s2): Deceleration gradient. s' (cm / s) (TDI): Contraction velocity. e' (cm / s) (TDI): Early relaxation velocity. a' (cm / s) (TDI): Late relaxation velocity. E / e': LV filling pressure index. CO (ml / min): Cardiac output. HR (beats / min): Heart rate. SV (ml / beat): Stroke volume. *: At least p<0.05, control vs. STZ, or STZ plus drug vs. pre-STZ. Table 5. Hemodynamic parameters after IV infusion of CVie214-salt in STZ-diabetic rats [Table 5] Table 6. Hemodynamic parameters after IV infusion of CVie216-salt in STZ-diabetic rats [Table 6]

[0491] Receptor binding assay Binding of the radioligand to the panel of receptors was performed by Eurofin on crude membrane samples by using the appropriate reference standard (Eurofin, Taiwan, compound code CVie216-3 (1226840), study # TW04-0004235, quote # TW04-0004235-Q04, for Cvie Therapeutics Limited, Taiwan) according to published procedures. CVie216-salt was tested at a concentration of 10 μM. As shown in Table 7, no significant interactions were recorded with the panel of receptors. Table 7. Receptor binding assay of Cvie216-salt [Table 7-1] [Table 7-2] [Table 7-3] Note: bov = bovine; ham = hamster; hum = human; no items met the significance criteria of >50% stimulation or inhibition.

Claims

1. Formula (I) 【Chemistry 1】 [In the formula, X is selected from the group consisting of a carboxylic acid, a carboxylic acid ester, or a bioisoster thereof, a hydroxyl group, an ester, and an amine group, wherein the bioisoster consists of a sulfate, a sulfonic acid, a phosphate, a phosphonate, or a nitrogen-containing heterocyclic ring, wherein the amine group optionally comprises a primary amine, a secondary amine, or a cyclic amine; n is 1, 2, 3, 4, or 5; The dashed line at C3-C1′ represents an optional exocyclic double bond C═C at position C3-C1′; The dashed line at C2-C3 represents the optional endocyclic double bond C═C; Y at C6 is hydroxyl (OH) in the α- or β-configuration, or hydroxymethyl (CH ２ OH); Z at C7 is —H, or —OH in the α configuration, or a ketone, and the dashed line represents an optional carbonyl group (C═O) at Z. or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

2. 2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein X is selected from the group consisting of a carboxylic acid, a carboxylic acid ester, a primary amine, a secondary amine, and a cyclic amine.

3. 2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein X is a carboxylic acid or a carboxylic acid ester.

4. the below described: (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (E)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one; (Z)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxyandrostan-17-one; (E)-3-(4-aminobutyl)-6α-hydroxyandrost-2-en-17-one hydroiodide; 3-[2-(piperidin-4-yl)ethyl]-6α-hydroxyandrost-2-en-17-one hydroiodide; (EZ)-3-(4-aminobutylidene]-6α-hydroxyandrostan-17-one; (E)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one; (Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxyandrostan-17-one; 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxyandrostan-17-one; Ethyl (6α-hydroxy-17-ketoandrostan-3β-yl)acetate; 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid; 4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid; 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; Ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyrate; Ethyl 6-(6α-hydroxy-17-oxoandrostan-3-yl)caproate; 6-(6β-hydroxy-17-oxoandrostan-3-yl)caproic acid; (E,Z)-3-(5-N-methylaminopentylidene]-6α-hydroxymethylandrostane-7,17-dione; (E,Z)-3-[2-(pyrrolidin-3yl)ethylidene]-6α-hydroxymethylandrostane-7,17-dione; (E,Z)-3-[2-(azetidin-3-yl)ethylidene]-6α-hydroxymethylandrostane-7,17-dione; (E,Z)-3-[2-(piperidin-4-yl)ethylidene]-6α-hydroxymethylandrostane-7,17-dione; (E,Z)-3-(5-N-methylaminopentylidene)-6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(azetidin-3-yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione; 3β-[2-(azetidin-3-yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(pyrrolidin-3yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione; 3β-[2-(pyrrolidin-3yl)ethyl]6α-hydroxymethyl-7α-hydroxyandrostan-17-one; 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethylandrostane-7,17-dione; and, 3β-[2-(piperidin-4-yl)ethyl]-6α-hydroxymethyl-7α-hydroxyandrostan-17-one, 4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, selected from the group consisting of:

5. the below described: (E)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (Z)-4-(6α-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (E)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; (Z)-4-(6β-hydroxy-17-oxoandrostan-3-ylidene)butyric acid; Ethyl (6α-hydroxy-17-ketoandrostan-3β-yl)acetate; 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid; 4-(6β-hydroxy-17-oxoandrostan-3-yl)butyric acid; 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid; Ethyl 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyrate; Ethyl 6-(6α-hydroxy-17-oxoandrostan-3-yl)caproate; and, 6-(6β-hydroxy-17-oxoandrostan-3-yl)caproic acid, 4. The compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, selected from the group consisting of:

6. 2. The compound of claim 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, selected from the group consisting of 4-(6α-hydroxy-17-oxoandrostan-3-yl)butyric acid and 2-(6β-hydroxy-17-oxoandrostan-3-yl)acetic acid.

7. 7. The compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the pharmaceutically acceptable salt is selected from chloride, bromide, sulfate, phosphate, nitrate, fumarate, succinate, oxalate, malate, tartrate, maleate, citrate, methanesulfate, and benzoate.

8. A pharmaceutical composition comprising one or more of the compounds according to any one of claims 1 to 7, or pharmaceutically acceptable salts, solvates, or hydrates thereof, in combination with at least one pharmaceutically acceptable vehicle and / or excipient.

9. 10. A pharmaceutical composition for treating heart failure comprising one or more of the compounds according to any one of claims 1 to 7, or pharmaceutically acceptable salts, solvates, or hydrates thereof, in combination with at least one pharmaceutically acceptable vehicle and / or excipient.

10. 10. The pharmaceutical composition of claim 8 or 9, formulated for intravenous injection, intramuscular injection, enteral administration, parenteral administration, or inhalation.

11. 10. The pharmaceutical composition of claim 8 or 9, formulated for oral administration.

12. 12. The pharmaceutical composition of any one of claims 8 to 11, administered at a dose of 1 mg / kg to 20 mg / kg, optionally wherein the dose may be 1 mg / kg to 10 mg / kg.

13. ACE inhibitors, AIRB, diuretics, Ca ２＋ further comprising one or more additional therapeutically active ingredients selected from the group consisting of channel blockers, beta-blockers, digitalis, NO donors, vasodilators, SERCA2a stimulators, neprilysin (NEP) inhibitors, myosin filament activators, recombinant relaxin-2 mediators, recombinant NP protein, activators of soluble guanylate cyclase (sGC), and beta-arrestin ligands for the angiotensin II receptor; wherein a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is administered simultaneously with, or before or after, said one or more additional therapeutically active ingredients. The pharmaceutical composition according to any one of claims 8 to 12.

14. Use of a compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, for the manufacture of a medicament for treating heart failure.

15. One or more additional therapeutically active ingredients are used in combination with the compound of any one of claims 1 to 7, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, wherein the one or more additional therapeutically active ingredients are selected from the group consisting of an ACE inhibitor, an AIRB, a diuretic, a Ca ２＋ channel blockers, beta-blockers, digitalis, NO donors, vasodilators, SERCA2a stimulators, neprilysin (NEP) inhibitors, myosin filament activators, recombinant relaxin-2 mediators, recombinant NP protein, activators of soluble guanylate cyclase (sGC), and beta-arrestin ligands of the angiotensin II receptor, 15. The use according to claim 14.

16. 10. The method of claim 1, further comprising administering to said patient a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, in combination with one or more additional therapeutically active ingredients, wherein the additional therapeutically active ingredients are selected from the group consisting of an ACE inhibitor, an AIRB, a diuretic, a Ca ２＋ A therapeutic agent for heart failure, which is one or more selected from the group consisting of a channel blocker, a β-blocker, digitalis, an NO donor, a vasodilator, a SERCA2a stimulator, a neprilysin (NEP) inhibitor, a myosin filament activator, a recombinant relaxin-2 mediator, a recombinant NP protein, an activator of soluble guanylate cyclase (sGC), and a β-arrestin ligand for angiotensin II receptor; Therapeutic agents wherein said compound, or a pharmaceutically acceptable salt, solvate, or hydrate thereof, is administered simultaneously with, or before or after, said additional therapeutically active ingredient.

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