Benzodihydropyranol compounds used to treat heart failure

Abstract

This invention relates to certain benzodihydropyranol, quinone, or hydroquinone compounds and their derivatives for the treatment of heart failure with reduced ejection fraction (HFrEF). Specifically, this invention relates to benzodihydropyranol compounds selected from S-(6-hydroxy-2,5,7,8-tetramethyl-2-yl)(piperazin-1-yl) methyl ketone and S-(6-hydroxy-2,5,7,8-tetramethyl-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl) methyl ketone, and their pharmaceutically acceptable salts.

Description

I. Technical Field

[0001] This invention relates to benzodihydropyranol compounds and their derivatives for the treatment or prevention of heart failure with reduced ejection fraction (HFrEF). II. Background Technology

[0002] Heart failure is a clinical diagnosis characterized by symptoms and signs including shortness of breath, fatigue, and elevated venous pressure, which are caused by significant cardiac dysfunction (Pearse and Cowie 2014).

[0003] There are different types of heart failure, which are generally classified as heart failure with reduced ejection fraction (HFrEF), heart failure with preserved ejection fraction (HFpEF), and congestive heart failure. These conditions are typically treated differently, and medications effective for one type of heart failure are usually not suitable for other types.

[0004] HFrEF is quantified by referring to left ventricular ejection fraction (LVEF), which is usually derived from echocardiography. Values ​​above 50%-60% are considered normal. Patients with heart failure with preserved ejection fraction also show values ​​≥50%. Values ​​below 40% are considered reduced LVEF (HFrEF), and patients with LVEF in the 40%-49% range are in the 'grey zone,' which is defined as heart failure with mildly reduced ejection fraction (HFmrEF). Patients with HFmrEF are most likely to have mild systolic dysfunction but also feature diastolic dysfunction.

[0005] It is important to differentiate patients with HF based on LVEF due to different underlying causes, demographics, comorbidities, and responses to treatment.

[0006] Asymptomatic structural or functional cardiac abnormalities (such as left ventricular (LV) systolic or diastolic dysfunction) are precursors to heart failure. However, abnormalities of valves, pericardium, endocardium, heart rhythm, and conduction can also lead to heart failure (and usually more than one abnormality is present).

[0007] Patients with heart failure and reduced ejection fraction have left ventricular systolic dysfunction, often accompanied by diastolic dysfunction. (Yancy et al., JACC, Vol. 62, No. 16, 2013)

[0008] Identifying the underlying cardiac problem causing heart failure is crucial for treatment, as precise pathology determines the specific treatment to be used (e.g., valve repair or replacement for valvular disease, specific drug treatment for HF with reduced EF, reducing heart rate in tachycardia-induced cardiomyopathy, etc.). (ESC Guidelines 2016).

[0009] WO2020 / 096862 describes a cardiac device for remodeling the ventricles, the device including a force-distributing means configured to extend from a first ventricular wall to a second ventricular wall and a first plurality of anchoring means configured to fix the force-distributing means to a first region of tissue at the first ventricular wall as a percutaneous treatment for heart failure with reduced ejection fraction.

[0010] RU2422136 discloses a method for treating chronic heart failure with reduced left ventricular ejection fraction using β-blockers, diuretics, recombinant human interleukin, and ACE inhibitors.

[0011] Although these methods are available, new methods or compounds are still needed to treat heart failure with reduced ejection fraction.

[0012] One object of the present invention is to provide compounds for the treatment or prevention of heart failure with reduced ejection fraction (HFmrEF or HFrEF). III. Summary of the Invention

[0013] The above objectives are achieved by providing certain benzodihydropyranol, quinone, or hydroquinone compounds for such treatments.

[0014] The above objective is achieved by the present invention by providing compounds of formula (I) and (II), hydroquinone analogs of formula (II) or pharmaceutically acceptable salts thereof for the treatment or prevention of heart failure with reduced ejection fraction;

[0015]

[0016] - Where R1 represents hydrogen or a prodrug fraction that can be removed from living tissue.

[0017] -and among them

[0018] ○R2 and R3 together with the N atom to which they are attached form a saturated or unsaturated, non-aromatic, optionally substituted 5-8 membered ring having one to four N, O or S atoms, wherein R2 and R3 together contain 3-12 carbon atoms;

[0019] ○ Alternatively, R2 is a hydrogen atom or an alkyl group having 1-6 carbon atoms, and R3 is an alkyl group optionally substituted with nitrogen or oxygen, wherein the alkyl group contains 3-12 carbon atoms, the alkyl group in R3 contains one or more non-aromatic cyclic structures capable of containing nitrogen or oxygen atoms in the ring and capable of containing straight-chain and / or branched substituents, and one or more olefinic unsaturated groups.

[0020] For the purposes of this invention, compounds according to formula (II) include hydroquinone (i.e., hydroquinone) analogs, although quinone derivatives are preferred in consideration of stability.

[0021] The compound of formula II is one of the metabolites of the compound of formula I. Therefore, the compound of formula I is a prodrug of the compound of formula II, wherein both...

[0022] In a preferred embodiment, nitrogen can be an amine, quaternary ammonium, guanidine, or imine, and oxygen can be a hydroxyl, carbonyl, or carboxylic acid; and / or oxygen and nitrogen can together form an amide, urea, or carbamate group.

[0023] In a preferred embodiment, R1 in formula (I) is hydrogen or an ester group having 2-6 carbon atoms formed together with the 6-oxygen.

[0024] In a preferred embodiment of the compound according to formula (I) or formula (II), R2 and R3 together with the N atom to which they are attached form a saturated ring containing additional N atoms, said ring being unsubstituted or substituted with an alcohol or an alkanolic group having 1-4 carbon atoms, such as hydroxyethyl.

[0025] In another preferred embodiment, R2 is a hydrogen atom and R3 comprises a saturated cyclic structure having 4-7 carbon atoms and one nitrogen atom, the ring being substituted by an alkyl group, an alcohol group or a group having 1-4 carbon atoms that may contain an oxygen, carboxylic acid or amine group.

[0026] In another preferred embodiment, the compound is a compound according to Formula II, and R2 is a hydrogen atom, and R3 comprises a cyclic structure having 4-6 carbon atoms and having a nitrogen atom, the ring being unsubstituted or substituted with an alcohol or an alkanol group having 1-4 carbon atoms such as hydroxyethyl, and preferably optionally substituted with methyl, ethyl or alcohol-substituted methyl or ethyl.

[0027] In another preferred embodiment, the compound is a compound according to Formula I, R2 is a hydrogen atom, and R3 comprises a saturated cyclic structure having 4-7 carbon atoms and having one nitrogen atom, the ring being unsubstituted or substituted with an alcohol or an alkanolic group having 1-4 carbon atoms such as hydroxyethyl, and preferably optionally substituted with methyl, ethyl or alcohol-substituted methyl or ethyl.

[0028] According to another preferred embodiment, the compound is (6-hydroxy-2,5,7,8-tetramethyl-2-yl)(piperazin-1-yl) methyl ketone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidin-3-yl)-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethyl-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl) methyl ketone (SUL-109), or a pharmaceutically acceptable salt thereof, as a racemic mixture or as one of its enantiomers.

[0029] Although the S enantiomer is effective, in the most preferred embodiment, the compound is the (2R)-enantiomer of SUL-121, namely (2R)-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl)methyl ketone (SUL-150) or a pharmaceutically acceptable salt thereof, because the R enantiomer appears to be even more effective.

[0030] In a preferred embodiment of the invention, the compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da.

[0031] In a preferred embodiment of the invention, the compound according to formula (I) or formula (II) is used to treat or prevent heart failure with reduced ejection fraction (HFmrEF), wherein the ejection fraction is reduced to 50% or less, and even more preferably, the compound according to formula (I) or formula (II) is used to treat or prevent heart failure with reduced ejection fraction (HFrEF), wherein the ejection fraction is reduced to 40% or less. IV. Description of the attached drawings

[0032] Figure 1 This indicates that SUL-150 administration at least partially precludes the development of heart failure with reduced ejection fraction. Long-term doxorubicin administration in rats leads to heart failure with reduced ejection fraction, characterized by decreases in (A) heart rate, (B) ejection fraction, (C) stroke work, and (D) cardiac output. Prophylactic or therapeutic oral administration of SUL-150 precludes at least some of these changes in cardiac function. *p<0.05 relative to the sham treatment group. The relative value of Doxorubic star / medium is p<0.05.

[0033] Figure 2The results indicate that EGFP synthesis is associated with H9C2 cardiomyocyte size and protein synthesis. (A) Dyspamine increases H9C2 cardiomyocyte surface area and (B) EGFP expression in a dose-dependent manner. (C) H9C2 cardiomyocyte surface area is associated with EGFP expression. (D) Dyspamine increases protein synthesis in H9C2 cardiomyocytes in a dose-dependent manner. (E) H9C2 cardiomyocyte protein synthesis is associated with EGFP expression. (F) Dyspamine induces EGFP expression in H9C2 cardiomyocytes compared to control cardiomyocytes treated with the medium. The protein synthesis inhibitor brevidin A reduces EGFP expression in H9C2 cardiomyocytes.

[0034] Figure 3 This indicates that the SUL compound inhibits the norepinephrine-induced hypertrophic response in rat H9C2 cardiomyocytes. Norepinephrine dose-dependently induces EGFP expression in H9C2 cardiomyocytes (grey lines, Figures A-H). Pre-incubation with 30 μM (A) SUL-11, (B) SUL-99, (C) SUL-127, (D) SUL-13, (E) SUL-138, or (F) the primary metabolite of SUL-138, SUL-138M2, (G) SUL-150, or (H) SUL-151 reduces EGFP expression in cardiomyocytes.

[0035] Figure 4 This indicates that SUL-150 administration excluded doxorubicin-induced cardiac fibrosis. Long-term doxorubicin administration in rats leads to fibrosis, characterized by increased collagen deposition between cardiomyocytes. Prophylactic or therapeutic oral administration of SUL-150 excluded cardiac fibrosis resulting in lower collagen content in cardiac tissue.

[0036] Figure 5 This indicates that SUL-150 administration reduces cardiac oxidative stress. Long-term doxorubicin administration induces cardiac oxidative stress, as indicated by increased lipid peroxidation products (TBARS, A). Oral administration of SUL-150 in prophylactic or therapeutic regimens excluded the induction of cardiac oxidative stress and maintained lipid peroxidation product levels at baseline (A). Since oxidative stress can be caused by increased free radical production or decreased free radical scavenging activity, cardiac free radical scavenging activity was investigated (B). Neither doxorubicin nor SUL-150 administration altered cardiac free radical scavenging activity.

[0037] Figure 6SUL-150 administration indicates that it maintains cardiac energy levels and mitochondrial copy number. Long-term doxorubicin administration depletes cardiac ATP levels (normalized against ADP), suggesting mitochondrial dysfunction (A). Prophylactic or therapeutic administration of SUL-150 reduces cardiac energy loss. Long-term doxorubicin administration reduces cardiac mtDNA copy number (B), which may underlie ATP loss. SUL-150 alleviates the reduction in mtDNA copy number. In doxorubicin-treated rats, mtDNA copy number correlated with cardiac ejection fraction (EF), indicating that maintaining cardiac mitochondrial quality is fundamental to improved myocardial contractility (C).

[0038] Figure 7 This indicates that SUL-150 maintains the activity of respiratory complex IV under doxorubicin stress. H9C2 cardiac myoblasts were exposed to doxorubicin (1 μM) for 24 hours, after which their mitochondria were isolated and complex IV activity was assessed. Doxorubicin exposure reduced complex IV activity, which was mitigated by co-treatment of H9C2 cardiac myoblasts with SUL-150. V. Detailed Implementation

[0039] The object of the present invention is achieved by providing compounds for the treatment or prevention of heart failure with reduced ejection fraction (HFrEF) as shown above, or pharmaceutically acceptable salts thereof, for the treatment or prevention of HFrEF.

[0040] Treatment or prevention with benzodihydropyranol, quinone or hydroquinone compounds according to the invention is preferably part of a combination therapy having one or more other commonly used measures for treating heart failure.

[0041] R1 can be a substituent that is easily removed from the human body, making the compound a prodrug. R1 can be, for example, an amino acid derivative or an ester derivative, and typically has a molecular weight of less than 100 Daltons.

[0042] In a preferred embodiment, R1 in formula (I) is hydrogen or an ester group having 2-6 carbon atoms formed together with the 6-oxygen. The ester may contain one or more ether or alcohol groups. Suitable esters are acetates, butyrates, 3-hydroxybutyrates, etc.

[0043] In a preferred embodiment of the compound according to formula (I) or formula (II), R2 and R3 together with the N atom to which they are attached form a saturated ring having 3-6 carbon atoms and containing an additional N atom, the saturated ring being substituted with 1-4 carbon atoms that may contain oxygen, carboxylic acid or amine groups.

[0044] More preferably, R2 and R3 together with the N atom to which they are attached form a 5-7 membered ring containing an additional amine group, which is optionally substituted with methyl, ethyl or alcohol.

[0045] In another preferred embodiment, R2 is a hydrogen atom and R3 comprises a cyclic structure having 3-6 carbon atoms and one nitrogen atom.

[0046] More preferably, R2 is a hydrogen atom, and R3 comprises a 5-7 membered ring containing an additional amine group attached to the amide-nitrogen, and the ring is optionally substituted with a methyl or ethyl group that is substituted with a methyl, ethyl, or alcohol.

[0047] In either case, the ring (a cyclic structure formed by R2 and R3, or a cyclic structure with only R3) may be unsubstituted or substituted with an alkyl, alcohol, or alkanol group (such as hydroxyethyl) having 1-4 carbon atoms.

[0048] In a preferred embodiment of the invention, the compound according to formula (I) or formula (II) has a molecular weight of less than 500 Da.

[0049] In a preferred embodiment, the compound used according to the invention is a benzodihydropyranol compound according to formula I.

[0050] Certain benzodihydropyranol compounds have been described in WO2014 / 098586. The compounds described in detail have abbreviations, see SUL-XXX (XXX is a 2 or 3-digit number). Many of these compounds are racemic mixtures, although some enantiomers have also been tested. Suitable methods for preparing the benzodihydropyranol compounds according to the invention are described in WO2014 / 098586 or WO2014 / 011047.

[0051] WO 2017 / 060432 A1 discloses amide derivatives of 2-hydroxy-2-methyl-4-(3,5,6-trimethyl-1,4-benzoquinone-2-yl)-butyric acid and methods for preparing such compounds.

[0052] Hydrogenated quinone derivatives can be readily prepared by hydrogenation of the quinone structure.

[0053] According to another preferred embodiment, the compound is (6-hydroxy-2,5,7,8-tetramethyl-2-yl)(piperazin-1-yl) methyl ketone (SUL-121), ((S)-6-hydroxy-2,5,7,8-tetramethyl-N-((R)-piperidin-3-yl)-2-carboxamide hydrochloride (SUL-13), or (6-hydroxy-2,5,7,8-tetramethyl-2-yl)(4-(2-hydroxyethyl)piperazin-1-yl) methyl ketone (SUL-109), or a pharmaceutically acceptable salt thereof, as a racemic mixture or as one of its enantiomers.

[0054] In the most preferred embodiment, the compound is the R-enantiomer of SUL-121, namely R-(6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl) methyl ketone (SUL-150) or a pharmaceutically acceptable salt thereof.

[0055] The counterion in a pharmaceutically acceptable salt can be a counterion known in the art. Preferably, the compound has at least one basic nitrogen atom, which is a protonable amine. The counterion is preferably a halogen such as chloride, sulfate, citrate, formate, etc., and most preferably chloride.

[0056] The compound is effective as a racemic mixture or in substantially pure enantiomeric form. The compound has one or more chiral centers, typically one or two.

[0057] Preferably, the compound is substantially enantiomerically pure. Substantially enantiomerically pure means an enantiomer excess of about 95% or higher, more preferably about 98% or higher, and most preferably about 99% or higher. These amounts also apply when the compound contains more than one chiral center.

[0058] The compound is preferably used in an effective amount to achieve the treatment or prevention of HFrEF, where the ejection fraction is about 40% or less.

[0059] Treatment or prevention of heart failure includes improving symptoms and / or reducing the progression of heart failure, including improving cardiac functions such as heart rate and cardiac output.

[0060] Preferably, the compound according to the invention is used for the treatment or prevention of HFrEF in mammalian organs, wherein the mammal is preferably human.

[0061] HFrEF is quantified by referring to left ventricular ejection fraction (LVEF), which is usually derived from echocardiography. Values ​​above 50%-60% are considered normal. Patients with heart failure with preserved ejection fraction also show values ​​≥50%. Values ​​below 40% are considered reduced LVEF (HFrEF), and patients with LVEF in the 40%-49% range are in the 'grey zone,' which is defined as heart failure with mildly reduced ejection fraction (HFmrEF). Patients with HFmrEF are most likely to have mild systolic dysfunction but also feature diastolic dysfunction.

[0062] This invention provides compounds for treating HFrEF with a retained ejection fraction of 50% or less (HFmrEF) and about 40% or less (HFrEF). Preferably, the compounds for treatment are used to treat HFrEF with a retained ejection fraction of about 40% or less.

[0063] Effects are typically observed in bodily fluids at amounts of about 1 μM, but higher amounts are preferred. Preferred amounts are about 10 μM or higher, more preferably about 20 μM or higher in vivo or in vitro concentrations. Generally, concentrations of about 200 μM or lower should be sufficient and safe in humans.

[0064] For human use, this would mean (assuming a 30L distribution volume, 100% availability, and a concentration of about 1 μM) a dose of about 10 mg or more. A preferred amount would produce a concentration of about 10 μM, for which a dose of about 100 mg or more would be suitable. Therefore, preferably, a dosage form of about 20 mg or more, preferably 50 mg or more, and preferably 100 mg or more is suitable.

[0065] Typically, solid oral dosage forms contain up to about 500 mg of the compound, preferably about 450 mg or less, to allow for excipients.

[0066] For parenteral administration, such as intravenous administration, or for other forms of fluid administration, larger doses may be administered.

[0067] Examples of usable dosages are effective amounts of the compounds of the invention at a dose of 0.2 mg / kg or higher, such as preferably in the range of about 1 mg / kg to about 100 mg / kg, or about 2 mg / kg to about 40 mg / kg body weight, or about 3 mg / kg to about 30 mg / kg body weight, or about 4 mg / kg to about 15 mg / kg body weight. The compounds of the invention can be administered in a single daily dose, or the total daily dose can be administered in divided doses two, three, or four times daily.

[0068] The compounds described herein can be formulated into pharmaceutical compositions by combining them with additives such as pharmaceutically or physiologically acceptable excipients, carriers, and mediators.

[0069] Suitable pharmaceutically or physiologically acceptable excipients, carriers, and mediators include processing agents and drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, cyclodextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextran, hydroxypropyl-P-cyclodextrin, polyvinylpyrrolidone, low-melting-point waxes, and any combination of two or more of them. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991).

[0070] The pharmaceutical composition preferably comprises a unit dose formulation, wherein the unit dose is a dose sufficient to have a therapeutic effect. The unit dose may be a dose administered periodically during the treatment or suppression of a condition.

[0071] The compounds of the present invention can be administered in dose-unit formulations containing desired, conventionally non-toxic, pharmaceutically or physiologically acceptable carriers, adjuvants, and mediators via enteral, oral, parenteral, sublingual, inhalation (e.g., as a nebulizer or spray), rectal, or topical administration. As used herein, parenteral administration includes subcutaneous, intravenous, intramuscular, intratarsal injection, or infusion techniques. The compounds are mixed with pharmaceutically acceptable carriers, adjuvants, and mediators suitable for the desired route of administration.

[0072] Oral administration is generally the preferred route of administration, and formulations suitable for oral administration are preferred formulations.

[0073] The compounds described herein may be administered in solid, liquid, or aerosol form, or in the form of tablets, pills, powder mixtures, capsules, granules, injections, creams, solutions, suppositories, enemas, colonic lavage solutions, emulsions, dispersants, food premixes, and other suitable forms. The compounds may also be administered in liposome formulations.

[0074] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions in non-toxic, non-enteric-acceptable diluents or solvents, such as solutions in propylene glycol. Acceptable media and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can be used in the preparation of injections.

[0075] Suppositories for rectal administration of drugs can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycol, which is solid at room temperature but liquid at rectal temperature, and thus melts and releases the drug in the rectum.

[0076] Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be mixed with at least one inert diluent such as sucrose, lactose, or starch. These dosage forms may also contain substances other than inert diluents, such as lubricants like magnesium stearate. In the case of capsules, tablets, and pills, the dosage form may also contain a buffer. Tablets and pills may also be prepared using enteric coating.

[0077] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, cyclodextrins, as well as sweeteners, flavoring agents, and aromatizers.

[0078] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the carrier and specific mode of administration of the active ingredient. Typically, a selected unit dose is prepared and administered to provide a defined final drug concentration in the blood, tissue, organ, or other target area of ​​the body. The effective amount for a given situation can be readily determined through routine experiments and is within the skill and judgment of an average clinician or technician.

[0079] The present invention will be further illustrated using the following embodiments. In these embodiments, reference is made to the accompanying drawings.

[0080] VI. Examples

[0081] Example 1

[0082] The efficacy of the compounds according to the invention for the treatment or prevention of HFrEF was tested in rats. Doxorubicin (an anthracycline antibiotic used in cancer chemotherapy) was used to induce heart failure with reduced ejection fraction in preclinical rat models (Christiansen et al., (2006) Eur. J. Cardiothorac. Surg. 30: 611-616; Ertunc et al., (2009) Pharmacology 84: 240-248; Hayward et al., (2007) J. Am. Ass. Lab. Animal Sci. 46: 20-32).

[0083] experiment

[0084] SUL-150 (250 mg) was dissolved in 100% EtOH (1.02 ml) and further diluted in distilled water (68.7 ml) to obtain a clear 10 mM solution. The SUL-150 solution was evenly sprayed onto standard food pellets (1.25 kg). The food pellets were air-dried overnight. Fresh food pellets were prepared weekly. The food pellets contained approximately 200 mg / kg of SUL-150, and the daily dietary intake of a 350 g rat was 25 g, resulting in a daily dose of approximately 5 mg of SUL-150.

[0085] Male outbred Wistar rats (10-12 weeks old) weighing 250-300 g were kept in a 12-hour light / dark cycle and given a standard diet containing distilled water at will. Thirty rats were administered doxorubicin (2 mg / kg) weekly via intraperitoneal injection for 9 weeks without treatment (standard diet, 10 rats), or received a diet supplemented with SUL-150 as a preventative measure (before the first doxorubicin administration, 10 rats) or as a treatment (starting after the sixth doxorubicin administration, 10 rats). A sham control group (8 rats) was administered saline weekly via intraperitoneal injection for 9 weeks and was given a standard diet. Cardiac function was assessed in all rats 12 weeks after the first doxorubicin administration.

[0086] Cardiac function tests (PV loop assessment) were performed under anesthesia (O2 containing isoflurane (FiO2 100%)), followed by endotracheal intubation. Rats were placed on a mechanical rodent ventilator (Harvard Apparatus, Holliston, Massachusetts) with a tidal volume of 10 mL / kg and a respiratory rate of 70 breaths per minute. A small window was opened below the sternum, and a 2F microtip pressure conduction catheter (SPR-838; Millar Instruments, Houston, Tx) was inserted directly into the left ventricle (LV) through a small incision. After stabilization, the signal was recorded using a pressure-volume conductance system (MPVSSUL-Ultra, Millar Instruments, Houston, Tx) connected to a data acquisition system (PowerLab, AD Instruments, Colorado Springs, Co).

[0087] Under steady-state conditions, mean arterial pressure (MAP), LV stroke work (LVSW), stroke volume (SV), LV end-diastolic volume (LVEDV), LV end-systolic pressure (LVESP), the ratio between mean arterial pressure and LV end-systolic pressure (MAP-LVESP), LV ejection fraction (LVEF), heart rate (HR), the maximum slope of LV systolic pressure increment (dP / dt max), and the time constant of LV pressure decay (tau) were obtained.

[0088] For preload actions, the inferior vena cava is compressed and data are collected to assess preload-compensated stroke work (PRSW), dP / dt-end-diastolic volume relationship (dP / dt-EDV), and the slopes of the end-systolic and end-diastolic PV relationships (ESPVR and EDPVR).

[0089] Volume calibration was performed using fresh heparinized warm blood from each animal. Additionally, 50 mL of 7.5% hypertonic saline was injected at the end of each experiment for parallel conductivity-volume calibration.

[0090] Data were analyzed in GraphPad Prism 8.0 (GraphPad Software Inc, Ca). All results are expressed as mean ± SD (standard deviation) or median and interquartile range. Differences between groups were assessed by ANOVA, followed by paired comparisons with the sham treatment group and the carboxyl treatment group. Multiple comparisons were performed using FDR-corrected p-values.

[0091] result

[0092] Figure 1This indicates that long-term doxorubicin administration in rats leads to the development of heart failure, as indicated by decreases in heart rate, ejection fraction, and stroke work, ultimately resulting in a severe reduction in cardiac output (*indicating p<0.05 relative to the sham treatment group). Figure 1 Further evidence indicates that administration of SUL-150 via food particles prior to doxorubicin administration (prevention group) essentially preserved cardiac function and largely maintained ejection fraction and cardiac output. The indication was p<0.05 relative to the doxorubicin treatment group. Compared with the non-treatment group (p<0.05 compared with the doxorubicin treatment group), administration of SUL-150 via food particles (treatment group) after 6 cumulative doxorubicin administrations significantly improved cardiac function, and the parameters were not different from those obtained in the prevention group, demonstrating that SUL-150 is a therapeutic agent for heart failure.

[0093] Table 1 shows that doxorubicin-induced heart failure manifests as heart failure with reduced ejection fraction (HFrEF), characterized by decreased systolic and diastolic capacity, indicating remodeling or fibrosis within cardiac tissue.

[0094] The application of SUL-150 in preventive or therapeutic programs to maintain systolic capacity (i.e., dP / dTmax, systolic efficiency) and diastolic capacity (i.e., Tau) does not wish to be bound by theory, suggesting that SUL-150 inhibits cardiac fiber development or increases systolic energy.

[0095] in conclusion

[0096] Doxorubicin-induced heart failure is characterized by reduced cardiac contractility and consequently decreased cardiac output. Treatment with 6-benzodihydropyranol SUL-150 significantly improves cardiac contractility and thus cardiac output.

[0097]

[0098]

[0099] Example 2

[0100] Example 2 demonstrates in in vitro experiments that several different compounds according to the present invention have improved efficacy compared to Trolox and some other Trolox derivatives not according to the present invention.

[0101] Ventricular hypertrophy is a severe exacerbation of hemodynamic stress conditions such as hypertension and valvular disease, and it is directly associated with heart failure with reduced ejection fraction (HFrEF).

[0102] Ventricular hypertrophy in heart failure can be attributed to increased cardiac mass and asymmetric thickening of the interventricular septum. Histological markers include increased cardiomyocyte size (i.e., cardiomyocyte hypertrophy), myocyte disorganization (myofibril disorganization), and perivascular and interstitial fibrosis, which are common phenotypes in many cardiomyopathy cases.

[0103] Pathological cardiomyocyte hypertrophy is characterized by a shift in gene expression profiles, including the replacement of the adult major α-isotype (MHC-α) with fetal cardiac myosin heavy chain-β (MHC-β), the upregulation of skeletal α-actin (SKA), and atrial natriuretic factor (ANF) genes. Additionally, the shift in cardiomyocytes to a carbohydrate-dependent energy mechanism rather than fatty acid oxidation necessitates altered expression levels of metabolic genes. Interestingly, both pathophysiological adaptations are associated with increased transcriptional activity downstream of CREB, JNK, NFκB, and NFAT.

[0104] In vitro assays for studying cardiomyocyte hypertrophy and screening putative inhibitors of cardiomyocyte hypertrophy combine well-characterized hypertrophy inducers (e.g., phenylephrine or IL-6) with transcriptomic screening of typical genes upregulated during cardiomyocyte hypertrophy and quantification of cardiomyocyte surface area. However, these assays are laborious, time-consuming, and impractical for rapidly screening a larger group of small molecules.

[0105] An alternative method for indirectly quantifying protein synthesis and cardiomyocyte size is the quantification of enhanced green fluorescent protein (EGFP) expression under the control of standard cytomegalovirus (CMV) enhancer / promoter elements [Vettel, 2012]. The CMV promoter contains multiple functional binding sites for CREB, NFκB, and NFAT. Therefore, in vitro cardiomyocyte expression of EGFP can serve as a high-throughput platform for screening small molecules that inhibit cardiomyocyte hypertrophy [Vettel, 2012], and may have clinical efficacy in the treatment of heart failure.

[0106] The following compounds were tested (Table 2). Table 2

[0107]

[0108]

[0109] Experimental Design

[0110] H9C2 cardiomyocyte culture and differentiation

[0111] Rat H9C2 cardiac myoblasts (ATCC CRL-1446) were maintained in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (Sigma-Aldrich) and passaged when the cultures reached 70% confluence. Prior to experiments, H9C2 myoblasts were differentiated into cardiomyocytes by serum reduction (to 1%) and stimulation with 20 nM retinoic acid for 5 days. For all experiments, a concentration of 0.6·10⁻⁶ was used. 5 cells / cm 2 Inoculate differentiated H9C2 cardiomyocytes.

[0112] Cell size determination

[0113] H9C2 cardiomyocytes were starved of serum for 24 hours, and then exposed to different concentrations of phenylephrine (dose range 2.10). -5 Up to 1.10 -11 M) 24h. Wash cells in ice-cold PBS and fix them in PBS containing 2% paraformaldehyde for 10 min at room temperature. Incubate the fixed cells with 5 μM rhodamine-conjugated phalloidin (ThermoFisher Scientific) and wash thoroughly with PBS. Take random fluorescence images using a Zeiss AxioObserver Z1 microscope and analyze cell size using CellProfiler software [McQuin, 2018].

[0114] Quantification of EGFP synthesis as a substitute for cell size.

[0115] H9C2 cardiomyocytes (0.6·10) 5 cells / cm 2 H9C2 cardiomyocytes were infected with CMV-copGPF (MOI 10) in serum-free medium for 24 h. The cells were then pre-incubated with 30 μM SUL compound under standard culture conditions for 30 min, followed by inoculation with phenylephrine (dose range 2.10). -5 Up to 1.10 -11 M) Restimulated for 24 h. Then, H9C2 cardiomyocytes were lysed in 100 μl of soft lysis buffer per well (25 mM Tris, 2 mM dithiothreitol, 2 mM EDTA, 1% Triton X-100, pH 7.4), and the fluorescence intensity of the obtained supernatant was recorded in a CLARIOStar Plus plate reader (BMG Labtech) equipped with a FITC filter bank (excitation 488 nm, 10 nm bandwidth; emission 515, with 20 nm bandwidth).

[0116] Statistical assessment

[0117] All experiments were performed in triplicate under each condition and the average was taken. Data from two separate experiments were used for evaluation in GraphPad Prism 8.0 (GraphPad Software Inc, Ca). Mean H9C2 cardiomyocyte size was correlated with mean EGFP level using linear regression. Phenylephrine-induced EGFP synthesis was normalized using a baseline level of 0% and a maximum EGFP record of 100%. All datasets were normalized to mediator controls. A 4-parameter nonlinear regression was used to determine the efficacy of phenylephrine-induced GFP synthesis. The efficacy of the SUL compound in inhibiting phenylephrine-induced EGFP synthesis was calculated as 100% - E. max Emax is the maximum effect caused by norepinephrine.

[0118] result

[0119] EGFP synthesis is associated with H9C2 cardiomyocyte hypertrophy.

[0120] According to the established protocol, H9C2 cardiac myoblasts were differentiated into cardiomyocytes, and an increased dose of phenylephrine (1.10 g / L) was used. -11 Up to 2.10 -5 M) stimulation to induce cardiomyocyte hypertrophy for 24 hours. Dose-dependent phenylephrine (ECG) 50 It is 7.4·10 -10 The surface area of ​​H9C2 cardiomyocytes was 791 ± 263 μm compared to that of control cardiomyocytes treated with the medium. 2 Increase exposure to 2.10 -5 3278±296 μm of methyl-D-phenylephrine in cardiomyocytes 2 ( Figure 2 A). Similarly, phenylephrine dose-dependently increased EGFP fluorescence (ECG) in H9C2 cardiomyocytes transformed with CMV-EGFP lentiviral particles. 50 5.2·10 -9 M)( Figure 2 B). Increased surface area of ​​H9C2 cardiomyocytes is associated with EGFP expression (R). 2 The value was 0.5601, p < 0.001; Figure 2 C). Similar to an increase in cell surface area, phenylephrine dose-dependently increases protein synthesis in H9C2 cardiomyocytes (EC). 50 It is 5.5·10 -10 ; Figure 2 D), which is also associated with increased EGFP fluorescence (R). 2 The value was 0.5734, p < 0.0001; Figure 2E). The addition of brevidin A, a broadly effective protein synthesis inhibitor, inhibited the increase in EGFP fluorescence induced by norepinephrine. Figure 2 F) indicates that EGFP is newly synthesized after stimulation with norepinephrine. This verifies the experimental setup.

[0121] Sul compounds inhibit the hypertrophic response of H9C2 cardiomyocytes.

[0122] Dose-dependent induction of EGFP expression in H9C2 cardiomyocytes by phenylephrine (P-phenylephrine) Figure 3 (AH, gray line). Pre-culture of H9C2 cardiomyocytes with 30 μM of SUL-11, SUL-99, SUL-127, SUL-13, SUL-138, SUL-138M2, SUL-150, or SUL-151 reduced norepinephrine-induced EGFP expression. However, the compounds according to the invention (SUL-13, SUL-138, SUL-150, or SUL-151) clearly showed increased potency and efficacy (Table 2). Notably, the primary metabolite of SUL-138, SUL-138M2, exhibited inhibitory efficacy against SUL-138, suggesting that SUL-138 could serve as a prodrug in this experiment. Furthermore, the R-enantiomers and S-enantiomers of (6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)(piperazin-1-yl) methyl ketone, SUL 150 and SUL-151, respectively, exhibited considerable efficacy in inhibiting EGFP expression in H9C2 cardiomyocytes (respectively...). Figure 2 G and H).

[0123] The results are further summarized in Table 3.

[0124]

[0125] Example 3

[0126] The following experiments were designed following the discovery of the unexpected results in Example 1. These findings can be used to understand why the claimed SUL-type compound is effective in treating heart failure with reduced ejection fraction.

[0127] In this Example 3, it was shown that DOX-induced heart failure overlaps with cardiac fibrosis (increased collagen deposition) and oxidative stress (increased lipid peroxidation), which may originate from mitochondrial dysfunction (reduced cardiac mtDNA copy number and ATP production).

[0128] SUL-150, administered in both prophylactic and therapeutic models, alleviated these pathological processes and maintained cardiac mitochondrial function at homeostatic levels. In isolated mitochondria, SUL-150 reduced DOX-induced decreases in respiratory complex IV activity, which is considered a potential mechanism for the observed therapeutic effect.

[0129] Experimental Design

[0130] Preparation of SUL-150 food pellets

[0131] Dissolve SUL-150 (250 mg) in 100% EtOH (1.02 ml) and further dilute in distilled water (68.7 ml) to obtain a clear 10 mM solution. Spray the SUL-150 solution evenly onto standard food pellets (1.25 kg). Air-dry the food pellets overnight. Prepare fresh food pellets weekly. Approximately 200 mg / kg of food pellets. -1 SUL-150 was administered to a 350g rat with a daily dietary intake of 25g, and the daily dose of SUL-150 was approximately 5mg. -1 .

[0132] Animal program

[0133] Male outbred Wistar rats (10-12 weeks old) weighing 250-300 g were kept in a 12-hour light / dark cycle and given a standard diet containing distilled water at will. Thirty rats were administered doxorubicin (2 mg / kg) weekly via intraperitoneal injection. -1 Rats were administered standard food diet (10 rats) for 9 consecutive weeks without treatment, or received a diet supplemented with SUL-150 as a preventative measure (before the first doxorubicin administration, 10 rats) or as treatment (starting after the sixth doxorubicin administration, 10 rats). Sham control rats (8 rats) received weekly intraperitoneal injections of saline for 9 consecutive weeks and were on a standard diet. Cardiac function was assessed in all rats 12 weeks after the first doxorubicin administration.

[0134] Experimental protocol

[0135] For the results of the cardiac function test (PV ring assessment), refer to Example 1 above.

[0136] Cardiac fibrosis

[0137] Cardiac tissue samples for histopathology were fixed in 3.6% formalin and embedded in paraffin. 4 μm thick sections of cardiac tissue were prepared and stained with Sirius red, then counterstained with Weighert hematoxylin (both Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. Samples were imaged on a NanoZoomer S60 digital slide scanner (Hammamatsu Photonics), and left ventricular interstitial fibrosis (i.e., non-perivascular fibrosis) was quantified using Aperio ImageScope (Leica Biosystems, Nussloch, Germany).

[0138] Cardiac oxidative stress

[0139] Heart tissue samples were homogenized in ddH2O using a TissueRuptor II (Qiagen, Hilden, Germany), followed by sonication at 20 kHz for 3 × 1 min (Sonopuls 2000, Bandelin, Berlin, Germany) and centrifuged at 14000 g to precipitate insoluble proteins. The supernatant was used to assess the free radical scavenging activity of ABTS radical decolorization, as described by Re et al.

[14] , based on lipid peroxidation by Ohkawa et al.

[15] by assessing reactivity to thiobarbituric acid.

[0140] Heart mitochondrial copy number

[0141] Heart tissue samples were placed in a solution containing 50 U / ml -1 RNase I and 100 U·ml -1Homogenize the proteinase K (all from ThermoFisher, Walthham, MA) in lysis buffer (100 mM NaCl, 10 mM EDTA, 20 mM Tris-HCl containing 0.5% SDS, pH 7.4). After incubation overnight at 55°C, total DNA was precipitated with 2-propanol. Aliquots of 5 ng total DNA were amplified on a ViiA7 real-time PCR system (ThermoFisher, Waltham, MA) using iTaq Universal SYBR (Bio-Rad, Hercules, CA) primers specific to mitochondrial DNA (MT-ND1; sense 5′-CCTCCTAATAAGCGGCTCCT-3′, antisense 5′-GGCGGGGATTAATAGTCAGA-3′) or nuclear DNA (NDUFA1; sense 5′-ATGGCCCGAACCAAGCAGACC-3′, antisense 5′-TTAAGCTCTCTCCCCCCGTATCCG-3′). The mtDNA copy number is calculated as: mtDNA = 2 × 2 Cq(NDUFA1)–Cq(MT–ND1) .

[0142] Cardiac ATP / ADP ratio

[0143] Heart tissue samples were homogenized in Tris-saturated phenol (pH 7.4) using TissueRuptor II (Qiagen, Hilden, Germany) to extract adenosine, which was then separated by centrifugation via chloroform:water (1:1 v / v). Nucleotide separation and ATP and ADP concentrations were measured by HPLC. Separation was performed by injecting 100 μL of sample into a reversed-phase C18 column. The column temperature was maintained at 25 °C. The mobile phase was 70% acetonitrile:30% 75 mmol L⁻¹ KH₂PO₄ (v / v), and the flow rate was 1 mL / min. Eluted nucleotides were detected at a wavelength of 260 nm. The nucleotide concentration of the eluent was calculated using a calibration curve based on the peak area of ​​each standard nucleotide. ATP concentration was normalized relative to ADP concentration in each sample.

[0144] Respiratory complex IV activity

[0145] H9C2 cardiac myoblasts were exposed to 1 μM DOX for 24 h under standard cell culture conditions, with or without 1 μM SUL-150. Cells were then centrifuged using a density gradient centrifugation method. Mitochondrial isolation kit (Cayman Chemical #701010, Ann Arbor, MI) was used to isolate mitochondria according to the manufacturer's instructions and assess the activity of mitochondrial complex IV (cytochrome c oxidase) by measuring the oxidation rate of cytochrome c, which was reflected by the increase in absorbance at 550 nm (Cayman Chemical #700990, Ann Arbor, MI).

[0146] Statistical assessment

[0147] Data were analyzed in GraphPad Prism 8.0 (GraphPad Software Inc, Ca). All results are expressed as mean ± SD or median and interquartile range. Differences between groups were assessed by ANOVA, followed by paired comparisons with the sham treatment and carboxyl treatment groups. Multiple comparisons were performed using FDR-corrected p-values.

[0148] result

[0149] SUL-150 administration appears to reduce cardiac fibrosis in a rat model of doxorubicin-induced heart failure.

[0150] Long-term doxorubicin administration in rats leads to the development of heart failure, as indicated by decreases in heart rate, ejection fraction, and stroke work, ultimately resulting in a severe reduction in cardiac output. Furthermore, long-term doxorubicin administration induces a fibrotic response in the left ventricular interstitium, as indicated by increased collagen deposition. Figure 4 Before doxorubicin administration (prevention group) or after 6 cumulative doxorubicin administrations (treatment group), SUL-150 was administered via food particles to maintain cardiac function (as shown above) and block the fiber response. Figure 4 ).

[0151] SUL-150 administration appears to reduce cardiac oxidative stress without affecting cardiac antioxidant capacity.

[0152] Heart failure is associated with increased oxidative stress in cardiac tissue, which may be caused by an imbalance between the capacity to scavenge free radicals and the production of free radicals. Cardiac oxidative stress is evident after long-term doxorubicin administration, such as through increased lipid peroxidation products (TBARS). Figure 5 As indicated in A). SUL-150 is used in preventative or treatment regimens to reduce cardiac lipid peroxidation ( Figure 5 A) suggests a decrease in cardiac oxidative stress.

[0153] Long-term doxorubicin administration ( Figure 5 B) and prophylactic or therapeutic application of SUL-150 ( Figure 5B) Keeping cardiac free radical scavenging activity unchanged suggests that increased free radical production, rather than decreased scavenging capacity, is the basis for the observed increase in oxidative stress.

[0154] SUL-150 administration maintains cardiac mitochondrial copy number and normalizes cardiac energy status.

[0155] Heart failure is associated with cardiac energy loss, the development of mitochondrial dysfunction, and the loss of mitochondrial mass through mitophagy, leading to contractile dysfunction. In fact, long-term doxorubicin administration reduces the amount of cardiac ATP available for contraction. Figure 6 A) This can be alleviated by preventative or therapeutic application of SUL-150.

[0156] Mitochondrial DNA (mtDNA) copy number can be used as a surrogate marker of mitochondrial quality, and a decrease in mitochondrial quality may underlie energy expenditure following prolonged doxorubicin administration. Prolonged doxorubicin administration reduces cardiac mtDNA copy number. Figure 6 B), which was ruled out by the administration of SUL-150. It is noteworthy that in animals treated with doxorubicin, mtDNA copy number was positively correlated with cardiac ejection fraction (r). 2 =0.454, p=0.033; Figure 6 C) indicates that a higher mitochondrial load corresponds to better cardiac contractility.

[0157] SUL-150 alleviates doxorubicin-induced reduction in respiratory complex IV activity in isolated cardiac mitochondria.

[0158] SUL compounds have been shown to increase mitochondrial function through activation of respiratory complex IV, which can be inhibited by doxorubicin. In fact, when H9C2 cardiac myoblasts were exposed to doxorubicin (1 μM, 24 h), the activity of respiratory complex IV was significantly reduced. Figure 7 In H9C2 cardiac myoblasts, co-incubation with doxorubicin and SUL-150 (both 1 μM, 24 h) maintained the activity of respiratory complex IV, which may explain the increased ATP production observed in SUL-150-treated rats. Interestingly, in untreated control H9C2 cardiac myoblasts, administration of SUL-150 (1 μM, 24 h) did not alter the activity of respiratory complex IV.

[0159] in conclusion

[0160] The chemotherapeutic agent doxorubicin is known to have cardiotoxic effects, ultimately leading to heart failure with reduced ejection fraction (HFrEF).

[0161] Example 1 shows that, despite the administration of doxorubicin, 6-benzodihydropyranol SUL-150 (prophylactic or therapeutic administration) maintains cardiac systolic function and thus cardiac output.

[0162] Example 2 shows that the claimed SUL-150 analogues are also effective in preventing ventricular hypertrophy; and noteworthyly, they are more effective than other Trolox-type compounds not according to the invention, suggesting that these claimed compounds may also be used to treat HFrEF.

[0163] Example 3 shows that doxorubicin-induced heart failure coincides with increased cardiac oxidative stress, energy expenditure, and mitochondrial mass loss. Prophylactic or therapeutic administration of SUL-150 may have inadvertently excluded these pathological changes by maintaining respiratory complex IV activity.

Claims

1. Use of the 2R-enantiomer of (6-hydroxy-2,5,7,8-tetramethyl-2-yl)(piperazin-1-yl) methyl ketone: (2R)-(6-hydroxy-2,5,7,8-tetramethyl-2-yl)(piperazin-1-yl) methyl ketone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prevention of heart failure with reduced ejection fraction.

2. The use according to claim 1, wherein the treatment or prevention is carried out in combination therapy employing one or more commonly used measures for treating heart failure.

3. The use according to claim 1 or 2, wherein the patient with heart failure and reduced ejection fraction has a preserved ejection fraction (HFmrEF) of 50% or less.

4. The use according to claim 3, wherein the patient with heart failure and reduced ejection fraction has a preserved ejection fraction (HFrEF) of about 40% or less.

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