Cardiovascular therapeutic agents

Abstract

The present invention relates to a novel cardiac therapeutic agent effective against atrial fibrillation (AF), a pharmaceutical composition containing the same, its use for treating cardiac disease, and a method for treating cardiac disease comprising using the same.

Description

[Technical Field]

[0001] The present invention relates to a novel cardiac therapeutic agent effective against atrial fibrillation (AF), stroke, and thromboembolism, a pharmaceutical composition containing the same, the use thereof for treating heart disease, and a method for treating heart disease including the use thereof. [Background technology]

[0002] Atrial fibrillation (AF) is the most common persistent arrhythmia, with its prevalence increasing globally due to the aging population. Approximately 2.3 million people in North America and 4.5 million in Europe suffer from AF. In the United States, AF accounts for over 750,000 hospitalizations and an estimated 130,000 deaths annually. AF consequently increases the risk of stroke and thromboembolism fivefold and doubles the mortality rate. The global AF market is estimated to reach $16 billion by 2020. Even today, drug therapy remains clinically important.

[0003] AF is characterized by an irregular and rapid heartbeat, which may occur without prior cardiac complications (isolated AF, 3%), or may be associated with underlying heart conditions such as congestive heart failure, coronary artery disease, hypertension, diabetes, or atherosclerosis.

[0004] Amiodarone (Figure 1A) and dronedarone (Figure 1B) are multiple benzofuran-derived cardiac ion channel blockers and FDA-approved antiarrhythmic drugs. Amiodarone is used to treat 45% of AF cases, but it has serious adverse effects, including thyroidopathy, pneumonia, interstitial pneumonia, and pulmonary fibrosis. Dronedarone avoids the thyroidopathy and pulmonary fibrosis associated with amiodarone, but clinical trials have reported a higher mortality risk with dronedarone compared to placebo, and a boxed warning has been issued for its use in patients with New York Heart Association (NYHA) class IV heart failure, or NYHA class II-III heart failure with recent decompensation or persistent AF. Surgical treatment with catheter ablation is an alternative, but it has a relatively low success rate (28% for the first procedure), is expensive, and is not accessible to patients in certain countries.

[0005] In the human heart, cytochrome P450 2J2 (CYP2J2) metabolizes arachidonic acid (AA) into epoxyeicosatrienoic acid (EET), which is involved in controlling cardiac rhythm.

[0006] The inventors have confidentially discovered a significant correlation between potent covalent inactivation of cardiac CYP2J2 by ​​reactive metabolite benzofuran derivatives such as dolonedarone and beat-to-beat variability (BBV) in cardiomyocytes. The inventors evaluated their observations in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM).

[0007] Using this information, the inventors developed a novel site-specific deuterated analog of doronedarone, which is referred to herein as poyendarone.

[0008] Deuteration processes involve atomic-scale modifications of target compounds, specifically the exchange of hydrogen (a proton with orbital electrons) for deuterium (a protein and neutron with orbital electrons). In pharmacology, deuteration is often applied to metabolically stabilize drug compounds and improve their pharmacokinetics. In this study, deuteration was used to modify the chemical reactivity of the electrophilic intermediate of dolonedarone to avoid CYP2J2 inhibition and improve its safety profile by mitigating significant off-target cardiac adverse effects. Counterintuitively, the pharmacokinetics of dolonedarone are preserved in poiendarone by avoiding random deuteration.

[0009] Advantageously, targeted deuteration results in the production of the molecule, poiendarone, which has the same favorable pharmacokinetics and anti-atrial fibrillation properties as its non-deuterated analog (i.e., dronedarone), but with a significantly reduced risk of arrhythmia induction. Consistently, based on these findings, the inventors also discovered that in a human cell model, i.e., hiPSC-CM, poiendarone does not inactivate recombinant CYP2J2 enzyme and does not inactivate CYP2J2, i.e., unlike dronedarone, does not produce BBV in hiPSC-CM. Furthermore, the inventors found that when tested in vivo, poiendarone produced a similar pharmacokinetic and cardiac hemodynamic profile to dronedarone, exhibiting anti-atrial fibrillation activity, but with less ventricular arrhythmia induction compared to dronedarone.

[0010] In summary, the inventors have developed a novel small molecule therapeutic agent for treating typical cardiac arrhythmias that possesses the favorable pharmacokinetics and antiarrhythmic pharmacology of FDA-approved benzofuran-derived drugs such as dronedarone, but without significant ventricular arrhythmia-inducing toxicity. Furthermore, when used in humans, poyendarone is expected to have minimal organ damage compared to amiodarone, and therefore protect at least the thyroid and lungs. [Overview of the project] [Problems that the invention aims to solve]

[0011] According to a first aspect of the present invention, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof is provided. [ka] Equation (I)

[0012] During the ceremony, R 1 , R 2 , and R 3 Each of these represents deuterium, n represents 2 or 3, Each R 4 C may be independently substituted with one or more of the following: nitro group, halogen group, amino group, amide group, cyano group, carboxyl group, sulfonyl group, hydroxyl group, ketone group, and aldehyde group. 1-6 Represents a hydrocarbyl group, R 5 is hydrogen or [ka] This represents, Each R 6 These independently represent hydrogen or halogen, However, each atom not designated as deuterium exists in its natural isotopic abundance, and each position designated as deuterium has at least 45% deuterium incorporation.

[0013] The compounds of the present invention not only exhibit the same favorable pharmacokinetics and anti-atrial fibrillation effects as their non-deuterated analogs (e.g., dronedarone), but also show lower toxicity in inducing ventricular arrhythmia.

[0014] In the compounds of the present invention, any atom not specifically defined as a particular isotope is present in its natural isotope abundance. For example, unless otherwise specified, when a position is specifically defined as “H” or “hydrogen”, that position is understood to have hydrogen with the isotope composition of its natural abundance. Also, unless otherwise specified, when a position is specifically defined as “deuterium”, that position has deuterium ([ 2 H) isotope at an abundance at least 3000-fold higher than the natural abundance of deuterium, which is 0.015% (i.e., an uptake of at least 45% of deuterium is required).

[0015] In the compounds of the present invention, the amount of deuterium present at each of the three specific sites R 1 、R 2 、and R 3 is higher than its natural isotope abundance. Preferably, each position defined as deuterium (i.e., R 1 、R 2 、and R 3 ) has an uptake of at least 90% (i.e., at least 6000-fold greater than the natural abundance of deuterium), more preferably at least 95%, and most preferably 100% deuterium.

[0016] As used herein, all percentages expressed with respect to the abundance of deuterium in the compounds of the present invention are mole percentages.

[0017] Deuteration can be achieved by exchanging protons with deuterium in the non-deuterated analog or by synthesizing the compound using deuterated starting materials.

[0018] In the compounds of the present invention, each R 4C may be independently substituted with one or more of the following: nitro group, halogen group, amino group, amide group, cyano group, carboxyl group, sulfonyl group, hydroxyl group, ketone group, or aldehyde group. 1-6 This represents a hydrocarbyl group. As used herein, the term "hydrocarbyl" refers to a group composed of carbon and hydrogen atoms. These include aliphatic (i.e., alkyl, alkenyl, or alkynyl) groups, as well as aromatic groups such as phenyl, or combinations thereof. The aliphatic group may be linear or branched, or may form or contain a non-aromatic ring structure. For ease of understanding, "C 1-6 Terms such as "hydrocarbyl" have the same meaning as the additional requirement that any such functional group contains a total of 1 to 6 carbon atoms.

[0019] As used herein, the term halogen refers to a fluoro group, a chloro group, a bromo group, or an iodine group.

[0020] Preferred mounting method, each R 4 Independently, the non-substituted C 1-6 Hydrocarbyl group, more preferably unsubstituted C 2-4 Represents a hydrocarbyl group. In a particularly preferred embodiment, the hydrocarbyl group is an alkyl group. In a particularly preferred embodiment, each R 4 It is a C4 alkyl group, typically n-butyl.

[0021] In the compound of the present invention, R 5 is hydrogen or [ka] It may also represent R. 5 Preferably [ka] Similarly, each R 6 R may represent hydrogen or a halogen group, typically iodine. However, in preferred embodiments, each R 6It is hydrogen. The halogen atom is (R 6 By removing the methanesulfonamide group, the compound becomes non-toxic to the lungs and thyroid, and such methanesulfonamide group is removed (R 5 By incorporating it, the lipid solubility of the compound is modified, further reducing tissue accumulation and systemic toxicity.

[0022] In the compound of the present invention, n is preferably 3.

[0023] Particularly suitable compounds of the present invention are compounds of formula (II), where R 1 , R 2 , and R 3 It is defined as above. [ka] Formula (II)

[0024] As described above, the compounds of the present invention are suitable for use in the treatment of cardiac arrhythmias, particularly atrial fibrillation. Furthermore, in contrast to non-deuterated analogs, these compounds have minimal toxicity in inducing ventricular arrhythmias.

[0025] Therefore, a second aspect of the present invention provides a compound according to the first aspect of the present invention for use in pharmaceuticals.

[0026] Furthermore, compounds according to a first aspect of the present invention for use in the treatment of heart disease are also provided. In a preferred embodiment, the heart disease is cardiac arrhythmia, and in a particularly preferred embodiment, atrial fibrillation.

[0027] In a third aspect, the present invention provides a method for treating a heart disease, the method comprising administering a therapeutically effective amount of a compound according to the first aspect of the present invention to a patient in need of such treatment.

[0028] In some cases, the heart condition being treated is a cardiac arrhythmia, preferably atrial fibrillation.

[0029] The compounds of the present invention are typically administered in pharmaceutical compositions, and therefore, further embodiments of the present invention provide pharmaceutical compositions comprising the compounds of the first embodiment of the present invention and pharmaceutically acceptable excipients or carriers.

[0030] The composition may be administered via any suitable route, such as orally, buccally, nasally, percutaneously, or parenterally, such as intravenously or intramuscularly. Preferably, the composition is for oral administration.

[0031] The formulations of the present invention for oral administration may be presented as individual units such as capsules, sachets, tablets, lozenges, or other formulations, each containing a predetermined amount of the active agent, as powders or granules, as aqueous or non-aqueous liquid solutions or suspensions of the active agent, as oil-in-water or water-in-oil liquid emulsions, as syrups or elixirs, or as boluses, etc.

[0032] In the case of compositions for oral administration (e.g., formulations including tablets, capsules, mucosal adhesives, etc.), the term "acceptable carrier" includes common excipients, such as binders such as syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose, and starch; fillers and carriers such as corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid; wetting agents / surfactants such as poloxamer, polysorbate, sodium doxate, and sodium lauryl sulfate; disintegrants such as starch or sodium starch glycolate; and vehicles such as lubricants such as magnesium stearate, sodium stearate, and other metal stearates, glycerol stearate, stearic acid, silicone fluids, talc wax, oil, and colloidal silica. Sweeteners and flavorings such as peppermint, wintergreen oil, and cherry flavoring can also be used. Coloring agents may be added to facilitate identification of the dosage form. Tablets may also be coated by methods well known in the art.

[0033] Tablets may be prepared by compression or molding, with one or more auxiliary components as optional. Compressed tablets may be prepared by compressing an active agent in a free-flowing form, such as a powder or granules mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant, in a suitable machine. Molded tablets may be prepared by molding a mixture of powder compounds moistened with an inert liquid diluent, in a suitable machine. Tablets may be optionally coated or notched, and may be formulated to provide sustained or sustained release of the active agent.

[0034] Some formulations may contain mucopolysaccharides, such as sodium hyaluronate, as mucoadhesives. Such compositions may be formulated, for example, as liquids, liquid syrups, softgels, liquid gels, fluid gels, or aqueous suspensions, and may contain one or more of the above additional excipients in addition to the active agent and mucoadhesive. Liquid formulations usually also contain a liquid carrier, which may be a solvent or suspension, such as water or saline solution, and may also contain a substance that increases their viscosity, such as sodium carboxymethylcellulose, sorbitol, or dextran.

[0035] Other formulations suitable for oral administration include lozenges containing the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles containing the active agent in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

[0036] Parenteral formulations are generally sterilized.

[0037] A fourth aspect of the present invention provides a compound represented by formula (III) or a pharmaceutically acceptable salt thereof. [ka] Formula (III) (In the formula, each R 7 (This represents deuterium.)

[0038] Throughout this specification and in the claims, the terms “comprise” and “contain” and their variations, such as “comprising” and “comprises,” mean “including but not limited to,” and do not exclude other parts, additives, components, integers, or processes. Throughout this specification and in the claims, unless the context specifically requires otherwise, the singular form includes the plural form. In particular, where the indefinite article is used, this specification should be understood to intend both the plural and the singular, unless the context specifically requires otherwise.

[0039] All references, including any patents or patent applications cited herein, are incorporated herein by reference. No reference is intended to constitute prior art. Furthermore, no prior art is intended to constitute part of the common general knowledge in the art.

[0040] Preferred features of each aspect of the present invention may be as described in relation to any of the other aspects.

[0041] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention extends to any novel features or any novel combination of features disclosed herein (including the appended claims and drawings). Accordingly, any features, integers, properties, compounds, or chemical parts described in relation to a particular aspect, embodiment, or example of the present invention should be understood to be applicable to any other aspect, embodiment, or example described herein, provided that they are not incompatible.

[0042] Furthermore, unless otherwise specified, any feature disclosed herein may be replaced by an alternative feature that serves the same or similar purpose.

[0043] The present invention will now be described by reference only to the following embodiments and drawings. [Brief explanation of the drawing]

[0044] [Figure 1] The chemical structures of (A) amiodarone and (B) dronedarone, FDA-approved antiarrhythmic drugs derived from benzofurans, are shown. Dronedarone lacks an iodine atom but has a methanesulfonamide group. In addition, dronedarone contains an N-dibutylamine moiety, while amiodarone contains an N-diethylamine moiety. Furthermore, dronedarone contains a propoxy(-O-CH2-CH2-CH2-) linker between the N-dibutylamine moiety and the phenyl group, while amiodarone contains an ethoxy(-O-CH2-CH2-) linker between the N-diethylamine moiety and the phenyl group. [Figure 2] This shows the cytochrome P450 (CYP450)-mediated AA metabolic pathway. AA is metabolized by CYP2J2 to multiple positional isomers of EET. EET is further metabolized by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acid (DHET). [Figure 3] This diagram shows the metabolism of doronedarone to quinone oxime-reactive metabolites by CYP2J2. The electrophilic sites of the reactive metabolites are indicated by asterisks (positions 4 and 6). [Figure 4] The chemical structure of the deuterated benzofuran derivative, poyendalone, is shown. "D" indicates the presence of a deuterium isotope. [Figure 5]The synthesis of the hydrochloride salt of the deuterated benzofuran derivative, poyendalone ("poyendalone HCl"), is shown. "D" indicates the presence of a deuterium isotope. Reagents and conditions: (a) Concentrated H2SO4, 48% HBr, 35% HCHO, 75°C, 6 hours. D1 yield: 76.5%; (b) Toluene, PPh3, reflux, 1 hour. D2 yield: 99.0%; (c) CHCl3, pyridine, valeroyl chloride, reflux, 2 hours; Toluene, triethylamine, reflux, 3 hours. D3 yield: 73.5%; (d) Dichloromethane, C6H5COCl(p-OCH3), SnCl4, room temperature, 24 hours. D4 yield: 93.5%; (e) Dichloromethane, AlCl3, reflux, 24 hours. D5 yield: 98.0%; (f) Acetone, anhydrous K2CO3, 1-chloro-3-di-n-butylaminopropane, reflux, overnight. D6 yield: 77.0%; (g) Fe, EtOH, H2O, concentrated HCl, 65°C, 3 hours. D7 yield: 80.3%; (h) Dichloromethane, pyridine, CH3SO2Cl, 35°C, 3 hours; (i) Methanol, hydrochloric acid, 0°C, 3 hours. D8 yield: 79.0%. [Figure 6] Representative plots showing the effect of each CYP2J2 siRNA treatment condition on CYP2J2 gene expression are shown. Calcium transients were captured by video 96 hours after siRNA transfection, followed by quantitative polymerase chain reaction (qPCR). A 40-80% knockdown of CYP2J2 was observed across all siRNA treatments (***: p<0.001, N=3). [Figure 7]The effect of each CYP2J2 siRNA treatment condition on the measured inter-beat variability in standard deviation experienced by individual clusters of cardiomyocytes is shown across the entire biological replicate. Each point represents the inter-beat variability of an individual cluster of cardiomyocytes measured during a 30-second window. The mean and 95% confidence interval are plotted. The number of individual clusters measured was: control: 60, siRNA1: 48, siRNA2: 66, siRNA3: 72, siRNA4: 86, pooled: 81. Since the distribution of the data determined using the Shapiro-Wilk normality test (**: p<0.01, ***: p<0.001, ****: p<0.0001) was not normal, significance was assessed using the non-parametric Mann-Whitney U test. [Figure 8] Representative raster plots of individual clusters of cardiomyocytes under each treatment condition are shown. Each line represents the occurrence of a contraction along a time axis of 30 seconds or less. The regularity of contractions can be visually confirmed in these graphs and quantified using the standard deviation between contraction occurrences. [Figure 9] The effect of CYP2J2 siRNA treatment on the measured inter-beat variability in standard deviation for individual clusters of cardiomyocytes is shown across the entire biological replicate. Each point represents the inter-beat variability of an individual cluster of cardiomyocytes measured during a 30-second window. The mean and 95% confidence interval are plotted. The number of individual clusters measured was 60 for controls and 353 for siRNA (including all four individual treatment conditions and the pooled condition). Since the distribution of the data determined using the Shapiro-Wilk normality test (****: p<0.0001) was not normal, significance was assessed using the non-parametric Mann-Whitney U test. [Figure 10]The time-dependent, concentration-dependent, and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent inactivation of CYP2J2 using astemizole as a probe substrate is shown. The figure shows that CYP2J2 is not inactivated in the absence of the inhibitor (0 μM drug) or in the absence of NADPH (no NADPH). However, in the presence of the inhibitor and NADPH, a concentration-dependent decrease in CYP2J2 activity is observed. The logarithm of the CYP2J2 activity percentage was plotted against the pre-incubation time in the presence of (A) doronedarone and (B) poyendarone. The reciprocal of the observed inactivation rate (kobs) was plotted against the reciprocal of the inhibitor concentration to form a Kitz-Wilson plot, and the kinact / KI ratio was calculated. A higher kinact / KI ratio indicates a greater ability of mechanism-based inactivation (MBI) of CYP2J2. Each point represents the average value and standard deviation (SD) of three experiments. [Figure 11] Using rivaroxaban as a probe substrate, (A) delonedarone and (C) poiendarone demonstrate time-dependent and concentration-dependent inactivation of CYP2J2. Using nonlinear regression, the inactivation reaction rate constants KI and kinact were calculated for (B) delonedarone and (D) poiendarone using the observed inactivation rates (kobs). [Figure 12] Using rivaroxaban as a probe substrate, (A) we demonstrate the time-dependent and concentration-dependent inactivation of CYP2J2 by ​​compound 2 (Table 3) and compound 3 (Table 3). Using nonlinear regression, the inactivation reaction rate constants KI and kinact were calculated for (B) compound 2 and (D) compound 3 using the observed inactivation rates (kobs). [Figure 13](A) Relative expression of CYP2J2 and sEH mRNA is shown. Total RNA was isolated from control hiPSC-CM and transcribed to cDNA using the Superscript II first-strand synthesis system. 5 ng of complementary DNA (cDNA) was amplified using a Quanifast PCR master mix with SYBR Green dye. The samples were run on a 3% agarose gel electrophoresis. HiPSC-CM expresses both CYP2J2 and sEH at 14 and 30 days after differentiation. (B) HiPSC-CM were co-treated with astemizole and the inhibitor for 24 hours, and the astemizole metabolite, O-desmethylastemizole, was measured using liquid chromatography-tandem mass spectrometry (LC / MS / MS). The percentage of CYP2J2 activity was calculated and compared between the inhibitor and the control. ***p<0.001, *p<0.05 [Figure 14] (A) Dronedarone is cytotoxic to cardiomyocytes (increased dead cell protease activity, decreased intracellular adenosine triphosphate (ATP), and decreased tetramethylrhodamine methyl ester perchlorate (TMRM) fluorescence), but its toxicity is relieved in a concentration-dependent manner with concentrated 14,15-EET. (B) Poyendarone is significantly less cytotoxic to cardiomyocytes (the IC50 for ATP reduction is reduced to 1 / 13th, and the EC50 for cytotoxicity is reduced to 1 / 23rd; the IC50 and EC50 for driponedarone are 3.1 μM and 1.2 μM, respectively). [Figure 15]This is a schematic diagram illustrating the difference between action potentials and extracellular field potentials. (A) Action potentials can be measured conventionally in single cells using whole-cell patch-clamp techniques to record the action of a single ion channel (e.g., the human ether-a-go-go-related gene (hERG) channel). (B) Field potentials were measured in a group of cells (e.g., embryoid bodies) using multiple electrodes (e.g., titanium nitride, 30 μm in diameter) to simultaneously measure the cumulative effect on all ion channels (e.g., hERG and L-type calcium channels) and ion exchangers (e.g., Na+ / Ca2+ exchanger (NCX)). (C) HiPSC-CMs were treated with dolonedarone, amiodarone, and poyendarone (i.e., deuterated dolonedarone) for 5 minutes, and extracellular field recordings were made for 180–300 milliseconds at baseline. Local activation maps were generated using Cardio2D software (Multichannel Systems, Reutlingen, Germany). Field potential duration The duration (FPD) was normalized to the number of systolic pulses using the Bazzet correction formula and plotted against drug concentration. The graph shows the dose-dependent increase in FPD of hiPSC-CM for dronedarone, amiodarone, and poyendarone. The dashed box represents the previously published effective therapeutic unbound plasma concentration (ETUPC) for dronedarone and amiodarone. For comparison, the inventors assumed that the ETUPC of poyendarone was similar to that of dronedarone. [Figure 16]This shows the inhibition of NaV1.5 peak current by amiodarone. A. NaV1.5 channel current recorded from a holding potential of -120mV to -20mV. Bottom: Exemplary traces recorded in the presence of dimethyl sulfoxide (DMSO), at start (black) or 11 minutes later (red). Scale bars: Y=1000pA, X=10ms. B. Exemplary traces of NaV1.5 channel current formatted as A, in the presence of DMSO (black) and 11 minutes after 10μM amiodarone perfusion. C. Mean diary plot of residual NaV1.5 peak current % for each condition. Currents were normalized against a stable baseline peak current before being averaged. D. Dose-response curves for amiodarone inhibition of NaV1.5 current. [Figure 17] This shows the inhibition of NaV1.5 peak current by dolonedarone. A. NaV1.5 channel current recorded from a holding potential of -120mV to -20mV. Bottom: Exemplary traces recorded in the presence of DMSO, at the start (black) or after 11 minutes (red). Scale bars: Y=1000pA, X=10ms. B. Exemplary traces of NaV1.5 channel current formatted as A, in the presence of DMSO (black) and after 11 minutes of dolonedarone 5μM perfusion. C. Mean diary plot of residual NaV1.5 peak current % for each condition. Current was normalized against a stable baseline peak current and then averaged. D. Dose-response curve for dolonedarone inhibition of NaV1.5 current. [Figure 18]This shows the inhibition of NaV1.5 peak current by poiendalone. A. NaV1.5 channel current recorded from a holding potential of -120mV to -20mV. Bottom: Exemplary traces recorded in the presence of DMSO, at the start (black) or 11 minutes later (red). Scale bars: Y=1000pA, X=10ms. B. Exemplary traces of NaV1.5 channel current formatted as in A, in the presence of DMSO (black) and 11 minutes after 6μM poiendalone perfusion. C. Mean diary plot of residual NaV1.5 peak current % for each condition. Current was normalized against a stable baseline peak current and then averaged. D. Dose-response curve for poiendalone inhibition of NaV1.5 current. [Figure 19] This shows the inhibition of CaV1.2 peak current by amiodarone. A. CaV1.2 channel current recorded from a holding potential of -80mV to 0mV. Bottom: Exemplary traces recorded in the presence of DMSO, at the start (black) or after 10 minutes (red). Scale bars: Y=100pA, X=100ms. B. Exemplary traces of CaV1.2 channel current formatted as in A, in the presence of DMSO (black) and after 5μM amiodarone perfusion. C. Mean diary plot of residual CaV1.2 peak current % for each condition. Current was normalized against a stable baseline peak current and then averaged. D. Dose-response curve for amiodarone inhibition of CaV1.2 current. [Figure 20] This shows the inhibition of CaV1.2 peak current by dolonedarone. A. CaV1.2 channel current recorded from a holding potential of -80mV to 0mV. Bottom: Exemplary traces recorded in the presence of DMSO, at the start (black) or after 10 minutes (red). Scale bars: Y=100pA, X=100ms. B. Exemplary traces of CaV1.2 channel current formatted as in A, in the presence of DMSO (black) and after perfusion of 2μM dolonedarone. C. Mean diaplot of residual CaV1.2 peak current % for each condition. Current was normalized against a stable baseline peak current and then averaged. D. Dose-response curve for dolonedarone inhibition of CaV1.2 current. [Figure 21]This shows the inhibition of CaV1.2 peak current by poyendarone. A. CaV1.2 channel current recorded from a holding potential of -80mV to 0mV. Bottom: Exemplary traces recorded in the presence of DMSO, at the start (black) or after 10 minutes (red). Scale bars: Y=100pA, X=100ms. B. Exemplary traces of CaV1.2 channel current formatted as in A, in the presence of DMSO (black) and after perfusion of 0.5μM poyendarone. C. Mean diaplot of residual CaV1.2 peak current % for each condition. Current was normalized against a stable baseline peak current and then averaged. D. Dose-response curve for poyendarone inhibition of CaV1.2 current. [Figure 22] This shows the inhibition of Kv11.1 tail current by amiodarone. A. Kv11.1 current was induced from a holding potential of -80mV to a 2.5-second pulse of 20mV. The voltage was then returned to -60mV and the tail current was recorded. Bottom: Exemplary traces recorded in the presence of DMSO (black) or 0.2μM amiodarone (red). Scale bars: Y=200pA, X=500ms. B. Average diary plot of residual Kv11.1 tail current % for each condition. The current was normalized against a stable baseline peak tail current and then averaged. C. Dose-response curve for amiodarone inhibition of Kv11.1 current. [Figure 23] This shows the inhibition of the Kv11.1 tail current by delonedarone. A. The Kv11.1 current was induced from a holding potential of -80mV to a 2.5-second pulse of 20mV. The voltage was then returned to -60mV and the tail current was recorded. Bottom: Exemplary traces recorded in the presence of DMSO (black) or 0.2μM delonedarone (red). Scale bars: Y=200pA, X=500ms. B. Average diary plot of residual Kv11.1 tail current % for each condition. The current was normalized against a stable baseline peak tail current and then averaged. C. Dose-response curve for delonedarone inhibition of Kv11.1 current. [Figure 24]This shows the inhibition of Kv11.1 tail current by poiendarone. A. Kv11.1 current was induced from a holding potential of -80mV to a 2.5-second pulse of 20mV. The voltage was then returned to -60mV and the tail current was recorded. Bottom: Exemplary traces recorded in the presence of DMSO (black) or 0.2μM poiendarone (red). Scale bars: Y=200pA, X=500ms. B. Average diary plot of residual Kv11.1 tail current % for each condition. The current was normalized against a stable baseline peak tail current and then averaged. C. Dose-response curve for poiendarone inhibition of Kv11.1 current. [Figure 25] Representative Poincaré plots for control, 10 nM delonedarone, 10 nM amiodarone, and 10 nM poyendarone are shown. Each point represents the inter-beat interval (IBI) between consecutive beats. Inset: Electrocardiogram (ECG) showing RR interval or inter-beat interval IBI. The scales of the Poincaré plots for delonedarone and poyendarone are compared to show the absence of BBV associated with poyendarone. [Figure 26] (A) A simplified schematic diagram of in vivo dog experiments conducted with two doses of poiendarone, 0.3 mg / kg and 3.0 mg / kg, is shown. Plasma samples were analyzed for poiendarone using validated LC / MS / MS methods. (B) Poiendarone exhibits a two-compartment model similar to that of dolonedarone, and both plasma clearance (CL) and volume of distribution (V) values ​​are equivalent between the two compounds. This is consistent with our reasoning that the major debutylation of poiendarone is conceptually unaffected by deuteration. [Figure 27] (A, B) shows the in vitro metabolism of doronedarone and poyendarone by CYP3A4, (C, D) by CYP3A5, and (E, F) by human liver microsomes (HLM), respectively. [Figure 28]The simulated pharmacokinetic profiles of doronedarone (left column) and poyendarone (red dotted line in the right column), validated against clinical data for the following administration methods. Administration methods are: (A, B) intravenous, (C, D) single oral dose (fasted), (E, F) single oral dose (fed), (G, H) multiple oral doses (fasted), and (I, J) multiple oral doses (fed). The black lines represent predicted doronedarone concentrations over time, while the gray lines represent the concentration-time profiles at the 95th and 5th percentiles. Red triangles represent clinical data. The red dotted line represents the simulated concentration-time profile of poyendarone using the same clinical trial parameters as for doronedarone. All studies were obtained from the FDA review package for doronedarone. [Figure 29] The LIN2890 clinical trial data

[27] show the validation of multiple oral doses of the doronedarone physiologically-based pharmacokinetic (PBPK) model with (A) MBI and (C) MBI. Enlarged portions of the graphs are shown in (B) and (D), respectively, with axis titles removed for readability. Red triangles indicate clinical concentrations. [Figure 30] This shows the time course of changes in sinoatrial rate (SAR) and mean blood pressure (MBP). The data is presented as mean ± standard error of the mean (SEM) (n=4). [Figure 31]This graph shows the time course of changes in inter-atrial conduction time (IACT, top) at atrial pacing periods of 400ms (IACT(CL400)), 300ms (IACT(CL300)), and 200ms (IACT(CL200)); atrial effective refractory period (AERP, center) at basic atrial pacing periods of 400ms (AERP(CL400)), 300ms (AERP(CL300)), and 200ms (AERP(CL200)); and ventricular effective refractory period (VERP, bottom) at basic ventricular pacing period of 400ms (VERP(CL400)). Data are presented as mean ± SEM (n=4). The blacked-out symbols represent statistically significant differences from each control value (C) at p<0.05. [Figure 32] Typical tracings of the right atrium (RA) and left atrium (LA) during and after burst pacing are shown for the baseline control before drug administration (control, top) and 20 minutes after administration of 3 mg / kg of poiendarone hydrochloride (20 minutes after 3 mg / kg of poiendarone hydrochloride, bottom). The duration of atrial fibrillation was reduced from 6.9 seconds to 1.8 seconds after administration of poiendarone hydrochloride. [Figure 33] This graph shows the time course of changes in the duration (Af-duration) and cycle length (Af-cycle length: CL) of atrial fibrillation. Data are presented as mean ± SEM (n=4). Blacked-out symbols indicate statistically significant differences from each control value (C) at p<0.05. [Figure 34] Figure 5 shows the 1H NMR spectrum of 2-(bromomethyl)-4-nitrophenol(3,5,6-d3)(D1) prepared according to the schematic synthesis scheme. [Figure 35]Figure 5 shows the 1H NMR spectrum of 2-((bromotriphenylphosphoranyl)methyl)-4-nitrophenol(3,5,6-d3)(D2) prepared according to the schematic synthesis scheme. [Figure 36] Figure 5 shows the 1H NMR spectrum of 2-butyl-5-nitro-1-benzo(4,6,7-d3)furan(D3) prepared according to the schematic synthesis scheme. [Figure 37] Figure 5 shows the 1H NMR spectrum of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-methoxyphenyl)methanone(D4) prepared according to the schematic synthesis scheme. [Figure 38] Figure 5 shows the 1H NMR spectrum of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-hydroxyphenyl)methanone(D5) prepared according to the schematic synthesis scheme. [Figure 39] Figure 5 shows the 1H NMR spectrum of (2-butyl-5-nitro-1-benzo(4,6,7-d3)-furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl)methanone(D6) prepared according to the schematic synthesis scheme. [Figure 40] Figure 5 shows the 1H NMR spectrum of (5-amino-2-butyl-1-benzo(4,6,7-d3)-furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl)methanone(D7) prepared according to the synthetic scheme schematically shown. [Figure 41] Figure 5 shows the 1H NMR spectrum of methanesulfonamide, N-(2-butyl-3-(4-(3-(dibutylamino)propoxy)benzoyl)-1-benzo(4,6,7-d3)-furan-5-yl) hydrochloride (D8), prepared according to the synthetic scheme schematically shown. [Figure 42] Figure 5 shows the 13C NMR spectrum of methanesulfonamide, N-(2-butyl-3-(4-(3-(dibutylamino)propoxy)benzoyl)-1-benzo(4,6,7-d3)-furan-5-yl) hydrochloride (D8), prepared according to the synthetic scheme schematically shown. [Figure 43] The synthesis of the deuterated doronedarone derivative "Compound 3" is shown. "D" indicates the presence of a deuterium isotope. Reagents and conditions: (a) Concentrated H2SO4, 48% HBr, 35% HCHO, 75°C, 6 hours, DD1: 76.0%. (b) Toluene, PPh3, reflux, 1 hour, DD2: 98.0%. (c) CHCl3, pyridine, valeroyl chloride, reflux, 2 hours; Toluene, triethylamine, reflux, 3 hours, DD3: 75.0%. (d) Dichloromethane, C6H5COCl(p-OCH3), SnCl4, room temperature, 24 hours, DD4: 95.0%. (e) Dichloromethane, AlCl3, reflux, 24 hours, DD5: 98.0%. (f) Acetone, anhydrous K2CO3, 1-chloro-3-di-n-butylaminopropane, reflux, overnight, DD6: 79.0%. (g) Fe, EtOH, H2O, concentrated HCl, 65°C, 3 hours, DD7: 82.0%. (h) Dichloromethane, pyridine, CH3SO2Cl, 35°C, 3 hours. (i) Methanol, hydrochloric acid, 0°C, 3 hours, DD-8: 80.0%. [Figure 44] Figure 43 shows the 1H NMR spectrum of 2-(bromomethyl)-4-nitrophenol(2,3,5,6-d4)(DD1) prepared according to the schematic synthesis scheme. [Figure 45] Figure 43 shows the 1H NMR spectrum of 2-((bromotriphenylphosphoranyl)methyl)-4-nitrophenol(2,3,5,6-d4)(DD2) prepared according to the synthetic scheme schematically shown. [Figure 46] Figure 43 shows the 1H NMR spectrum of 2-butyl-5-nitro-1-benzo(4,6,7-d3)furan (DD3) prepared according to the schematic synthesis scheme. [Figure 47] Figure 43 shows the 1H NMR spectrum of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-methoxyphenyl(2,3,5,6-d4))methanone (DD4) prepared according to the schematic synthesis scheme. [Figure 48]Figure 43 shows the 1H NMR spectrum of ((2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-hydroxyphenyl(2,3,5,6-d4))methanone (DD5) prepared according to the schematic synthesis scheme. [Figure 49] Figure 43 shows the 1H NMR spectrum of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl(2,3,5,6-d4))methanone (DD6) prepared according to the schematic synthesis scheme. [Figure 50] The 1H NMR spectrum of (5-amino-2-butyl-1-benzo(4,6,7-d3)furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl(2,3,5,6-d4))methanone (DD7), prepared according to the synthetic scheme schematically shown in Figure 43, is shown. [Figure 51] Figure 43 shows the 1H NMR spectrum of methanesulfonamide, N-(2-butyl-3-(4-(3-(dibutylamino)propoxy)benzoyl(2,3,5,6-d4))-1-benzofuran-5-yl)(DD8), prepared according to the synthetic scheme schematically shown. [Figure 52] Figure 43 shows the 13C NMR spectrum of methanesulfonamide, N-(2-butyl-3-(4-(3-(dibutylamino)propoxy)benzoyl(2,3,5,6-d4))-1-benzofuran-5-yl)(DD8), prepared according to the synthetic scheme schematically shown. [Modes for carrying out the invention]

[0045] Table 1. List of siRNA target sequences and qPCR primers.

[0046] Table 2. Compound-dependent mass spectrometry (MS) parameters of O-desmethylasterimizole and buspirone.

[0047] Table 3. MBI of CYP2J2-mediated metabolism of rivaroxaban.

[0048] Table 4. Forward and reverse sequences of primers for the human CYP2J2, EPHX2, and GAPDH genes.

[0049] Table 5. Optimal conditions for liquid chromatography-tandem mass spectrometry (LC-MS / MS) for the quantification of doronedarone, poyendarone, and N-desethylamiodarone (NDEA) (internal standard (IS)) in canine plasma. (A): Source-dependent parameters, (B): Compound-dependent parameters. Q1: Mass of parent ion, Q3: Mass of daughter ion, DP: Declustering potential, EP: Inlet potential, CE: Collision energy, CXP: Cell exit potential.

[0050] Table 6. Results of substrate depletion and time-dependent and concentration-dependent inactivation.

[0051] Table 7. Parameters used in PBPK modeling.

[0052] Table 8. Calculated Log P (cLogP, calculated using ChemSketch), water solubility (measured in general-purpose buffer (pH 7.4) using a multiscreen HTS PCF filter plate 34), effective permeability (measured using a parallel artificial membrane permeability assay), and in vitro metabolic half-life (T1 / 2, measured using recombinant CYP2J2, NADPH, and 1 μM drug) comparing poiendarone and dolonedarone.

[0053] Table 9. Comparison of the effects of antiarrhythmic drugs on the atrial effective refractory period (ΔAERP) and ventricular effective refractory period (ΔVERP). Atrial selectivity was measured based on the ratio of ΔAERP to ΔVERP (ΔAERP / ΔVERP), and the order was poyendarone = dronedarone > amiodarone > bepridil > dl-sotalol.

[0054] Table 10. Comparison of the effects of antiarrhythmic drugs on the terminal repolarization period (ΔTRP), early repolarization period (ΔJ-Tpeakc), and late repolarization period (ΔTpeak-Tend). ΔTRP indicates the risk of reentrant ventricular arrhythmias, with the order being dronedarone > bepridil > dl-sotalol > amiodarone > poyendarone.

[0055] Materials and methods All reagents and solvents, unless otherwise specified, are general-purpose or analytical grade and were purchased from Merck (formerly Sigma-Aldrich). Silica gel plates (pre-coated 60F) 254 The reaction was continuously monitored by thin-layer chromatography (TLC) on a Merck plate. Column chromatography was performed using silica gel 60 (Merck, 70-230 mesh). To determine the mass-to-charge ratio (m / z) using an ABSciex 2000 LC / MS / MS mass spectrometer (ionization source: electrospray ionization (ESI) probe), the compound was dissolved in methanol for high-performance liquid chromatography (HPLC). 1 ¹H NMR spectra were determined using a Bruker DPX ultrashield NMR (400 MHz) spectrometer in solutions of deuterated chloroform (CDCl3), deuterated dimethyl sulfoxide (DMSO-d6), and deuterated methanol (MeOH-d4). Chemical shifts were expressed as parts per million (δ) downfield relative to tetramethylsilane (TMS) as an internal standard, and the J value (coupling constant) was expressed in Hertz. The following abbreviations were used: s: single line, d: double line, t: triple line, m: multiline.

[0056] Details of the synthesis of the hydrochloride salt of poyendalone ("poyendalone HCl") are provided below.

[0057] Figure 5 shows the details of the experiment for the synthesis of boyendalone HCl. Synthesis of 2-(bromomethyl)-4-nitrophenol(3,5,6-d3)(D1). A mixture of 4-nitro(2,3,5,6-d4)phenol (1.25 g, 0.00873 M) and 48% HBr solution (15.8 ml) was mixed with concentrated sulfuric acid (0.121 ml) at room temperature, followed by the addition of 35% formalin solution (0.760 g, 0.0244 M, 2.8 equivalents). The reaction mixture was heated at 75°C for 6 hours with stirring. Subsequently, the reaction mixture was slowly poured into an ice water mixture and stirred for 1 hour. The resulting solid was filtered, washed with cold water, and dried under vacuum to obtain a beige solid. Toluene (50 ml) was added to the solid, and the mixture was heated at 85°C for 1 hour with stirring. The mixture was cooled to 5°C and stirred at the same temperature for 1 hour. The resulting solid was filtered, washed with toluene, and then dried under vacuum to obtain a white solid as the product. Yield: 76.5% 1 ¹H NMR (400 MHz, DMSO-d6): δ 4.70 (s, 2H), 11.61 (s, 1H). The synthesized product 1 The 1H NMR spectrum is shown in Figure 34.

[0058] Synthesis of 2-((bromotriphenylphosphoranyl)methyl)-4-nitrophenol(3,5,6-d3)(D2). 1.5 g (0.00638 M) of 2-(bromomethyl)-4-nitrophenol(3,5,6-d3)(D1) was dissolved in toluene (10 ml). 1.67 g of triphenylphosphine (0.00638 M) was added to this solution, and the mixture was refluxed for 1 hour. After the reaction was complete, the mixture was cooled, and the resulting precipitate was filtered. The filtrate was evaporated to dryness, stirred with toluene (10 ml) for 30 minutes, and filtered to obtain a solid. Both solids were combined and dried under vacuum to obtain a white solid. Yield: 99.0%. 1 ¹H NMR (400 MHz, CDCl3): δ 4.76-4.80 (d, J=14.0 Hz, 2H), 7.57-7.66 (m, 12H), 7.78-7.82 (m, 3H). The synthesized product 1 The 1H NMR spectrum is shown in Figure 35.

[0059] Synthesis of 2-butyl-5-nitro-1-benzo(4,6,7-d3)furan (D3). 1.65 g (0.00332 M) of 2-((bromotriphenylphosphoranyl)methyl)-4-nitrophenol(3,5,6-d3)(D2) was dissolved in 10 ml of CHCl3. To this solution, 0.522 g (0.0066 M, 2 equivalents) of pyridine was added. Then, 0.500 g (0.00405 M, 1.25 equivalents) of valeroyl chloride was slowly added to the mixture while stirring at room temperature. The mixture was refluxed for 2 hours, then 25 ml of toluene was added, and approximately half of the solvent was evaporated under reduced pressure. Then, 1.0 g (0.00996 M, 3 equivalents) of triethylamine was added, and the mixture was refluxed for a further 3 hours. The resulting reaction mixture was cooled, the formed triphenylphosphine oxide was filtered, washed with ethyl acetate, and the filtrate was concentrated under vacuum. The resulting viscous residue was purified by column chromatography using petroleum ether:toluene (95:5) to obtain a colorless residue. Yield: 73.5%. 1 H NMR(400MHz,CDCl3):δ 0.85-0.89(t,J=7.20Hz,3H),1.28-1.38(m,2H),1.70-1.78(m,2H),2.87-2.91(t,J=7.60Hz,2H),7.53-7.55(d,J=8.80Hz,1H). 1 The 1H NMR spectrum is shown in Figure 36.

[0060] Synthesis of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-methoxyphenyl)methanone(D4). 2-butyl-5-nitro-1-benzo(4,6,7-d3)furan(D3) (1.4 g, 0.00629 M) was dissolved in 15 ml of dichloromethane (7 ml). To this solution, 4-methoxybenzoyl chloride (1.61 g, 0.00945 M, 1.5 equivalents) and tin(IV) chloride (4.1 g, 0.0157 M, 2.5 equivalents) were slowly added over 1 hour at 0-5°C, and the mixture was stirred at room temperature for 24 hours. After cooling the reaction mixture to 0-5°C, water (10 ml) was slowly added, and the mixture was stirred for approximately 30 minutes. The aqueous layer was separated and extracted with dichloromethane (3 × 10 ml). The combined organic layers were evaporated under reduced pressure. The obtained crude compound was purified by column chromatography using petroleum ether: RINKAN (85:15) to obtain a white solid as the product. Yield: 93.5%. 1 H NMR(400MHz,CDCl3):δ 0.86-0.89(t,J=7.40Hz,3H),1.29-1.38(m,2H),1.71-1.78(m,2H),2.88-2.92(t,J=7.60 Hz,2H),3.90(s,3H),6.96-6.99(d,J=8.80Hz,2H),7.80-7.83(d,J=8.80Hz,2H).Synthesized products 1 The 1H NMR spectrum is shown in Figure 37.

[0061] Synthesis of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-hydroxyphenyl)methanone (D5). 1.02 g (0.00286 M) of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-methoxyphenyl)methanone (D4) was dissolved in 50 ml of dichloromethane. AlCl3 (2.29 g, 0.0172 M, 6 equivalents) was slowly added over 1 hour with stirring at 0-5°C. The mixture was refluxed for 24 hours, cooled to room temperature, and then to 0-5°C. Water (10 ml) was slowly added, and the mixture was stirred for approximately 30 minutes. The organic layer was separated and concentrated under vacuum to obtain the residue, which was purified by column chromatography using petroleum ether:siRNA (90:10) to obtain a yellowish oily substance. Yield: 98.0%. 1 H NMR(400MHz,CDCl3):δ 0.87-0.90(t,J=7.20Hz,3H),1.30-1.39(m,2H),1.72-1.80(m,2H),2.90-2.94(t,J =7.60Hz,2H),6.94-6.96(d,J=8.80Hz,2H),7.77-7.80(d,J=8.80Hz,2H). 1 The 1H NMR spectrum is shown in Figure 38.

[0062] Synthesis of (2-butyl-5-nitro-1-benzo(4,6,7-d3)-furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl)methanone (D6). 0.800 g (0.0023 M) of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-hydroxyphenyl)methanone (D5) was dissolved in 20 ml of acetone. To this solution, 3.23 g (0.0023 M, 1 equivalent) of anhydrous K2CO3 and 4.79 g (0.0023 M, 1 equivalent) of 1-chloro-3-di-n-butylaminopropane were added at room temperature. The reaction mixture was refluxed at 60°C overnight. The reaction mixture was cooled and evaporated under reduced pressure. Water (20 ml) was added to the resulting solid, stirred for 5 minutes, and extracted with dichloromethane (3 × 20 ml). The organic layer was evaporated under reduced pressure to obtain a crude residue, which was further purified by column chromatography using petroleum ether:siRNA(70:30) to obtain a yellowish oily residue. Yield: 77.0%, 1H NMR (400MHz, DMSO-d6): δ 0.80-0.84(m,9H),1.22-1.27(m,6H),1.30-1.37(m,4H),1.64-1.72(m ,2H),1.82-1.85(t,J=6.40Hz,2H),2.33-2.36(t,J=7.00Hz,4H),2.51- 2.53(m,2H),2.82-2.86(t,J=7.60Hz,2H),4.12-4.15(t,J=6.00Hz,2H),7.08-7.10(d,J=8.80Hz,2H),7.81-7.83(d,J=8.80Hz,2H). 1 The 1H NMR spectrum is shown in Figure 39.

[0063] Synthesis of (5-amino-2-butyl-1-benzo(4,6,7-d3)-furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl)methanone (D7). A mixture of 0.114 g (0.00022 M) of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4(3(dibutylamino)propoxy)phenyl)methanone (D6), 0.106 g (0.00133 M, 6 equivalents) of iron powder, 0.5 ml of ethanol, and 0.25 ml of water was stirred at room temperature for 10 minutes. The reaction mixture was cooled to 15°C and 0.5 ml of concentrated HCl was added. The reaction mixture was stirred at 65°C for 3 hours, cooled, poured into an ice water mixture, and stirred for 30 minutes. The aqueous layer was extracted with dichloromethane (3 × 5 ml). The pH of the organic layer was adjusted to 8-9 using an aqueous ammonia solution. Both the organic and aqueous layers were separated. The aqueous layer was extracted with dichloromethane (3 x 5 ml), and both organic layers were combined. The mixture was dried over Na₂SO₄ and removed under reduced pressure to obtain a colorless oily substance. Yield: 80.3%. 1 H NMR(400MHz,CDCl3):δ 0.87-0.91(m,9H),1.25-1.37(m,6H),1.42-1.49(m,4H),1.71-1.79(m,2H),1.99-2.02(m,2H),2.47-2.50(m,4H),2.65-2.69(m,2H) ),2.89-2.93(t,J=7.60Hz,2H),4.11-4.14(t,J=6.20Hz,2H),6.96-6.98(d,J=8.80Hz,2H),7.80-7.82(d,J=8.80Hz,2H). 1 The 1H NMR spectrum is shown in Figure 40.

[0064] Methanesulfonamide, N-(2-butyl-3-(4-(3-(dibutylamino) Synthesis of propoxy)benzoyl)-1-benzo(4,6,7-d3)-furan-5-yl) hydrochloride (D8). 0.100 g (0.000207 M) of (5-amino-2-butyl-1-benzo(4,6,7-d3)furan-3-yl)(4-(3(dibutylamino)propoxy)phenyl)methanone (D7) was dissolved in 3 ml of anhydrous dichloromethane and 0.0197 g (0.000249 M, 1.2 equivalents) of pyridine and 0.028 g (0.000249 M, 1.2 equivalents) of methanesulfonyl chloride were slowly added over 5 minutes at 35°C. The resulting mixture was stirred at the same temperature for 3 hours and then cooled to room temperature. Next, this mixture was washed with 2 × 5 ml of water, 2 × 5 ml of 5% NaHCO3 solution, and 1 × 5 ml of water. The organic phase was separated and concentrated, and further purified by column chromatography using petroleum ether:toluene (10:90) to obtain a brown oily residue in 80% yield. 5 ml of methanol was added to this residue, and a solution of hydrochloric acid (0.100 ml) dissolved in 0.4 ml of methanol was added over 20 minutes. The reaction mixture was stirred at 0°C for 3 hours, the resulting solid was filtered, washed with methanol, and dried to obtain the labeled compound as a light brown solid. 31 H 41 D3N2O5S[M+H] + The calculated ESI-MS (m / z) value was 559.77. Yield: 79.0%. 1 1H NMR (400MHz, MeOH-d4):δ 0.83-0.88(m,3H),0.89-0.93(t,J=7.20Hz,6H),1.27-1.33(m,6H),1.43-1 .47(m,4H),1.69-1.77(m,2H),1.94-1.99(m,2H),2.45-2.49(m,4H),2.64-2 .68(t,J=7.40Hz,2H),2.86-2.89(t,J=7.60Hz,2H),2.96(s,3H),4.12-4.16 (t,J=6.00Hz,2H),7.03-7.06(d,J=8.8Hz,2H),7.78-7.87(d,J=9.2Hz,2H), 13¹³C NMR (400MHz, MeOH-d4): 13.7, 19.5, 20.7, 23.2, 24.7, 26.7, 28.7, 31.1, 38.8, 51.2, 54.0, 66.3, 115.6, 117.9, 128.9, 129.7, 132.3, 132.7, 133.3, 135.5, 152.7, 163.9, 165.5, 167.5, 192.5. Synthesized product 1 1H NMR and 13 The 13C NMR spectra are shown in Figures 41 and 42, respectively.

[0065] Figure 43 shows the details of the experimental synthesis of the deuterated doronedarone derivative "Compound 3". Synthesis of 2-(bromomethyl)-4-nitrophenol(2,3,5,6-d4)(DD-1). A mixture of 4-nitrophenol(2,3,5,6-d4) (1.25 g, 0.00873 M) and 48% HBr solution (15.8 ml) was mixed with concentrated sulfuric acid (0.121 ml) at room temperature, followed by the addition of 35% formalin solution (0.760 g, 0.0244 M, 2.8 equivalents). The reaction mixture was heated at 75°C for 6 hours with stirring. Subsequently, the reaction mixture was slowly poured into an ice water mixture and stirred for 1 hour. The resulting solid was filtered, washed with cold water, and dried under vacuum to obtain a beige solid. Toluene (50 ml) was added to the solid, and the mixture was heated at 85°C for 1 hour with stirring. The mixture was cooled to 5°C and stirred at the same temperature for 1 hour. The resulting solid was filtered, washed with toluene, and then dried under vacuum to obtain a white solid as the product. Yield: 76.0% 1 ¹H NMR (400 MHz, DMSO-d6): δ 11.64 (s, 1H). The synthesized product 1 The 1H NMR spectrum is shown in Figure 44.

[0066] Synthesis of 2-((bromotriphenylphosphoranyl)methyl)-4-nitrophenol(2,3,5,6-d4)(DD-2). 1.5 g (0.00638 M) of 2-(bromomethyl)-4-nitrophenol(2,3,5,6-d4)(DD-1) was dissolved in toluene (10 ml). 1.67 g of triphenylphosphine (0.00638 M) was added to this solution, and the mixture was refluxed for 1 hour. After the reaction was complete, the mixture was cooled, and the resulting precipitate was filtered. The filtrate was evaporated to dryness, stirred with toluene (10 ml) for 30 minutes, and filtered to obtain a solid. Both solids were combined and dried under vacuum to obtain a white solid. Yield: 98.0%. 1 ¹H NMR (400 MHz, CDCl3): δ 4.70-4.74 (d, J=14.0 Hz, 2H). The synthesized product 1 The 1H NMR spectrum is shown in Figure 45.

[0067] Synthesis of 2-butyl-5-nitro-1-benzo(4,6,7-d3)furan (DD-3). 1.65 g (0.00334 M) of 2-((bromotriphenylphosphoranyl)methyl)-4-nitrophenol 2,3,5,6-d4) (DD-2) was dissolved in 10 ml of CHCl3. To this solution, 0.522 g (0.00663 M, 2 equivalents) of pyridine was added. Then, 0.500 g (0.00417 M, 1.25 equivalents) of valeroyl chloride was slowly added to the mixture while stirring at room temperature. The mixture was refluxed for 2 hours, then 25 ml of toluene was added, and approximately half of the solvent was evaporated under reduced pressure. Then, 1.0 g (0.01002 M, 3 equivalents) of triethylamine was added, and the mixture was refluxed for a further 3 hours. The resulting reaction mixture was cooled, the formed triphenylphosphine oxide was filtered, washed with ethyl acetate, and the filtrate was concentrated under vacuum. The resulting viscous residue was purified by column chromatography using petroleum ether:toluene (95:5) to obtain a colorless residue. Yield: 75.0% 1 H NMR (400MHz, CDCl3): δ 0.75-0.95(t,J=7.20Hz,3H),1.15-1.36(m,2H),1.45-1.67(m,2H),2.30-2.80(t,J=7.60Hz,2H),5.82(s,1H).1 The 1H NMR spectrum is shown in Figure 46.

[0068] Synthesis of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-methoxyphenyl(2,3,5,6-d4))methanone (DD-4). 2-butyl-5-nitro-1-benzo(4,6,7-d3)furan (DD-3) (1.4 g, 0.00630 M) was dissolved in 15 ml of dichloromethane (7 ml). To this solution, 4-methoxybenzoyl(d4) chloride (1.61 g, 0.00958 M, 1.5 equivalents) and tin(IV) chloride (4.1 g, 0.0157 M, 2.5 equivalents) were slowly added over 1 hour at 0-5°C, and the mixture was stirred at room temperature for 24 hours. After cooling the reaction mixture to 0-5°C, water (10 ml) was slowly added, and the mixture was stirred for approximately 30 minutes. The aqueous layer was separated and extracted with dichloromethane (3 × 10 ml). The combined organic layer was evaporated under reduced pressure. The resulting crude compound was purified by column chromatography using petroleum ether:toluene (85:15) to obtain a white solid as the product. Yield 95.0%. 1 H NMR (400MHz, CDCl3): δ 0.86-0.89(t,J=7.40Hz,3H),1.29-1.38(m,2H),1.71-1.79(m,2H),2.88-2.92(t,J=7.60Hz,2H),3.90(s,3H). 1 The 1H NMR spectrum is shown in Figure 47.

[0069] Synthesis of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-hydroxyphenyl(2,3,5,6-d4))methanone (DD-5). To a solution of 1.02 g (0.000283 M) of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-methoxyphenyl(2,3,5,6-d4)methanone (DD-4)) dissolved in 50 ml of dichloromethane, AlCl3 (2.29 g, 0.0169 M, 6 equivalents) was slowly added over 1 hour with stirring at 0-5°C. The mixture was refluxed for 24 hours, cooled to room temperature, and then cooled to 0-5°C. Water (10 ml) was slowly added, and the mixture was stirred for approximately 30 minutes. The organic layer was separated and concentrated under vacuum to obtain the residue, which was purified by column chromatography using petroleum ether:siRNA (90:10) to obtain a yellowish oily substance. Yield: 98.0%. 1 ¹H NMR (400 MHz, CDCl3): δ 0.87-0.90 (t, J=7.20 Hz, 3H), 1.30-1.39 (m, 2H), 1.72-1.80 (m, 2H), 2.90-2.94 (t, J=7.60 Hz, 2H). Synthesized product 1 The 1H NMR spectrum is shown in Figure 48.

[0070] Synthesis of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl(2,3,5,6-d4))methanone (DD-6). 0.800 g (0.00231 M) of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-hydroxyphenyl)(2,3,5,6-d4))methanone (DD-5) was dissolved in 20 ml of acetone. To this solution, 3.23 g (0.0023 M, 1 equivalent) of anhydrous K2CO3 and 4.79 g (0.00231 M, 1 equivalent) of 1-chloro-3-di-n-butylaminopropane were added at room temperature. The reaction mixture was refluxed overnight at 60°C. The reaction mixture was cooled and evaporated under reduced pressure. Water (20 ml) was added to the obtained solid, stirred for 5 minutes, and extracted with dichloromethane (3 × 20 ml). The organic layer was evaporated under reduced pressure to obtain a crude residue, which was further purified by column chromatography using petroleum ether:siRNA (70:30) to obtain a yellowish oily residue. Yield: 79.0%. 1 H NMR(400MHz,DMSO-d6):δ 0.81-0.84(m,9H),1.22-1.27(m,6H),1.30-1.37(m,4H),1.64-1.70(m,2H),1.83-1.86(t,J=6.40Hz,2H),2.33- 2.37(t,J=7.00Hz,4H),2.50-2.53(m,2H),2.82-2.86(t,J=7.60Hz,2H),4.12-4.16(t,J=6.00Hz,2H).Synthesized products 1 The 1H NMR spectrum is shown in Figure 49.

[0071] Synthesis of (5-amino-2-butyl-1-benzo(4,6,7-d3)furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl(2,3,5,6-d4))methanone (DD-7). A mixture of 0.114 g (0.00023 M) of (2-butyl-5-nitro-1-benzo(4,6,7-d3)furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl(2,3,5,6-d4))methanone (DD-6), 0.106 g (0.00138 M, 6 equivalents) of iron powder, 0.5 ml of ethanol, and 0.25 ml of water was stirred at room temperature for 10 minutes. The reaction mixture was cooled to 15°C and 0.5 ml of concentrated HCl was added. The reaction mixture was stirred at 65°C for 3 hours, cooled, poured into an ice water mixture, and stirred for 30 minutes. The aqueous layer was extracted with dichloromethane (3 × 5 ml). The pH of the organic layer was adjusted to 8-9 using an aqueous ammonia solution. Both the organic and aqueous layers were separated. The aqueous layer was extracted with dichloromethane (3 × 5 ml), and both organic layers were combined, dried over Na₂SO₄, and removed under reduced pressure to obtain a colorless oily substance. Yield: 82.0% 1 H NMR(400MHz,CDCl3):δ 0.87-0.91(m,9H),1.26-1.37(m,6H),1.42-1.49(m,4H),1.71-1.79(m,2H),1.99-2.02(m,2H),2.47- 2.51(m,4H),2.66-2.69(m,2H),2.89-2.93(t,J=7.60Hz,2H),4.11-4.14(t,J=6.20Hz,2H).Synthesized products 1 The 1H NMR spectrum is shown in Figure 50.

[0072] Synthesis of methanesulfonamide, N-(2-butyl-3-(4-(3-(dibutylamino)propoxy)benzoyl(2,3,5,6-d4))-1-benzofuran-5-yl)(DD-8). 0.100 g (0.000206 M) of (5-amino-2-butyl-1-benzo(4,6,7-d3)furan-3-yl)(4-(3-(dibutylamino)propoxy)phenyl(2,3,5,6-d4))methanone (DD-7) was dissolved in 3 ml of anhydrous dichloromethane and heated to a solution. 0.0197 g (0.000247 M, 1.2 equivalents) of pyridine and 0.028 g (0.000247 M, 1.2 equivalents) of methanesulfonyl chloride were slowly added over 5 minutes at 35°C. The resulting mixture was stirred at the same temperature for 3 hours and then cooled to room temperature. The mixture was then washed with 2 × 5 ml of water, 2 × 5 ml of 5% NaHCO3 solution, and 1 × 5 ml of water. The organic phase was separated and concentrated, and further purified by column chromatography using petroleum ether:alkyl (10:90) to obtain a brown oily residue in 80% yield. 5 ml of methanol was added to this residue, and a solution of hydrochloric acid (0.100 ml) dissolved in 0.4 ml of methanol was added over 20 minutes. The reaction mixture was stirred at 0°C for 3 hours, the resulting solid was filtered, washed with methanol, and dried to obtain the labeled compound as a light brown solid. 31 H 37 D7N2O5S[M+H] + ESI-MS (m / z) value calculated for this was 563.8. Yield: 80.0%. 1 H NMR(400MHz,MeOH-d4):δ 0.84-0.88(m,3H),0.89-0.93(t,J=7.20Hz,6H),1.27-1.34(m,6H),1.42-1.49(m,4H),1.69-1.76(m,2H),1.92-1.99(m,2H) ),2.45-2.49(m,4H),2.64-2.68(t,J=7.40Hz,2H),2.85-2.88(t,J=7.60Hz,2H),2.91(s,3H),4.12-4.15(t,J=6.00Hz,2H). 13¹³C NMR (400MHz, MeOH-d4): 13.9, 19.4, 20.9, 23.2, 24.6, 26.7, 28.7, 31.0, 38.8, 51.1, 54.0, 66.2, 115.6, 117.9, 128.9, 129.8, 132.3, 132.8, 133.2, 135.2, 152.8, 164.4, 165.3, 167.2, 192.2. Synthesized product 1 1H NMR and 13 The 13C NMR spectra are shown in Figures 51 and 52, respectively.

[0073] In vitro evaluation of CYP2J2 expression in relation to heart rate intervals (A) Cell culture. Human ES cell lines (H7 ESCs) containing the knock-in and constitutively expressed genetically encoded calcium indicator (GECI), GCaMP6s, were maintained on Matrigel-coated plates in StemMACS® iPS-Brew XF (Miltenyi Biotec) and passaged in aggregates using collagenase IV (1 mg / mL) enzyme treatment. For differentiation, ESCs were passaged as single cells using Accutase (Nacalai Tesque) and subjected to a previously established small molecule-based GiWi cardiomyocyte differentiation protocol. On day 7, the medium was changed with RPMI1640 (HyClone) supplemented with B27, including insulin (B27+) (Miltenyi Biotec), for maturation, and refreshed every 2 days. On day 21, glucose levels in the medium were slowly reduced to 0% by day 28 to promote metabolic selection of more mature cardiomyocytes dependent on β-oxidation. Cardiomyocytes (H7 CM) were used for siRNA transfection on day 28.

[0074] (B) CYP2J2 siRNA knockdown. A set of four self-delivery modified Accell®-siRNAs (Table 1) and delivery medium (catalog number B-005000) were obtained from Dharmacon®. Transfection was performed on adherent cells according to the supplier's protocol. Briefly, individual wells of H7 CM cells on day 28 were treated with 1 μM of individual siRNA or 0.25 μM of all four siRNAs (pooled condition) in Accell® siRNA delivery medium for 72 hours. After replacing the delivery medium with low-glucose B27+ medium for 24 hours, video analysis and RNA collection were performed for qPCR analysis.

[0075] (C) RNA isolation. For each treatment, approximately 5 × 10⁻⁶ 6 Cells were collected and dissolved in 500 μL of TRIzol reagent (Invitrogen). After allowing the sample to stand at room temperature for 5 minutes, 180 μL of chloroform (Kanto Chemical, Japan) was added, followed by centrifugation at 12,000 × g for 15 minutes at 4°C for phase separation. The aqueous phase was then transferred to a new tube containing equal volumes of isopropanol and GlycoBlue coprecipitant (Invitrogen, USA). The sample was incubated at room temperature for 20 minutes. The sample was pelletized by centrifugation at 12,000 × g for 15 minutes at 4°C. The RNA pellet was washed with 100% ethanol, air-dried, and reconstituted in nuclease-free water (Ambion, USA).

[0076] (D) Quantitative PCR. RNA sample (250 ng) was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) to obtain cDNA. 5 ng of cDNA was subjected to qPCR using FAST SYBR Green Mix (Applied Biosystems, USA). ΔΔC TA relative quantification method based on the above was used for qPCR analysis using the QuantStudio 5 384-well Block Real-Time PCR system (Applied Biosystems, USA). The threshold cycle was set to ≥35. The data is presented as a magnification change of the CT value normalized to β-ACTIN. Unless otherwise specified, the presented data are representative of two independent experiments with error bars indicating the standard deviation (SD).

[0077] The results are shown in Figure 6.

[0078] (E) Fluorescent Ca of GCaMP6s H7 CM 2+ Imaging and video analysis. Calcium transients of processed H7 CM were imaged using a Nikon ECLIPSE Ti-S fluorescence microscope and recorded at 15fps using an Andor Zyla 4.2 sCMOS camera. The video data was analyzed using Nikon's NIS-Elements AR and processed using RStudio v1.2.1335 to identify fluorescence peaks corresponding to single cardiac contractions. Related data were then computed using a computer to obtain pulse interval data and variability data.

[0079] The results are shown in Figures 7 and 8.

[0080] (F) Statistical analysis. Data points were plotted using GraphPad Prism 7, and statistical analysis was performed. The knockdown efficiency of siRNA treatment was evaluated using Student's t-test. The inter-pulsation variability between treatment groups was evaluated using the non-parametric Mann-Whitney U test. Statistical significance was defined as p<0.05.

[0081] The results are shown in Figure 9.

[0082] In vitro time-dependent, concentration-dependent, and NADPH-dependent inactivation of CYP2J2 by ​​doronedarone and poyendarone. (A) Reagents. Acetonitrile (ACN) for high-performance liquid chromatography (HPLC) was purchased from Tedia Company Inc. (Fairfield, Ohio). Dronedarone hydrochloride, astemizole, and buspirone hydrochloride were purchased from Sigma-Aldrich (St. Louis, Missouri). Human recombinant CYP2J2 Supersomes® (rCYP2J2), and an NADPH regeneration system consisting of NADPH A (NADP+ and glucose-6-phosphate) and NADPH B (glucose-6-phosphate dehydrogenase) were purchased from BD Gentest (U-Barn, Massachusetts). Poyendarone hydrochloride was synthesized in-house according to the synthesis described herein. Water was obtained using a Milli-Q water purification system (Millipore, Billerica, Massachusetts). All other reagents were for analytical use.

[0083] (B) Time-dependent, concentration-dependent, and NADPH-dependent inactivation of CYP2J2 using astemizole as a probe substrate with doronedarone and poiendarone. A primary incubation mixture (100 μL) containing various concentrations of doronedarone (0-1.0 μM) or poiendarone (0-10.0 μM), 20 pmol / mL of rCYP2J2, 100 mM potassium phosphate buffer (pH 7.4), and NADPH B was heated at 37°C for 3-5 minutes. The enzymatic reaction was initiated by adding NADPH A. At different pre-incubation times (0 min, 3 min, 8 min, 15 min, 22 min, 30 min, 45 min), 10 μL aliquots of the primary incubation mixture were transferred to 90 μL of pre-warmed secondary incubation mixture containing buffer, astemizole (15 μM), and an NADPH regeneration system to obtain 10-fold dilutions. The secondary incubation mixture was then further incubated at 37°C for 15 minutes, after which a 70 μL aliquot was taken and quenched using ice-cold ACN containing 0.1 μM buspirone hydrochloride (internal standard). The sample was centrifuged at 2755 g at 4°C for 30 minutes, and the supernatant was used for the determination of O-desmethylastemizole by liquid chromatography-tandem mass spectrometry (LC / MS / MS). As a negative control, NADPH A was replaced with 100 mM potassium phosphate buffer. The LC / MS / MS system consisted of an AB SCIEX QTRAP® 5500 tandem mass spectrometry (MS / MS) system (AB SCIEX, Framingham, Massachusetts) interfaced with an Agilent 1290 Infinity ultra-high pressure liquid chromatography (UHPLC) system (Agilent Technologies Inc., Santa Clara, California). The LC / MS / MS system was controlled by Analyst 1.4.2 software (Applied Biosystems) for peak integration of all chromatography data. ACQUITY UPLC BEH C 18Chromatographic separation was achieved using a 1.7 μM, 2.1 × 50 mm column (Waters, Milford, Massachusetts). The column and sample temperatures were maintained at 45°C and 4°C, respectively. The mobile phases used were 0.2% acetic acid (solvent A) of 5 mM ammonium acetate dissolved in water and 0.2% acetic acid (solvent B) of ACN. These were delivered at a flow rate of 0.6 mL / min. The elution conditions were optimized as follows: a linear gradient of 30–95% B (0–1.60 min), isocratic elution at 95% B (1.61–1.99 min), and isocratic elution at 30% B (2.00–2.50 min). Multiple reaction monitoring (MRM) transitions of mass-to-charge ratio (m / z) from 445 to 121 and 386 to 122 were performed in positive electrospray ionization (ESI) mode to detect O-desmethylasterizole and buspirone, respectively. The MS source conditions were: curtain gas: 25 psi, collision gas: medium, ion spray voltage: 5500 V, temperature: 550 °C, ion source gas 1: 50 psi, ion source gas 2: 55 psi. The compound-dependent mass spectrometry (MS) parameters for O-desmethylasterizole and buspirone are summarized in Table 2.

[0084] (C) Calculation of kinetic parameters. To calculate the kinetic parameters of inactivation, the mean of three sets of peak area ratios was normalized to 0 minutes relative to the pre-incubation time. The percentage of remaining probe activity was calculated, and the natural log activity was plotted against the pre-incubation time. The data were fitted to a linear regression. The slope of the graph indicates the observed rate of inactivation (K). obs The kinetic parameter K is determined. I and k inact , as well as the ability to deactivate, k inact / K I This was calculated using Equation 1 and a nonlinear least-squares regression method with GraphPad Prism 6.01 (San Diego, California). Here, k inact This represents the maximum deactivation rate constant, K I[I] is the concentration of the inhibitor at the maximum half-value of the inactivation rate constant, and [I] is the in vitro inactivator concentration.

number

[0085] The results are shown in Figure 10.

[0086] (D) Time-dependent and concentration-dependent inactivation of CYP2J2 by ​​doronedarone, poyendarone, and additional deuterated doronedarone analogs using clinically relevant probe substrate rivaroxaban. Inactivation of human recombinant CYP2J2 by ​​doronedarone, poyendarone, compound 2, and compound 3 was investigated using rivaroxaban as a probe substrate. Three incubations were performed in 96-well plates. Primary incubation mixtures containing various concentrations of inhibitors (0-20 μM) were pre-incubated at 37°C for 3 minutes in potassium phosphate buffer (100 mM, pH 7.4) with CYP450 enzyme (20 pmol / mL) and NADPH B. 5 μL of NADPH A was added to the primary incubation to initiate the enzymatic reaction. The final volume of the primary incubation mixture was 100 μL, and the organic solvent content was <1% v / v. At different pre-incubation time points (3 min, 8 min, 15 min, 22 min, 30 min, and 45 min) after the addition of NADPH A, 5 μL aliquots of the primary incubation were transferred to 95 μL of secondary incubation containing 50 μM rivaroxaban, NADPH A and NADPH B, and potassium phosphate buffer (100 mM, pH 7.4) to obtain 20-fold dilutions. The secondary incubation mixture was incubated with CYP2J2 at 37°C for 30 minutes, after which an 80 μL aliquot was taken and quenched in an equal volume of ice-cold ACN containing 4 μM dexamethasone as IS. The samples were then centrifuged at 3220 g at 4°C for 30 minutes, and the supernatant was transferred to a 96-well plate for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. The main rivaroxaban metabolites and morpholinone hydroxylated metabolites were quantified using LC-MS / MS analysis.

[0087] (E) Kinetic parameter of inactivation (K I and k inact ) calculation The average of the three sets of peak area ratios for each concentration and pre-incubation time was normalized to the peak area ratio of 0 μM at the same pre-incubation time. The amount of the metabolite of rivaroxaban formed during the secondary incubation (a measure of the remaining probe substrate activity) was calculated computationally, and the natural logarithm of this measure was plotted against the pre-incubation time for each inactivator concentration. Then, for each concentration, the data was fit to a linear regression model to obtain k obs (the apparent inactivation rate constant) as the negative slope of the linear regression. A plot of k obs against the inactivator concentration ([I]) was used to fit the inactivation kinetic parameters (K I and k inact ) based on Equation 1 to a non-linear least squares regression using GraphPad Prism 8. [Number]

[0088] In Equation 1, k inact = the maximum inactivation rate constant at infinite inactivator concentration (min -1 ), K (the apparent inactivation rate constant) as the negative slope of the linear regression. A plot of k obs against the inactivator concentration ([I]) was used to fit the inactivation kinetic parameters (K I and k inact ) based on Equation 1 to a non-linear least squares regression using GraphPad Prism 8. [Number]

[0088] In Equation 1, k inact = the maximum inactivation rate constant at infinite inactivator concentration (min -1 ), K I = the concentration of the inactivator at the maximum half-value rate constant of inactivation (μM), [I] = the in vitro inactivator concentration (μM). The MBI ability (k inact / K I ratio, μM-1.min-1) for human recombinant CYP2J2 was determined for each compound. The higher the k inact / K I ratio, the greater the MBI ability.

[0089] The results are shown in Figures 11 and 12 and summarized in Table 3.

[0090] In vitro expression of human CYP2J2 and sEH mRNA in hiPSC-CM, and in vitro inhibition of human CYP2J2 in hiPSC-CM by amiodarone, dronedarone, and poietarone (A) Reagents. ACN and dimethyl sulfoxide (DMSO) for high-performance liquid chromatography (HPLC) were purchased from Tedia Company Inc. (Fairfield, Ohio). Dronedarone hydrochloride, amiodarone hydrochloride, astemizole, and buspirone hydrochloride were purchased from Sigma-Aldrich (St. Louis, Missouri). The Milli-Q water purification system was obtained from EMD Millipore (Villerica, Massachusetts). Poyendarone hydrochloride was synthesized in-house according to the synthesis described herein. All other reagents were for analytical use. Stock solutions of driporone, poyendarone, astemizole, and buspirone were prepared in DMSO stored at -20°C.

[0091] (B) Cell lines and cultures. Human foreskin fibroblasts were reprogrammed to form human induced pluripotent stem cells (hiPSCs) using a virus-free method. The hiPSC cell lines were prepared as previously reported. 1 They were differentiated into cardiomyocytes (hiPSC-CM).

[0092] (C) Total RNA extraction and quantitative gene expression. Total RNA was isolated from 30-day-old hiPSC-CMs using the RNeasy Kit (Qiagen GmbH, Hilden, Germany). The isolated RNA was quantified using a NanoDrop® 2000 UV-Vis spectrophotometer (Thermo Fischer, Waltham, Massachusetts). 1 μg of total RNA was converted to cDNA using the Superscript III first-strand synthesis kit (Invitrogen). The cDNA template was used for PCR with the Quantifast Kit (Qiagen GmbH, Hilden, Germany), which contains SYBR Green dye as a DNA binder. PCR was performed using a Biorad (Applied Biosciences) thermocycler under the following conditions: 2 minutes at 55°C, 5 minutes at 95°C, followed by 10 seconds at 95°C and 30 seconds at 60°C for 40 cycles. Template control was not performed to confirm primer dimerization. Relative quantification was performed using the ΔCt method with GAPDH as an endogenous control. PCR products were mixed with 6× loading dye (Thermo Fischer, Waltham, Massachusetts) and loaded onto a 3% agarose gel prepared in 1× TAE buffer (Vivantis, Subang Jaya, Malaysia) containing 1× GelRed® nucleic acid stain (Biotium, Fremont, California). The GeneRuler Ultra Low Range DNA ladder (Thermo Fischer) was used as a molecular weight marker. Degraded cDNA samples were analyzed using Gel Doc® EZ (Bio Rad, Hercules, California) with Image Lab® (Bio Rad) image processing software. In human cardiomyocytes, CYP2J2 and sEH are encoded by the CYP2J2 gene and the EPHX2 gene. GAPDH was used as an endogenous control. The forward and reverse primer sequences for CYP2J2, EPHX2, and GAPDH are summarized in Table 4.

[0093] (D) Inhibition of CYP2J2-mediated astemizole metabolism. HiPSC-CM cells were seeded at a density of 100,000 cells / well in 12-well plates and differentiated for 30 days. After differentiation, hiPSC-CM cells were incubated in EB2 medium with dronedarone, amiodarone, or poyendarone (2 μM) and astemizole (1 μM) at 37°C for 24 hours under a humidified atmosphere of 5% CO2. The cells were then washed twice with 1 × phosphate-buffered saline (PBS) and removed using Accutase® cell detachment solution. The cells were then pelleted at 2000 g at 4°C for 10 minutes. The supernatant was discarded, and the pellet was resuspended in 100 μL of ice-cold ACN containing 0.1 μM buspirone as an internal standard. The cells were sonicated on ice for 15 minutes to induce complete cell lysis. The dissolved solution was centrifuged at 10,000 × g for 15 minutes at 4°C. The supernatant was collected and analyzed using LC / MS / MS.

[0094] (E) Measurement of residual CYP2J2 enzyme activity by LC / MS / MS. The LC / MS / MS system consisted of an Agilent 1290 Infinity ultra-high pressure liquid chromatography (UPLC) system (Agilent Technologies Inc., Santa Clara, California) interfaced with an AB SCIEX QTRAP® 5500 tandem mass spectrometry (MS / MS) system (AB SCIEX, Framingham, Massachusetts). The LC / MS / MS system was controlled by Analyst 1.4.2 software (Applied Biosystems) which performed peak integration of all chromatography. ACQUITY UPLC BEH C 18Chromatographic separation was achieved using a 1.7 μM, 2.1 × 50 mm column (Waters, Milford, Massachusetts). The column and sample temperatures were maintained at 45°C and 4°C, respectively. The mobile phases were 0.2% acetic acid in 5 mM ammonium acetate (solvent A) and 0.2% acetic acid in ACN (solvent B). These were delivered at a flow rate of 0.6 mL / min. The elution conditions were optimized as follows: a linear gradient of 30–70% B (0–1.60 min), isocratic elution at 70% B (1.61–1.99 min), and isocratic elution at 30% B (2.00–2.50 min). M / z ratio MRM transitions were performed in positive ESI mode to detect O-desmethylasterizole and buspirone (internal standard). Compound-dependent MS parameters are summarized in Table 2.

[0095] (F) Data analysis. Amiodarone, delonedarone, and poiendarone were compared using a two-way analysis of variance (ANOVA), followed by Tukey's post-hoc test. Each value was presented as mean ± SD using GraphPad Prism. * p<0.05, ** p<0.01, *** p<0.001, # Statistical significance was confirmed when p < 0.0001.

[0096] The results are shown in Figure 13.

[0097] Cytotoxicity measurement of poyendarone and dronedarone (A) Reagents. Dronedarone hydrochloride and all-trans retinoic acid (ATRA) were purchased from Sigma-Aldrich (St. Louis, Missouri). Poyendarone hydrochloride was synthesized in-house. 14,15-EET was purchased from Cayman Chemical (Ann Arbor, Michigan). Water was obtained using the Milli-Q water purification system (Millipore, Billerica, Massachusetts). All other reagents were for analytical use.

[0098] (B) H9c2 cell culture. The H9c2 cell line was purchased from American Tissue Type Collection (ATCC) (Manassas, Virginia). Growth medium (catalog number: 31600, Thermo Fisher Scientific, Waltham, Massachusetts) was prepared in low-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 1.5 g / L sodium bicarbonate, 25 mM hepes, 10% fetal bovine serum (FBS) (GE Healthcare, Singapore), 100 units / mL penicillin, 100 μg / mL streptomycin, and 250 ng / mL amphotericin B, for 75 cm³. 2 Cells were cultured in tissue culture flasks at 37°C under a humidified atmosphere of 5% CO2. Cells were nourished every 2-3 days, and subculture was performed when the culture density reached 70-80% to prevent loss of differentiation potential. For differentiation, H9c2 cells were seeded at a density of 100,000 cells / well into 12-well plates. Cells were maintained in low-glucose, high-serum growth medium for 1 day to allow for adhesion. Subsequently, cells were differentiated using 1 μM ATRA.

[0099] (C) Simultaneous reduction of cytotoxicity and intracellular ATP. H9c2 cells were seeded in white chimney plates and differentiated. To overcome the Crabtree effect, the medium was changed every 48 hours to DMEM containing 10 mM galactose, 2 mM glutamine (final concentration 6 mM), 5 mM hepes, 1 mM sodium pyruvate, 100 U / mL penicillin, 100 μg / mL streptomycin, 0.25 μg / mL amphotericin B, 1% FBS, 1 × insulin-transferrin-selenium, and 1 μM ATRA. Cells were treated with doronedarone or poyendarone (0-30 μM) in serum-free galactose medium for 6 hours. Cytotoxicity and intracellular ATP content were simultaneously measured using the manufacturer's protocol with the Mitochondrial ToxGlo® kit (Promega, Madison, Wisconsin). The Mitochondrial ToxGlo® assay measures cell viability using the fluorescent peptide bis-alanine-alanine-phenylalanine-R110 (bis-AAF-R110). Because bis-AAF-R110 is impermeable to living cells, it is a selective peptide for measuring protease activity in dead cells. Here, fluorescence derived from metabolized bis-AAF-R110 was measured at different concentrations of the test compound and compared to a control. As the concentration of the cytotoxic compound increased, the number of dead cells increased, resulting in a higher fluorescence signal. ATP was measured by adding an ATP detection reagent that induces cell lysis and generates a luminescence signal proportional to the amount of ATP present. Differentiated H9c2 cells were pre-treated in serum-free galactose medium with various concentrations (0–1 μM) of 14,15-EET for 2 hours. The medium was aspirated, cells were treated with delonedarone, and cytotoxicity and intracellular ATP were measured as described.

[0100] (D) Mitochondrial membrane potential (Δψ m Measurement of Δψ for each test compound using the mitochondrial permeable dye, tetramethylrhodamine methyl ester perchlorate (TMRM) (Thermo Fisher Scientific, Waltham, Massachusetts). mDissipation was measured. H9c2 cells were seeded in a 96-well plate at a density of 20,000 cells per well. Cells were treated with any of the delonedarones in serum-free low-glucose medium for 1 hour. The medium was discarded and the cells were washed twice with 1×PBS. TMRM dye (200 nM) was dissolved in serum-free medium and the plate was incubated at 37°C for 30 minutes. The medium was aspirated, the cells were washed with PBS, and fluorescence was measured at an excitation wavelength of 557 nm and an emission wavelength of 570 nm. TMRM concentration and cell density were optimized. Differentiated H9c2 cells were pretreated with 14,15-EET at various concentrations (0-1 μM) for 2 hours. The medium was aspirated, and the cells were then treated with delonedarone and Δψ was measured by AAD. m The dissipation was measured as described.

[0101] The results are shown in Figure 14.

[0102] Measurement of extracellular field potential duration (FPD) of HiPSC-CM induced by amiodarone, doronedarone, and poyendarone. (A) Electrophysiological measurements. Electrophysiological perturbations of hiPSC-CM were measured using a multi-electrode array (MEA) recording system (Multichannel Systems, Reutlingen, Germany). HiPSC-CM were treated with amiodarone, dolonedarone, or amiodarone (0-10 μM) administered to 2 mL of culture medium, and the extracellular field potential duration (FPD) was measured as described above. 2 As previously described, the Bazzet correction formula: cFPD = FPD / √(RR interval) normalizes the FPD measurement to the number of heartbeats in the contraction region (corrected FPD [cFPD]). 3 The corrected FPD curve was plotted using a single-phase attenuation algorithm.

[0103] The results are shown in Figure 15.

[0104] (B) Cell culture and transfection (Na V1.5 mA). HEK293FT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, and maintained in a 5% CO2 incubator at 37°C. For transfection, cells were seeded in a petri dish with a coverslip and grown overnight. Subsequently, 3.0 μg of wild-type or mutant hNa was added. V A 1.5-channel plasmid and 1.5 μg of β1 plasmid were simultaneously transfected into cells using lipofectamine 2000 (Invitrogen). The transfected cells were grown in a 5% CO2 incubator at 37°C for 24 hours prior to patch-clamp analysis, and then divided and seeded onto poly-D coated coverslips 24 hours before patch-clamp analysis.

[0105] (C) Whole cell patch clamp recording and data analysis. Na V 1.5 To record the current, the internal solution (pipette solution) contained, in mM units, 130 CsF, 5 NaCl, 5 ethyleneglycol bis(2-aminoethylether)tetraacetic acid (EGTA), 10 hepes, 2 MgCl2, 2 tetraethylammonium chloride (TEA-Cl), and pH 7.2 (adjusted with CsOH). The external solution contained, in mM units, 135 NaCl, 4.2 CsCl, 1.2 MgCl2, 1.8 CaCl2, 10 hepes, and glucose, and was adjusted to pH 7.4 with NaOH. Whole-cell current was obtained under fixed voltage using an Axopatch 200B or Multiclamp 200B amplifier (Molecular Device), and low-pass filtering was performed at 5-6 kHz. Typically, the series resistance was <5 MΩ after >70% compensation. Leak transients and volumetric transients were subtracted online using the P / 4 protocol. The dose-response curve was fitted using the log(inhibitor) versus response variable gradient equation, Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0106] The results are shown in Figures 16, 17, and 18.

[0107] (D) Cell culture and transfection (Ca V 1.2 current). HEK293FT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, and maintained in a 5% CO2 incubator at 37°C. For transfection, cells were seeded in petri dishes with coverslips and grown overnight. Subsequently, 1.7 μg of human Ca was transfected with lipofectamine 2000 (Invitrogen). V 1.2_1a8a cardiac mutants and 1.25 μg of human β2 and α2δ1 subunits were obtained. Transfected cells were grown in a 5% CO2 incubator at 37°C for 24 hours prior to patch-clamp analysis, and then divided and seeded onto poly-D coated coverslips 24 hours before patch-clamp analysis.

[0108] (E) Whole cell and patch clamp recording and data analysis. V1.2 To record the current, the internal solution (patch pipette solution) contained the following: 138 mM Cs-MeSO3, 5 mM CsCl, 5.0 mM EGTA, 10 mM hepes, 1 mM MgCl2, and 2 mg / ml Mg-ATP, with a pH of 7.3 (adjusted with CsOH) and a glucose concentration of 290 mOsm. The external solution contained the following: 10 mM hepes, 140 mM tetraethylammonium methanesulfonate, and 5 mM CaCl2 (pH adjusted to 7.4 with CsOH, and osmotic pressure adjusted to 290-310 with glucose). A pipette with a resistance of 1.5-2 MΩ was used. Hall cell current was obtained under fixed voltage using an Axopatch200B or Multiclamp 200B amplifier (Molecular Device), and low-pass filtering was performed at 1 kHz. Typically, the series resistance was <5 MΩ after >70% compensation. Leak transients and capacitive transients were subtracted online using the P / 4 protocol. The dose-response curve was fitted using the log(inhibitor) versus response variable gradient equation, Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0109] The results are shown in Figures 19, 20, and 21.

[0110] (F) Cell culture and transfection (K v 11.1 Current). HEK293FT cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin, and maintained in a 5% CO2 incubator at 37°C. For transfection, cells were seeded in a Petri dish and grown overnight. Subsequently, 2 μg of wild-type or mutant K was administered. v Cells were simultaneously transfected with a 11.1 channel plasmid and 1 μg of KCNE1 plasmid using lipofectamine 2000. Transfected cells were incubated in a 5% CO2 incubator at 37°C for 24 hours. 48 hours after transfection, cells were divided and seeded on poly-D-lysine coverslips one day prior to recording.

[0111] (G) Whole cell and patch clamp recording and data analysis. K v 11.1 To record the current, the internal solution (pipette solution) contained, in mM units, 130 k-gluconic acid, 10 kCl, 5 EGTA, 10 hepes, 1 mMgCl2, 0.5 mNa3GTP, 4 mMg-ATP, and sodium phosphocreatine at pH 7.4 (adjusted with KOH). The external solution contained, in mM units, 125 mNaCl, 2.5 mKCl, 25 mNa-gluconic acid, 1.0 mMgCl2, 1.8 mCaCl2, 10 hepes, and 11.1 mGlucose, and was adjusted to pH 7.4 with NaOH. Whole-cell current was obtained under fixed voltage using an Axopatch 200B or multiclamp 200B amplifier (Molecular Device), and low-pass filtering was performed at 1 kHz. Typically, the series resistance was <5 MΩ after >70% compensation. Leak transients and capacitive transients were subtracted online using the P / 4 protocol. v 11.1 A current was induced from a holding potential of -80mV to 20mV in a 2.5s pulse. The voltage was then returned to -60mV and the tail current was recorded. The dose-response curve was fitted using the log(inhibitor) versus response variable gradient equation, Y = Bottom + (Top - Bottom) / (1 + 10^((LogIC50 - X) * HillSlope)).

[0112] The results are shown in Figures 22, 23, and 24.

[0113] In vitro measurement of interpulse variability (BBV) in HiPSC-CM induced by amiodarone, doronedarone, and poiendarone. (A) Measurement of BBV. BBV calculation is performed using Kubios HRV 2.2 (Department of Applied Physics, University of Eastern Finland, Kuopio, Finland). 4 This was done using [method / tool]. BBV is visualized as a scatter plot known as a Poincaré plot. In these plots, each time interval between two consecutive beats [interval between beats (RR)] n)] followed by the interval (RR n+1 The plot is then generated against the given timeframe. The Poincaré plot descriptors, SD1 and SD2, define the degree of variation. SD1 represents a measure of short-term variation, while SD2 represents long-term variation. 5 .

[0114] The results are shown in Figure 25.

[0115] In vivo pharmacokinetic studies of doronedarone and poyendarone in dogs (A) Reagents. Acetonitrile (ACN) for high-performance liquid chromatography (HPLC) was purchased from Tedia Company Inc. (Fairfield, Ohio). Dronedarone hydrochloride was purchased from Sigma-Aldrich (St. Louis, Missouri). N-desethylamiodarone (NDEA) hydrochloride was purchased from Cayman Chemical (Ann Arbor, Michigan). Poyendarone hydrochloride was synthesized in-house according to the synthesis described herein. Water was obtained using the Milli-Q water purification system (Millipore, Billerica, Massachusetts). Dimethyl sulfoxide (DMSO) was purchased from VWR Life Science (Pennsylvania, USA).

[0116] (B) Plasma samples. For each compound (dronedarone or poiendarone), plasma samples were obtained from four male dogs weighing approximately 10 kg each, provided by Kitayama Labes Co., Ltd. (Nagano Prefecture, Japan). All experiments were approved by the Animal Experiment Committee of Toho University (No. 12-52-151) and conducted in accordance with the Animal Experiment Handling Regulations of Toho University. Plasma samples were obtained at 5, 10, 15, 20, 30, 45, and 60 minutes after intravenous (IV) bolus administration of 3.0 mg / kg of dronedarone or poiendarone. Plasma samples were stored at -80°C and thawed in ice before use.

[0117] (C) Sample preparation. Ice-cold NDEA internal standard (IS) solution was used for protein precipitation at a plasma:IS ratio of 1:3. The mixture was vortex-mixed for 1 minute, then centrifuged at 14,000 g at 4°C for 15 minutes. The supernatant was then transferred to a vial for LC / MS / MS analysis.

[0118] (D) Instruments. The LC / MS / MS system consisted of an Infinity ultra-high pressure liquid chromatography (UHPLC) system (Agilent Technologies Inc., Santa Clara, California) equipped with an AB SCIEX QTRAP® 5500 tandem mass spectrometry (MS / MS) system (AB SCIEX, Framingham, Massachusetts). All chromatographic peak integrations were performed using MultiQuant software version 1.4.0.18067 (Applied Biosystems).

[0119] (E)LC / MS / MS conditions. Chromatographic separation is performed using a gradient elution program, ACQUITY UPLC BEH C 18 The analysis was performed using a 1.7 μm, 2.1 × 50 mm column (Waters, Milford, Massachusetts). The mobile phases were 0.2% acetic acid (A) of 5 mM ammonium acetate dissolved in water and 0.2% acetic acid (B) of ACN. The elution conditions were a linear gradient from 30–95% B (0–1.60 min), isocratic at 95% B (1.61–1.99 min), and isocratic at 30% B (2.00–2.50 min)

[21] . Multiple reaction monitoring (MRM) transitions were optimized for NDEA (internal standard, IS), delonedarone, and poyendarone. Other MS conditions are listed in Table 5.

[0120] (F) Derivation of pharmacokinetic parameters using non-compartmental analysis (NCA). Individual pharmacokinetic parameters were estimated using NCA on IV bolus data (model 201) with WinNonlin® (Pharsight, USA). The area under the curve (AUC) from time 0 minutes to the last observation time and the AUC extrapolated to infinity (AUCinf) were calculated using the logarithmic trapezoid rule. The data points included in the calculation of lambda Z (λz) were first determined by WinNonlin® and then visually evaluated and corrected accordingly. Other parameters calculated included apparent total plasma clearance (CL) and apparent volume of distribution (V) based on the terminal phase. z ), apparent emission half-life (T 1 / 2 ), apparent volume (V) in steady state ss This includes the mean residence time (MRT) and the MRT extrapolated to infinity (MRTinf). ss This represents the apparent volume of distribution when plasma concentrations across all compartments reach steady-state conditions. Next, WinNonlin® was used to fit each drug into the compartment model and visualize the individual disposition models.

[0121] (G) Statistical analysis. Pharmacokinetic parameters were logarithmically transformed, and it was assumed that the distribution of the logarithmically transformed data was normal. In the case of one-way ANOVA, the null hypothesis was that the mean pharmacokinetic parameter estimates (CL and V) between doronedarone and poyendarone were equal. z The results showed no significant difference between the two values. All statistical tests were performed using IBM SPSS Statistics for Windows, version 25.0 (IBM Corp, Armonk, New York).

[0122] The results are shown in Figure 26.

[0123] Comparison of in vivo pharmacokinetics of poyendarone and dronedarone (A) Chemicals. Acetonitrile (ACN) for high-performance liquid chromatography was purchased from Tedia Company Inc. (Fairfield, Ohio). Dronedarone hydrochloride and dexamethasone were obtained from Sigma-Aldrich (St. Louis, Missouri). Poyendarone hydrochloride was synthesized in-house. Pooled human liver microsomes (HLM), recombinant human CYP450 supersomes, and NADPH A (NADP + A reduced nicotinamide adenine dinucleotide phosphate (NADPH) regeneration system consisting of glucose-6-phosphate and NADPH B (glucose-6-phosphate dehydrogenase) was obtained from BD Gentest (Uburn, Massachusetts). Water was obtained using a Milli-Q water purification system (Millipore, Billerica, Massachusetts). All other reagents were for analytical use.

[0124] (B) Metabolic stability studies. Metabolic stability experiments were conducted, and intrinsic clearance (CL) was determined by the substrate depletion method. int The values ​​were derived. 1 μM dolonedarone or poyendarone was pre-incubated at 37°C for 5 minutes with CYP3A4 (10 pmol / mL), CYP3A5 (10 pmol / mL), or HLM (0.5 mg / mL) in potassium phosphate buffer (100 mM, pH 7.4) and NADPH B. Then, NADPH A was added to start the reaction. The final incubation mixture volume was 100 μL, and the organic solvent content was <1% v / v. The reaction mixture was incubated at 37°C with gentle stirring. At each time point (0-60 minutes), 80 μL of the reaction mixture was quenched using an equal volume of ice-cold ACN containing 1 μM tamoxifen as an internal standard (IS). The samples were then centrifuged at 3220 g at 4°C for 30 minutes, and the supernatant was transferred to a 96-well plate for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0125] The results are shown in Figure 27.

[0126] (C)CLint Calculation of values. The average of three sets of peak area ratios for each compound was normalized relative to the peak area ratio of the same compound at time 0 to obtain the percentage of substrate remaining at each time point. This was plotted against the incubation time for each compound, and then the data was fitted to a single-phase decay model using GraphPad Prism software (version 8.02, GraphPad Inc., San Diego, California) to obtain an estimate of the efflux rate constant (k). Then, CL int This was calculated based on Equation 3.

number

[0127] In Equation 3, k = discharge rate constant (min -1 V = volume of incubation mixture (mL), P = amount of protein in the mixture (mg).

[0128] The results are shown in Table 6.

[0129] (D) Time-dependent and concentration-dependent inactivation of CYP3A4 and CYP3A5. Inactivation of human recombinant CYP3A4 and CYP3A5 by doronedarone and poyendarone was investigated using rivaroxaban as a probe substrate. Three incubations were performed in 96-well plates. Primary incubation mixtures containing various concentrations of inhibitors (0-20 μM) were pre-incubated at 37°C for 3 minutes in potassium phosphate buffer (100 mM, pH 7.4) with CYP3A4 or CYP3A5 (20 pmol / mL) and NADPH B. 5 μL of NADPH A was added to the primary incubation to initiate the enzymatic reaction. The final volume of the primary incubation mixture was 100 μL, and the organic solvent content was <1% v / v. At different pre-incubation time points after the addition of NADPH A (3 min, 8 min, 15 min, 22 min, 30 min, and 45 min), 5 μL aliquots of the primary incubation were transferred to 95 μL of secondary incubation containing 50 μM rivaroxaban, NADPH A and NADPH B, and potassium phosphate buffer (100 mM, pH 7.4) to obtain 20-fold dilutions. The secondary incubation mixture was incubated with CYP3A4 or CYP3A5 at 37°C for 2 minutes, after which an 80 μL aliquot was taken and quenched in an equal volume of ice-cold ACN containing 4 μM dexamethasone as the isolation solution (IS). The samples were then centrifuged at 3220 g at 4°C for 30 minutes, and the supernatant was transferred to a 96-well plate for liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis. The main rivaroxaban metabolites and morpholinone hydroxylated metabolites were quantified using LC-MS / MS analysis.

[0130] (E) Kinetic parameter of inactivation (K I and k inactThe calculation of k was performed. The mean of three sets of peak area ratios for each inhibitor concentration and pre-incubation time was normalized against the peak area ratio of 0 μM inhibitor at the same pre-incubation time. The amount of rivaroxaban metabolite formed during secondary incubation (a measure of residual probe substrate activity) was calculated by computer, and the natural logarithm of this measure was plotted against the inactivation pre-incubation time for each inactivator concentration. Then, for each concentration, the data was fitted to a linear regression model to obtain k obs The (apparent deactivation rate constant) was obtained as the negative slope of a linear regression. k with respect to the inactivator concentration ([I]) obs The plot shows that using GraphPad Prism 8, the nonlinear least-squares regression based on Equation 4 has an inactivation kinetic parameter (K, which will be explained below). I and k inact ) was fitted.

number

[0131] In equation 4, k inact = Maximum potential deactivation rate constant at infinite inactivator concentration (min -1 ), K I =Rate constant of the maximum half-value of inactivation (μM), [I] =In vitro inactivator concentration (μM).

[0132] The results are shown in Table 6.

[0133] (F) Development of physiologically-based pharmacokinetics (PBPK) models. PBPK models for dolonedarone and poyendarone were constructed using the Simcyp simulator (version 19.0.96.0, Sheffield, UK). Table 7 below lists important drug-dependent parameters implemented in Simcyp, based on information from the literature. The PBPK model for dolonedarone was constructed using a middle-out approach, combining data from in vitro experiments and observed clinical parameters.

[0134] The results are shown in Figure 28.

[0135] (G) Validation of the PBPK model. Using the Simcyp simulator, two scaling factors (milligrams of protein per gram of liver (MPPGL) and mean liver weight) were used to determine the CL. int Value and f u,mic The value is scaled to unbound liver-specific clearance CL u,int,H We obtained the following results. By applying a well-stirred model of hepatic clearance, we obtained hepatic blood clearance, CL. b,H This is then calculated, and subsequently converted into the blood-to-plasma concentration ratio (blood-to-plasma: B / P) and the percentage of metabolism (f m It was converted to plasma clearance by ).

[0136] The hypothetical population used was Sim-Healthy Volunteer, except that the maximum age was changed to 67 to match the age of the selective clinical trial used to validate the simulation. Simulated plasma concentration-time profiles were generated for each dosing regimen (e.g., intravenous, single oral, multiple oral), and compared with clinical data obtained by digitizing published mean plasma concentration-time data using WebPlotDigitizer (version 4.2). The validity of the predicted pharmacokinetic (PK) parameters was examined, where possible, in accordance with Abduljalil et al. 9 The range of validity generated was compared using the method outlined in [reference]. Where the success criterion could not be computer-calculated, a standard 0.5x vs. 2x ratio was used.

[0137] The results are shown in Figure 29.

[0138] Measurement of cLogP, water solubility, effective transmittance, and in vitro metabolic half-life of doronedarone and poyendarone. (A) Assay. The calculated Log P (cLogP) for delonedarone and poyendarone was calculated using ChemSketch. Multiscreen HTS The water solubility of each compound was measured in a general-purpose buffer (pH 7.4) using a PCF filter plate. The effective permeability of each compound was measured using a parallel artificial membrane permeability assay (PAMPA). Finally, the in vitro metabolic half-lives (T) of 1 μM doronedarone and 1 μM poyendarone were measured. 1 / 2 ) was measured using recombinant human CYP2J2.

[0139] The results are shown in Table 8.

[0140] Comparison of the effects of antiarrhythmic drugs on the refractory period of the atrial (AERP) and ventricular (VERP), and comparison of the effects of antiarrhythmic drugs on the early (J-Tpeakc) and late (Tpeak-Tend) repolarization periods. (A) Materials and Methods. Experiments were conducted on female dogs weighing approximately 10 kg for each drug (dronedarone, amiodarone, and poyendarone) (n=4). Animals were obtained through Kitayama Labes Co., Ltd. (Nagano Prefecture, Japan). All experiments were approved by the Toho University Animal Experiment Committee (No. 18-51-395) and conducted in accordance with the Toho University Animal Experiment Handling Regulations.

[0141] (B) General anesthesia and surgical preparation. The dogs were initially anesthetized with thiopental sodium (30 mg / kg, iv). After intubation with a cuffed endotracheal tube, anesthesia was maintained by inhaling vaporized halothane (1% v / v) in oxygen using a volume-restricted ventilator (SN-480-3, Shinano Seisakusho Co., Ltd. (Tokyo, Japan)). The tidal volume and respiratory rate were set to 20 mL / kg and 15 breaths / min, respectively. Six clinically available catheter-sheath sets (FAST-CATH® 406119, St. Jude Medical Daig Division, Inc., Minnesota, USA) were used, with two inserted into the left and right femoral arteries toward the abdominal aorta, two inserted into the right femoral vein, and the remaining two inserted into the left femoral vein toward the inferior vena cava. To prevent blood clotting, heparin calcium (100 IU / kg) was administered intravenously through the flush line of the catheter sheath placed in the right femoral vein.

[0142] (C) Cardiac hemodynamic variables. Left ventricular pressure was measured by placing a pigtail catheter in the left ventricle through the right femoral artery, while aortic pressure was measured through a flush line in the space between the inside of the catheter sheath and the outside of the pigtail catheter. Left ventricular end-diastolic pressure was defined as the left ventricular pressure at one time point in the peak of the R wave on the ECG. Maximum rate of increase in left ventricular pressure (LVdP / dt max ) and left ventricular end-diastolic pressure were obtained during sinus rhythm to estimate contractility and left ventricular preload, respectively. A thermodilution catheter (TC-504 NH, Nihon Kohden Corporation, Tokyo, Japan) was placed on the right side of the heart via the right femoral vein. Cardiac output was measured using a cardiac output computer (MFC-1100, Nihon Kohden Corporation) with a standard thermodilution method. Total peripheral vascular resistance was calculated using the basic formula: Total peripheral vascular resistance = Mean blood pressure / Cardiac output.

[0143] (D) Electrophysiological variables. Lead II electrocardiograms were obtained from limb electrodes. P wave duration, PR interval, QRS width, and QT interval were measured, and the QT interval was corrected using Van de Water's formula: QTc = QT - 0.087 × (RR - 1,000) (RR is obtained in ms). JT peakand T peak -T end The following was measured. If the end of the T wave was unclear, the inventors used a monophasic action potential (MAP) signal as a guide to estimate the end. The coefficient (JT) described above was used. peak c=JT peak / RR 0.58 (RR is obtained in seconds) and JT peak This was corrected. In previous QT / QTc studies, T peak -T end Since it has been shown that resting heart rate is dependent on minimum heart rate, peak -T end No correction was made for this.

[0144] A standard 6-French quadrupole catheter (Cordis-Webster Inc., Baldwin Park, California, USA) was placed in the non-coronary apex of the aortic valve via the left femoral artery to obtain a His bundle electromorphism. Another 6-French quadrupole catheter (Cordis-Webster Inc.) was placed in the sinoatrial node region of the right atrium via the right femoral vein, electrically paced, and a local electromorphism was recorded. A bidirectional MAP recording / pacing combination catheter (1675P, EP Technologies, Inc., California, USA) was placed in the endocardium of the interventricular septum of the right ventricle via the left femoral vein to obtain a MAP signal. The signal was amplified with a DC preamplifier (Model 300, EP Technologies, Inc.). The duration of the MAP signal was measured as the interval from the MAP elevation stroke to the desired repolarization level along a horizontal line corresponding to the diastolic baseline. The interval (ms) at 90% repolarization was defined as the MAP 90 This was defined as follows.

[0145] The heart was electrically stimulated by a cardiac stimulator (SEC-3102, Nihon Kohden Corporation) via pacing electrodes of a combination catheter placed in the right ventricle or electrodes of a catheter placed in the right atrium. The stimulation pulse was rectangular in shape, 1-2V (approximately twice the threshold voltage), and had a duration of 1ms. During sinus rhythm (MAP) 90(sinus) ) at 400ms (MAP 90(CL400) ) and 300ms (MAP 90(CL300) ) Ventricular MAP at pacing period length 90 The following were measured. The effective atrial refractory period (AERP) and effective ventricular refractory period (VERP) were evaluated using programmed electrical stimulation. The pacing protocol consisted of five basal stimuli with a period length of 400 ms, followed by premature stimuli at various coupling phases. Starting from end-diastole, coupling phases were shortened by 5 ms attenuation until additional stimuli could no longer elicit a response. AERP and VERP were defined as the shortest coupling phases at which a response could be produced. The duration of the terminal ventricular repolarization phase, which reflects the three-phase repolarization time of the action potential, was measured at the same site as the MAP. 90(CL400) and VERP (End-of-Life Repolarization Period = MAP) 90(CL400) The degree of electrical vulnerability of the ventricular muscle was estimated by calculating the difference between -VERP and -VERP.

[0146] (E) Experimental protocol. Aortic pressure, left ventricular pressure, electrocardiogram, right atrial electrophysiogram, His bundle electrophysiogram, and MAP signal were monitored using a polygraph system (RM-6000, Nihon Kohden Corporation) and analyzed using a real-time fully automated data analysis system (Win VAS 3 for Windows ver.1.1R24v, Physiotech, Tokyo, Japan). Using three recordings of a continuous complex, the mean for electrocardiogram display, MAP duration, and atrial-His (AH) and His-ventricular (HV) intervals were calculated. Cardiovascular variables were evaluated in the following order: Electrocardiogram, atrial and His bundle electrophysiograms, aortic pressure, left ventricular pressure, and MAP signal were recorded under sinus rhythm. Cardiac output was then measured three times. Next, the MAP signal was recorded during ventricular pacing with period lengths of 400 ms and 300 ms. Finally, VERP and AERP were measured. All of the above parameters were usually obtained within 2 minutes at each time point.

[0147] After the initial assessment, a low dose of 0.3 mg / kg of poiendarone hydrochloride was intravenously infused over 30 seconds through a catheter sheath placed in the left femoral vein, and each variable was evaluated at 5, 10, 15, 20, and 30 minutes after the start of administration (n=4). Next, a high dose of 3 mg / kg of poiendarone hydrochloride was infused in the same manner, and each variable was observed at 5, 10, 15, 20, 30, 45, and 60 minutes after the start of administration. The inventors selected a current dose of poiendarone hydrochloride and compared its electropharmacological effect with that of dolonedarone hydrochloride. 6 I directly compared it to that.

[0148] The results are shown in Tables 9 and 10.

[0149] Atrial electropharmacological properties of poyendarone as an anti-atrial fibrillation agent in a canine model of paroxysmal AF (A) Animals. The experiment was conducted using four female beagle dogs weighing approximately 10 kg each. The animals were obtained through Kitayama Labes Co., Ltd. (Nagano Prefecture, Japan). All experiments were approved by the Toho University Animal Experiment Committee (No. 19-52-395) and conducted in accordance with the Toho University Animal Experiment Handling Regulations.

[0150] (B) Creation of dogs with chronic atrioventricular block. Catheter ablation technique of the atrioventricular node has been described previously. 11、14The procedure was as follows: The dog was anesthetized with pentobarbital sodium (30 mg / kg, iv). After intubation with a cuffed endotracheal tube, respiration was controlled with room air using a volume-restricted ventilator (SN-480-3, Shinano Seisakusho Co., Ltd. (Tokyo, Japan)). The tidal volume and respiratory rate were set to 20 mL / kg and 15 breaths / min, respectively. Heparin calcium (100 IU / kg, iv) was administered to prevent blood clotting. Surface lead II electrocardiogram was continuously monitored. A 4 mm quadrupole electrode catheter with a large tip (D7-DL-252, Cordis-Webster Inc., California, USA) was placed in the right femoral vein and inserted into a catheter sheath (FAST-CATH® 406119, St. Jude Medical Daig The electrode was inserted via Division, Inc. (Minnetonka, Minnesota, USA) and positioned across the tricuspid valve under the guidance of a bipolar electrogram from the distal electrode pair. The optimal site for atrioventricular node ablation, i.e., a dense atrioventricular node, was determined based on intracardiac electrograms in which very small His amplitudes were recorded and the atrial / ventricular voltage ratio was >2. Power for atrioventricular node ablation was obtained from an electrosurgical generator (MS-1500, Izumi Medical & Scientific Industries Co., Ltd., Tokyo, Japan) delivering continuous unmodulated radiofrequency energy at a frequency of 500 kHz. After determining the appropriate position, 20 W of radiofrequency energy was delivered for 10 seconds from the tip electrode to an indifferent patch electrode placed on the animal's back, and then continued for 30 seconds if a junctional ectopic complex was induced. The endpoint of this procedure was the development of complete atrioventricular block with the occurrence of a stable ventricular intrinsic escape rhythm. All surgical procedures described above were performed under sterile conditions. The electropharmacological effects and anti-atrial fibrillation effects of poiendarone were investigated. 11、14 Up until then, appropriate care was taken of the animals.

[0151] (C) Surgical preparation for a paroxysmal atrial fibrillation model. A paroxysmal atrial fibrillation model has been previously reported. 12、13Preparations were made as described above. More than three months after induction of atrioventricular block, dogs (n=4) were anesthetized with pentobarbital sodium (30 mg / kg, iv). After intubation with a cuffed endotracheal tube, the dogs were mechanically resuscitated with 0.5-1.5% isoflurane in oxygen using a volume-limiting ventilator (SN-480-3, Shinano Seisakusho Co., Ltd. (Tokyo, Japan)). The tidal volume and respiratory rate were set to 20 mL / kg and 15 breaths / min, respectively. Surface lead II electrocardiograms were obtained from limb electrodes. Four clinically available catheter-sheaths (FAST-CATH®, St. Jude Medical Daig Division, Inc.) were used, with two inserted into the right femoral vein and the remaining two inserted into the left femoral vein toward the inferior vena cava. An indwelling needle (Surflo® 18G, Terumo Corporation, Tokyo, Japan) was placed in the right femoral artery to measure arterial pressure.

[0152] Three sets of standard 6-French quadrupole electrode catheters (Cordis-Webster Inc.) were used. The first was placed in the upper right atrium via the right femoral vein to electrically pace the sinoatrial node region and simultaneously obtain a right atrial electrocardiogram. The second was placed in the esophagus via an oscillating tube to record a left atrial electrocardiogram. The third was placed in the interatrial septum of the right atrium via the left femoral vein to electrically induce paroxysmal atrial fibrillation as described below. The optimal site for each catheter was determined by the temporal relationship between the bipolar atrial electrocardiogram from the distal electrode pair and the P wave of the electrocardiogram. A standard 4-French quadrupole electrode catheter (401993, St. Jude Medical Daig Division, Inc.) was placed in the endocardium of the right ventricle via the right femoral vein to electrically drive the right ventricle.

[0153] (D) Measurement of electrophysiological variables. The heart was electrically stimulated by a cardiac stimulator (SEC-3102, Nihon Kohden Corporation) through pacing electrodes of a catheter placed in the sinoatrial node region or the right ventricle. The stimulation pulse was set to a rectangular shape, with an amplitude of 2-2.5V (approximately twice the threshold voltage) and a duration of 1ms. The interatrial conduction time (IACT) was set to 400ms (IACT). (CL400) ), 300ms (IACT(CL300) ), and 200ms (IACT (CL200) The effective refractory period (AERP) was defined as the difference in the temporal position between the left and right atrial electrophysiograms measured with a pacing period of 400 ms (AERP). The effective refractory period (AERP) and effective refractory period (VERP) were evaluated by programmed electrical stimulation to the sinoatrial node region and the right ventricle, respectively. The pacing protocol was defined as 400 ms (AERP) for AERP. (CL400) ), 300ms (AERP (CL300) ), and 200ms (AERP (CL200) ), and VERP 400ms (VERP (CL400) The stimuli consisted of a basal stimulus of 5 beats with a period length of ) followed by premature stimuli with various coupling periods. The coupling periods were shortened by a 5 ms decay until additional stimuli could no longer elicit a response. AERP and VERP were defined as the shortest coupling periods that could induce a stimulus response.

[0154] (E) Induction of paroxysmal atrial fibrillation. Using a stimulator (SEN-7203, Nihon Kohden Corporation) equipped with an isolation unit (SS-201J, Nihon Kohden Corporation) [2, 3], the atrial septum was electrically paced for 10 seconds with a period length of 60 ms (1,000 bpm) through the distal electrode pair of the catheter (= burst pacing). The stimulation pulse to induce atrial fibrillation was set to a rectangular shape, with an amplitude of 60 V and a duration of 10 ms. Atrial fibrillation was defined as a period of rapid and irregular atrial rhythm that resulted in an irregular baseline on the electrocardiogram. The duration of atrial fibrillation was measured from induction to termination on the right atrial electrophysiogram, while the period length of atrial fibrillation was determined using the left atrial electrophysiogram.

[0155] (F) Experimental protocol. The left and right atrial electrograms, electrocardiogram, and arterial pressure were monitored with a polygraph system (RM-6000, Nihon Kohden Corporation) and analyzed with a real-time fully automatic data analysis system (WinVAS3 version 1.1R24, Physiotec Co., Ltd., Tokyo, Japan). For each measurement of the IACT variable, the average of three recordings of consecutive complexes was used. The electropharmacological variables were evaluated in the following order. First, with spontaneous sinus rhythm, the left and right atrial electrograms, electrocardiogram, and arterial pressure were recorded. Second, the sinoatrial node region was electrically paced at cycle lengths of 400 ms, 300 ms, and 200 ms to measure IACT. Third, AERP was evaluated at basic pacing cycle lengths of 400 ms, 300 ms, and 200 ms, and VERP was measured at a basic pacing cycle length of 400 ms. Fourth, paroxysmal atrial fibrillation was induced by a burst pacing protocol and repeated 10 times at each time point. If atrial fibrillation was converted to atrial flutter or maintained for >30 seconds, it was terminated by rapid atrial pacing and its duration was considered 30 seconds.

[0156] After the basic evaluation, through a catheter sheath placed in the left femoral vein, a low dose of 0.3 mg / kg of pindo-lol hydrochloride was intravenously infused over 30 seconds, and each variable was evaluated 10 minutes, 20 minutes, and 30 minutes after the start of administration (n = 4). Then, a high dose of 3 mg / kg of pindo-lol hydrochloride was infused in the same manner, and each variable was observed 10 minutes, 20 minutes, 30 minutes, 45 minutes, and 60 minutes after the start of administration.

[0157] (G) Pindo-lol hydrochloride and drugs. Pindo-lol hydrochloride was dissolved in 100% ethanol at a concentration of 20 mg / mL to prepare injection solutions of 0.3 mg / 15 μL / kg and 3 mg / 150 μL / kg. The other drugs used were pentobarbital sodium (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), isoflurane (Isoflurane Inhalation Solution, Pfizer Inc., Tokyo, Japan), and heparin calcium (Caprocin (registered trademark), Sawai Pharmaceutical Co., Ltd., Osaka Prefecture, Japan).

[0158] (H) Statistical analysis. Data are presented as mean ± SEM. Differences within parameters were estimated by one-way repeated measures analysis of variance (ANOVA), followed by contrast tests as post-hoc tests for mean comparisons. A p-value < 0.05 was considered statistically significant.

[0159] The results are shown in Figures 30, 31, 32, and 33.

[0160] result Amiodarone (Figure 1A) is associated with severe pulmonary and thyroid damage. This is primarily due to the presence of iodine atoms in the molecule, its lipophilicity, and widespread tissue accumulation. Dronedarone (Figure 1B) is a non-iodinated analog of amiodarone that does not have the aforementioned systemic toxicity due to lower tissue accumulation caused by the addition of a methanesulfonamide group. However, it exacerbates heart failure and increases mortality in patients with persistent AF and NYHA class III and class IV heart failure. Due to these fatal side effects, the United States Food and Drug Administration (USFDA) has issued a black-bordered warning about its cardiotoxic effects.

[0161] Unfortunately, clinical development of another amiodarone analog, celivaron, has been discontinued due to low efficacy.

[0162] Arachidonic acid (AA) is an endogenous ω-6 polyunsaturated fatty acid that acts as a precursor to several biologically important lipids, including prostaglandins and thromboxanes. AA is primarily metabolized by cyclooxygenases, lipoxygenases, and CYP450 enzymes. Extrahepatic CYP2J2, an epoxygenase primarily expressed in the human heart, metabolizes AA into four positional isomers of cardioprotective epoxyeicosatrienoic acid (EET) (Figure 2). EETs help maintain cardiac homeostasis through their vasodilatory, anti-inflammatory, anti-apoptotic, and ion channel modulating activities. EETs are further metabolized by soluble epoxide hydrolase (sEH) to less potent dihydroxyeicosatrienoic acid (DHET) (Figure 2). Changes in cardiac CYP2J2 and subsequent perturbations in AA metabolism are responsible for the onset, persistence, and exacerbation of cardiac hypertrophy. Since hypertrophic response typically precedes heart failure, perturbations of EET may accelerate the progression from cardiac hypertrophy to heart failure. Conversely, cardiac-specific overexpression of CYP2J2 mitigates several pathological conditions, including arrhythmic susceptibility in cardiac hypertrophy, endoplasmic reticulum stress in heart failure, and doxorubicin-induced cardiotoxicity due to increased EET production. Interestingly, overexpression of CYP2J2 reduces QT prolongation caused by the pan-epoxygenase inhibitor MS-PPOH. This further emphasizes the role of EET as an ion channel modulator. Therefore, perturbations in the cardiac AA metabolic pathway manifest as homeostasis in a dysregulated heart.

[0163] The inventors previously reported that amiodarone and dolonedarone inhibit human cardiac CYP2J2 by ​​mechanistic inactivation (MBI) and reversible inhibition. 7The inventors have elucidated that the MBI of CYP2J2 is mediated by quinone-oxime metabolites (Figure 3). To alleviate the MBI of CYP2J2, site-directed deuteration of dolonedarone was attempted. Deuteration is a chemical process in which at least one hydrogen atom in a molecule is replaced by deuterium. Because deuterium has a large atomic mass, the cleavage energy of the carbon-deuterium (CD) bond is relatively larger than the cleavage energy of the carbon-hydrogen (CH) bond (341.4 kJ / mol).

[0164] The inventors conducted a deuteration experiment and produced a certain product. That product was... 1. Reduces the MBI capacity of CYP2J2 and reduces AA metabolic perturbation.

[0165] 2. To maintain the pharmacokinetic and pharmacodynamic properties of dolonedarone, and to alleviate its ventricular arrhythmia-inducing properties and associated exacerbation of heart failure.

[0166] As a result of our research, we have developed a product containing site-specific deuteration of dolonedarone at positions 4, 6, and 7 of the benzofuran ring (i.e., poyendarone, Figure 4).

[0167] Poyendarone exhibits similar physicochemical properties, effective permeability, and metabolic half-life to its non-deuterated analog, doronedarone.

[0168] Except for the substitution of deuterium atoms at positions 4, 6, and 7 of the benzofuran ring, poiendarone (Figure 4) and dronedarone (Figure 1B) are structurally identical. As expected, deuteration does not alter the lipophilicity (cLogP), water solubility, and effective permeability of poiendarone compared to dronedarone (Table 8). This means that both drugs could potentially fall into the same class in the Biopharmaceutical Classification System (BCS) as defined by the USFDA. Furthermore, the metabolic half-life (T) of poiendarone is also shown. 1 / 2) is equivalent to that of doronedarone. This means that poyendarone maintains the metabolic stability of doronedarone. In summary, our findings indicate that poyendarone potentially preserves the favorable pharmacokinetic properties of doronedarone.

[0169] Downregulation of CYP2J2 increases the cardiac interval, confirming that CYP2J2 inhibition supports the arrhythmic effect of dolonedarone. Human CYP2J2 was knocked down in human cardiomyocytes (H7 CM) using four different siRNAs (Table 1). All siRNAs significantly knocked down CYP2J2, with siRNA1 being the most effective (Figure 6).

[0170] Knockdown of cardiac CYP2J2 using four siRNAs increased the pulse interval in individual clusters of cardiomyocytes (Figure 7). Based on the pulse generation graph, the pulse of control cardiomyocytes was observed to be regular (Figure 8). However, during CYP2J2 knockdown, the pulse of cardiomyocytes was clearly irregular. When data points related to siRNA were combined, the increase in pulse interval was statistically significant (Figure 9). In summary, these results highlight the central role of CYP2J2 in maintaining cardiac rhythm control and confirm that CYP2J2 inhibition supports the arrhythmic effect of dolonedarone.

[0171] Mechanistic inactivation (MBI) of recombinant human CYP2J2: Poyendarone << Dronedarone The inventors previously reported doronedarone and amiodarone as potent mechanistic inactivators of CYP2J2. As is readily apparent to those skilled in the art, the MBI ability of a drug against the enzyme is substrate-dependent. In our preliminary study using astemizole as a probe substrate, we confirmed that poyendarone's MBI ability against CYP2J2 was 1 / 62nd that of doronedarone (Figure 10), and the MBI ability measured as a kinact / KI ratio was 0.008 min for each. -1 μM -1and 0.5 min -1 μM -1 This finding supports the inventors' assumption that deuterated quinone oximes are less reactive. Furthermore, poyendarone has one-ninth the potency of amiodarone (data not shown).

[0172] To further investigate the MBI (metabolic bisphosphonate) activity of poyendarone, dronedarone, and two other deuterated analogs of dronedarone, clinically relevant rivaroxaban was used as a probe substrate. Consistent with the prior astemizole-specific MBI data (which demonstrates the considerably low MBI activity of poyendarone), rivaroxaban-specific MBI could not be measured for poyendarone. Specifically, the MBI activity of poyendarone, dronedarone, compound 1, and compound 2 is summarized in Table 3. Notably, only dronedarone showed CYP2J2 inactivation (Figures 11A, 11B), while poyendarone did not induce CYP2J2 MBI (Figures 11C, 11D). Compound 2 inactivated CYP2J2 in a time-dependent and concentration-dependent manner (Figures 12A, 12B), while compound 3 did not (Figures 12C, 12D). These MBI data, obtained using differentially deuterated doronedarone analogs, demonstrate that deuteration of the benzofuran ring is important for mitigating the MBI of CYP2J2.

[0173] Inhibition of CYP2J2 in hiPSC-CM: Poyendarone << Dronedarone hiPSC-cardiomyocardial cells (hiPSC-CMs) are electrophysiologically similar to adult human cardiomyocytes. Due to their spontaneous beating ability, hiPSC-CMs are a desirable model for investigating the antiarrhythmic activity of established novel compounds. Furthermore, hiPSC-CMs are a suitable model for investigating the torsadogenic risk of drugs. Here, we investigated whether CYP2J2 is expressed in hiPSC-CMs and whether our test drugs inhibit CYP2J2 in hiPSC-CMs.

[0174] As far as the inventors know, they are the first group to demonstrate the expression of CYP2J2 and sEH in hiPSC-CMs (Figure 13A). This finding supports the established knowledge that CYP2J2 is highly expressed in primary human cardiomyocytes as well as in heart tissue. Thus, hiPSC-CMs are a metabolic means for investigating CYP2J2 biological events in vitro. Using this means, the inventors demonstrated that CYP2J2 is actually active and is potently inhibited by dronedarone (98% inhibition), but not by amiodarone (Figure 13B). The lack of CYP2J2 inhibition by amiodarone is consistent with previous reports using primary human cardiomyocytes 8 and is important in that the inhibition of CYP2J2 by pioendron is significantly lower (50% inhibition) (Figure 13B).

[0175] Pioendron is significantly less cytotoxic to cardiomyocytes than dronedarone.

[0176] When there was no pretreatment with EET, protease activity increased by approximately 150 - 200%, and significant cell death was shown when H9c2 cells were treated with dronedarone (Figure 14A). When there was pretreatment of H9c2 cells with 14,15-EET, the bis-AAF-R110 fluorescence signal decreased in a concentration-dependent manner, and mitigation of cell cytotoxicity by EET was confirmed. Similarly, 14,15-EET mitigated the decrease in intracellular ATP levels in a concentration-dependent manner.

[0177] Dissipation of Δψ was measured using the TMRM fluorescent dye. Dronedarone showed potent dissipation of Δψ (IC m = 0.5 μM). Here, H9c2 cells were pretreated with 14,15-EET and subsequently treated with 5 μM dronedarone. For pretreatment with 14,15-EET, a concentration-dependent mitigation of Δψ m dissipation was observed (Figure 14A). 50 dissipation was observed (Figure 14A). m dissipation was observed (Figure 14A).

[0178] After exposing H9c2 cells to dolonedarone for 6 hours, the fluorescence signal increased in a concentration-dependent manner, indicating the cytotoxicity (EC) of dolonedarone against H9c2 cells. 50 A concentration of 1.21 μM was confirmed (Figure 14B). Simultaneously, intracellular ATP levels were induced by doronedarone and decreased in a concentration-dependent manner (IC). 50 =3.10 μM) (Figure 14B). Poyendarone is significantly less cytotoxic to H9c2 cells (EC 50 =27.63μM). As a result, poyendarone is equivalent to doronedarone (IC). 50 Poyendarone does not reduce the ATP level of H9c2 cells as strongly as (41.52 μM). Therefore, based on its experimental capabilities, poyendarone is not expected to cause cytotoxicity in cardiomyocytes at therapeutic plasma concentrations (sub-μM).

[0179] Poyendarone exhibits ion channel blocking activity similar to that of doronedarone and amiodarone in hiPSC-CM.

[0180] HiPSC-CMs have the unique advantage of growing indefinitely and spontaneously beating during culture due to the expression of all major cardiac ion channels, gap junction proteins, and ion exchangers. Therefore, a large number of drugs can be tested for their ion channel inhibitory properties. Unlike conventional patch-clamp methods (Figure 15A), in this study we used an MEA system (Figure 15B) to measure the "comprehensive" effect of drugs on extracellular field potential duration (FPD), which reflects cardiac action potentials. The advantage of MEA lies in profiling cell populations rather than single cells, thereby avoiding bias. Notably, poyendarone shows a similar concentration-dependent effect on extracellular field potential duration (FPD) using a multi-electrode array (MEA) assay of electrophysiologically related hiPSC-CMs (Figures 15B, 15C).

[0181] Furthermore, based on patch-clamp experiments, poyendarone, doronedarone, and amiodarone were found to be effective in human cardiac Na. V1.5 (Figures 16, 17, and 18), Ca V 1.2 (Figures 19, 20, and 21), and K v Similar inhibitory activity has been demonstrated for 11.1 (Figures 22, 23, and 24). These pieces of evidence confirm the anti-AF pharmacology of poyendarone.

[0182] Poyendarone has a lower risk of inducing arrhythmias compared to delonedarone in hiPSC-CM.

[0183] Class III antiarrhythmic drugs are effective in treating atrial arrhythmias, but are known to potentially increase the risk of life-threatening ventricular arrhythmias. Therefore, it is important to distinguish between the arrhythmic and antiarrhythmic effects of potassium channel blockers. Conventionally, hERG inhibition and QT prolongation are used as indicators of drug-induced arrhythmias. Specifically, QT prolongation accompanied by instability can predict drug-induced arrhythmias, while QT prolongation without instability is antiarrhythmic. Instability can be assessed in hiPSC-CM by measuring BBV (beat-beat variability). BBV can be visualized graphically using a Poincaré plot. A Poincaré plot is a chart in which each RR interval or interval between beats (IBI) is plotted relative to its preceding interval, illustrating the correlation between consecutive intervals (Figure 25). IBIs for control and poyendarone are clustered around the centroid of an ellipse, aligned along the longitudinal axis, and defined as a cigar-shaped plot (Figure 25). This indicates the least variation between pulses. However, in the case of delonedaron, the IBI does not converge at the centroid of the ellipse but diffuses throughout the plot (Figure 25).

[0184] Poyendarone has similar in vivo plasma pharmacokinetics to doronedarone.

[0185] Similar elimination profiles and primary pharmacokinetic parameters (clearance and volume of distribution) were observed between poyendarone and dronedarone administered at 3.0 mg / kg. No acute injury or death was observed in vivo (Figure 26).

[0186] Poyendarone has similar simulated in vivo plasma pharmacokinetics to doronedarone.

[0187] To better understand Poyendaron's PK, we constructed a PBPK model. For accurate CL inputs to each PBPK model, we used the following method. int Value and k inact / K I By deriving these values, the elimination of each drug and the time dependence of the predicted plasma concentration-time profile are mechanistically explained. Furthermore, generating these in vitro human data ensures that the predicted profiles can be generalized as much as possible to different target populations.

[0188] Study of metabolic stability. The percentage of substrate remaining in the CYP3A4, CYP3A5, or HLM reaction mixture decreased exponentially as incubation time progressed. 1 / 2 And k were 16.73 min and 0.04144 min in CYP3A4. -1 For CYP3A5, the results were 17.37 min and 0.03989 min. -1 In HLM, the values ​​were 10.75 min and 0.06446 min. -1 (Figures 27A, 27C, and 27E, respectively). Poyendaron's T 1 / 2 And k were 7.551 min and 0.0918 min in CYP3A4. -1 For CYP3A5, the results were 25.14 min and 0.02757 min. -1 In HLM, the values ​​were 10.71 min and 0.06472 min. -1 These were the results (Figures 27B, 27D, and 27F, respectively). The CL obtained for doronedarone across all enzyme systems was also shown. intThe values ​​were similar to those for poyendarone (Table 6). CL of the poyendarone experiment int The values ​​(CYP3A4: 9.18 μL / min / pmol, CYP3A5: 2.757 μL / min / pmol) were similar to those of delonedarone (CYP3A4: 8.288 μL / min / pmol, CYP3A5: 3.989 μL / min / pmol), as expected. This is because the deuteration site is far from the main metabolic site (N-butyl chain) in delonedarone. In addition, the CL of the delonedarone experiment... int The values ​​are those reported by Hong et al. using a similar experimental setup (CYP3A4: 6.442 μL / min / pmol, CYP3A5: 2.604 μL / min / pmol). 10 This is similar to the previous example and supports the validity of those used in PBPK modeling.

[0189] Time-dependent and concentration-dependent inactivation of CYP3A4, CYP3A5, and CYP2J2. Dronedarone and poyendarone inactivated CYP3A4 and CYP3A5 in a time-dependent and concentration-dependent manner using rivaroxaban as a probe substrate (Table 6). The concentration dependence of inactivation was calculated from various concentration levels of the inactivator. obs The saturation reaction rate was observed, and as the inactivator concentration increased, it approached the maximum rate of inactivation. Dronedarone and Poyendarone showed similar k for CYP3A5. inact / K I It had a value, but for CYP3A4, the k of poyendarone inact / K I The value is k of Dronedaron inact / K I The value was higher than expected (a difference of 2.4 times) (Table 6).

[0190] Development and validation of a PBPK model. Our PBPK model successfully characterized clinical data of dolonedarone not only for intravenous and single oral administration, but also for multiple oral administrations (Figure 28). For multiple oral administrations, the AUC was reproduced within approximately 12% error in the fasted state and within approximately 34% error in the fed state. Predicted PK parameters were compared with clinical data based on relevant success criteria. It is also interesting to note that when the effects of MBI are not simulated (Figures 29C, 29D), satisfactory fitting of simulated multiple oral administration data to clinical data (Figures 29A, 29B) is lost. This highlights the importance of integrating accurate MBI data into PBPK modeling in order to successfully predict the time-dependent PK of dolonedarone.

[0191] Dronedarone and poyendarone produce similar MBI capabilities against CYP3A5 (at 0.00634 μM each). -1 min -1 and 0.00793 μM -1 min -1 (Table 6), the effect on CYP3A4 is not as significant (0.00505 μM each). -1 min -1 and 0.0123 μM -1 min -1 (Table 6). CYP3A4 is the primary metabolic enzyme for dronedarone compared to CYP3A5. This may also apply to poiendarone, as its metabolic hotspot is not deuterated. In summary, we infer that poiendarone has a more potent MBI effect on its key metabolic enzyme, resulting in greater autoinhibition of CYP3A4 and therefore a higher systemic exposure. However, based on read-across PBPK modeling, simulated multiple oral doses of poiendarone differed only slightly from those of dronedarone. Indeed, assuming that the usual dose of dronedarone is 400 mg twice daily with food, and assuming poiendarone has the same capacity as dronedarone, dose adjustments for poiendarone would not be required.

[0192] Herein, this finding demonstrates for the first time an experimental framework for read-across PBPK modeling to predict the clinical PK profile of poyendarone based on a validated PBPK model of dronedarone. Based on extended systemic exposure to poyendarone and assuming an exposure-efficacy relationship equivalent to that of dronedarone, a drug regimen can subsequently be planned for a first-in-human clinical trial of poyendarone.

[0193] Poyendarone retains its potential anti-atrial fibrillation properties. Similar to dronedarone and amiodarone, poyendarone prolongs both AERP and VERP, resulting in 1.8–2.7 times higher atrial selectivity (Table 9). This highlights its potential clinical efficacy for atrial arrhythmias.

[0194] Poyendarone avoids the potential arrhythmic effects associated with dronedarone. Poyendarone has the lowest risk of reentrant ventricular arrhythmias based on its minimal effect on the end-TRP (ΔTRP). The absence of early repolarization prolongation (ΔJ-Tpeakc) and minimal late repolarization prolongation (ΔTpeak-Tend) highlights the potentially low risk of torsades de pointes associated with poiendarone (Table 10).

[0195] Poyendarone possesses favorable atrial electropharmacological properties as an anti-atrial fibrillation agent in a canine model of paroxysmal atrial fibrillation. During the experiment, no animals exhibited lethal ventricular arrhythmias or hemodynamic collapses that led to death.

[0196] Effects on sinoatrial heart rate and mean blood pressure. Figure 30 summarizes the changes in sinoatrial heart rate and mean blood pressure over time. The baseline control values ​​(C) before drug administration were 96±12 bpm and 83±12 mmHg, respectively. Low doses of 0.3 mg / kg and high doses of 3 mg / kg had little effect on either of these variables.

[0197] Effects on IACT. Figure 31 summarizes the changes in IACT over time. (CL400) IACT (CL300) , and IACT (CL200) The baseline control values ​​(C) before drug administration were 44±2ms, 47±1ms, and 55±2ms, respectively. At low doses, these IACT values ​​did not change at any pacing cycle length. At high doses, the IACT values ​​for 10 minutes and 30-60 minutes were different. (CL400) 10 minutes of IACT (CL300) , and 10-60 minute IACT (CL200) It was extended.

[0198] Effects on AERP and VERP. Figure 31 summarizes the changes in AERP and VERP over time. AERP (CL400) , AERP (CL300) , AERP (CL200) , and VERP (CL400) The baseline control values ​​(C) before drug administration were 150±11ms, 145±12ms, 139±14ms, and 233±6ms, respectively. At low doses, 20 minutes of AERP were used. (CL400) Although the interval was prolonged, no significant changes were detected in other variables. At high doses, the interval between AERP was 10-60 minutes. (CL400) and AERP (CL300) While the extension was extended, AERP (CL200) or VERP (CL400) No significant changes were detected.

[0199] Effects on the duration and period length of atrial fibrillation. Typical follow-up graphs of left and right atrial electrocardiograms, electrocardiograms, and arterial pressure during and after burst pacing are shown in Figure 32, and the time course of changes in the duration and period length of atrial fibrillation is summarized in Figure 33. The baseline control values ​​(C) before drug administration were 4.0 ± 0.9 s and 155 ± 15 ms, respectively. At low doses, these variables did not change. At high doses, the duration (10-60 minutes) tended to shorten, while the period length tended to lengthen, but this was not statistically significant.

[0200] In summary, poyendarone has atrial-selective anti-atrial fibrillation properties for paroxysmal atrial fibrillation in dogs.

[0201] summary Our compelling in vitro and in vivo evidence confirms that site-targeted deuteration is a viable strategy for optimizing the safety and efficacy of benzofuran-derived antiarrhythmic drugs such as dronedarone.

[0202] The site-directed deuterated compound, poyendalone, exhibits the following characteristics:

[0203] • Similar physicochemical permeability and metabolic stability as doronedarone. • Cardiac ion channel blocking activity equivalent to that of doronedarone. • Similar favorable pharmacokinetics to that of doronedarone, and • Selectivity and activity for in vivo atrial fibrillation treatment equivalent to that of delonedarone.

[0204] Furthermore, unlike dolonedarone, the site-directed deuterated compound, poyendarone, does not cause the following:

[0205] • Inactivation of human cardiac CYP2J2, • Mitochondrial dysfunction in cardiomyocytes (which instead leads to a safer cytotoxic / ATP depletion profile, meaning a reduced risk of cardiotoxicity compared to dolonedarone), ·BBV in hiPSC-CM, and • Potential risk of inducing cardiac arrhythmias in vivo.

[0206] Therefore, site-directed deuterated benzofuran-derived antiarrhythmic drugs, particularly poiendarone, are viable and valuable compounds for the treatment of AF. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] TIFF2022541739000019.tif72168 [Table 9] [Table 10] [Table 11]

[0207] References 1.A.Mehta、YYChung、A.Ng、F.Iskandar、S.Atan、H.Weiらによる「Pharmacological response of human cardiomyocytes derived from virus-free induced pluripotent stem cells」、Cardiovasc.Res.91(2011)577-586.doi:10.1093 / cvr / cvr132. 2.A.Mehta、Y.Chung、GLSequiera、P.Wong、R.Liew、W.Shimによる「Pharmacoelectrophysiology of viral-free induced pluripotent stem cell-derived human cardiomyocytes」、Toxicol.Sci.131(2013)458-69.doi:10.1093 / toxsci / kfs309. 3.L.Zwi、O.Caspi、G.Arbel、I.Huber、A.Gepstein、I.-H.Park、L.Gepsteinによる「Cardiomyocyte differentiation of human induced pluripotent stem cells」、Circulation.120(2009)1513-23.doi:10.1161 / CIRCULATIONAHA.109.868885. 4.MPTarvainen、J.-P.Niskanen、JALipponen、PORanta-aho、PAKarjalainenによる「Kubios HRV-Heart rate variability analysis software」、Comput.Methods Programs Biomed.113(2014)210-20.doi:10.1016 / j.cmpb.2013.07.024. 5.A.Voss、S.Schulz、R.Schroeder、M.Baumert、P.Caminalによる「Methods derived from nonlinear dynamics for analyzing heart rate variability.Philos.Trans.A.Math.Phys.Eng.Sci.367(2009)277-96.doi:10.1098 / rsta.2008.0232. 6.Y.Motokawa、Y.Nakamura、MHNagasawa、A.Goto、K.Chiba、NJLubna、HINakaseko、K.Ando、ATNaito、H.Yamazaki、A.Sugiyama Cardiovasc.Toxicol.18(2018)242-51.doi:10.1007 / s12012-017-9434-y. 7. A. Karkhanis, HYLam, G. Venkatesan, SKKoh, CLLChai, L. Zhou, Y. Hong, P. Kojodjojo, and ECY Chan active metabolites.Biochem.Pharmacol.107(2016)67-80.doi:10.1016 / j.bcp.2016.03.005. 8. EAEvangelista, R. Kaspera, NAMokadam, JP Jones, and RATotah Cardiomyocytes.Drug.Metab.Dispos.41(2013)2087-94.doi:10.1124 / dmd.113.053389. 9.K.Abduljalil、T.Cain、H.Humphries、A.Rostami-Hodjeganによる「Deciding on success criteria for predictability of pharmacokinetic parameters from in vitro studies:An analysis based on in vivo observations」、Drug.Metab.Dispos.42(2014)1478-84.doi:10.1124 / dmd.114.058099. [ PubMed ] [ Cross Ref ] 10.Y.Hong, YMFChia, RHYeo, G.Venkatesan, SKKoh, CLLChai, L.Zhou, P.Kojodjojo, and ECYChan dronedarone.Mol.Pharmacol.89(2016)1–13.doi:10.1124 / mol.115.100891. [ PMC free article ] [ PubMed ] [ Cross Ref ] 11. A. Sugiyama, Y. Ishida, Y. Satoh, S. Aoki, M. Hori, Y. Akie, Y. Kobayashi, and K. Hashimoto Jpn J Pharmacol.88(2002)341–50.doi:10.1254 / jjp.88.341. 12.K.Wang、A.Takahara、Y.Nakamura、K.Aonuma、M.Matsumoto、A.Sugiyamaによる「In vivo electropharmacological effects of amiodarone and candesartan on atria of chronic atrioventricular block dogs」、J.Pharmacol.Sci.103(2007)207–13.doi:10.1254 / jphs.FP0060945. 13. H. Iwasaki, A. Takahara, Y. Nakamura, Y. Satoh, T. Nagai, N. Shinkai, and A. Sugiyama J.Pharmacol.Sci.110(2009)410-14.doi:10.1254 / jphs.09061sc. 14.A.Sugiyamaによる「Sensitive and reliable proarrhythmia in vivo animal models for predicting drug-induced torsades de pointes in patients with remodelled hearts」、Br.J.Pharmacol.154(2008)1528-37.doi:10.1038 / bjp.2008.240.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 Formula (I) During the ceremony, R １ , R ２ , and R ３ represent deuterium, n represents 2 or 3; Each R ４ C may be independently substituted with one or more of a nitro group, a halogen group, an amino group, an amide group, a cyano group, a carboxyl group, a sulfonyl group, a hydroxyl group, a ketone group, or an aldehyde group. １－６ represents a hydrocarbyl group, R ５ is hydrogen or 【Chemistry 2】 represents Each R ６ each independently represents hydrogen or halogen; provided that each atom not specified as deuterium is present at its natural isotopic abundance and each position specified as deuterium has at least 45% deuterium incorporation.

2. 2. The compound of claim 1, wherein each position defined as deuterium has at least 90% deuterium incorporation.

3. 3. The compound of claim 2, wherein each position defined as deuterium has 100% deuterium incorporation.

4. Each R ４ is C １－６ 4. A compound according to any one of claims 1 to 3, which represents an alkyl chain.

5. R ５ but 【Transformation 3】 5. The compound according to claim 1, wherein

6. Each R ６ 6. A compound according to any one of claims 1 to 5, wherein represents hydrogen.

7. 7. The compound of any one of claims 1 to 6, wherein the compound is a compound of formula (II) or a pharmaceutically acceptable salt thereof: 【Chemistry 4】 Formula (II)

8. 8. A pharmaceutical composition comprising a compound according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient or carrier.

9. 8. A compound according to any one of claims 1 to 7 or a composition for use in medicine.

10. 10. A compound according to any one of claims 1 to 7 for use in the treatment of cardiac disease.

11. 11. The compound of claim 10, wherein the cardiac disease is cardiac arrhythmia.

12. 12. The compound of claim 11, wherein the cardiac arrhythmia is atrial fibrillation.

13. 10. A method for treating cardiac disease, said method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound according to any one of claims 1 to 7 or a pharmaceutically acceptable salt form thereof.

14. 14. The method of claim 13, wherein the cardiac disease is cardiac arrhythmia.

15. 15. The method of claim 14, wherein the cardiac arrhythmia is atrial fibrillation.

16. 10. A compound according to any one of claims 1 to 7 for use in the manufacture of a medicament for treating heart disease.

17. A compound of formula (III) or a pharmaceutically acceptable salt thereof: 【Transformation 5】 Formula (III) In the formula, each R ７ represents deuterium.

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