HK1207073B - Agents for treating disorders involving modulation of ryanodine receptors - Google Patents

Description

Agents for treating modulation disorders involving modulation of ryanodine receptors

Technical Field

The present invention relates to 1, 4-benzothiazepine derivatives and their use in the treatment of disorders and diseases associated with ryanodine receptors (RyRs) that modulate calcium channels functioning in cells. The invention also discloses pharmaceutical compositions comprising these compounds and their use in the treatment of diseases and disorders associated with RyRs, particularly cardiac disorders, musculoskeletal disorders and Central Nervous System (CNS) disorders.

Background

As specialized intracellular calcium (Ca) in Sarcoplasmic Reticulum (SR) cells²⁺) Storing functioning, etc. RyRs are open and closed in SR to regulate Ca²⁺Released from the SR into the intracellular cytoplasm of the cell. Ca²⁺Release from SR into cytoplasm increases cytosolic Ca²⁺And (4) concentration. The probability of opening of RyRs means that RyR opens at any given time and is thus able to make Ca²⁺Possibility of release from SR into cytoplasm.

There are 3 types of ryrs, all of which are highly homologous: RyR1, RyR2, and RyR 3. RyR1 is found primarily in skeletal muscle and other tissues, RyR2 is found primarily in the heart and other tissues, and RyR3 is found in the brain and other tissuesIt is found in tissue. RyR is a tetramer. The portion of the RyR complex is formed by the binding of 4 RyR polypeptides to 4 FK506 binding proteins (FKBPs) (calcium channel stabilizing proteins), particularly FKBP12 (calcium channel stabilizing protein 1) and FKBP12.6 (calcium channel stabilizing protein 2). Calchannel stabilizing protein 1 binds to RyR1 and RyR3, while calchannel stabilizing protein 2 binds to RyR 2. Calcium channel stabilizing proteins bind to RyR (1 molecule/RyR subunit), stabilize RyR function, facilitate coupled gating between adjacent RyRs, and prevent aberrant activation of channels by stabilizing channel closure (Ca)²⁺Leakage).

Ryanodine receptor 2 and cardiac disorders

In the striated muscle of the heart, RyR2 is the predominant Ca required for excitation-contraction (EC) coupling and muscle contraction²⁺The channel is released. Myocardial cell membrane activation voltage-gated Ca during EC coupling, zero phase of action potential²⁺A channel. Ca²⁺Influx via open voltage-gated channels thereby initiating Ca²⁺Released from the SR via RyR 2. This process is called Ca²⁺-induced Ca²⁺And (4) releasing. RyR 2-mediated Ca²⁺-induced Ca²⁺Releasing contractile proteins that subsequently activate heart cells, resulting in myocardial contraction.

Phosphorylation of RyR2 by Protein Kinase A (PKA) is an important component of the "fight or escape" response by increasing Ca release for a given trigger²⁺The amount of (a) increases the cardiac EC coupling gain. This signaling pathway provides a mechanism by which activation of the Sympathetic Nervous System (SNS) leads to increased cardiac output in response to stress. Phosphorylation of RyR2 by PKA leads to partial dissociation of the calchannel stabilizing protein 2 from the channel, thus leading to increased open probability and Ca²⁺Increased release from the SR into the intracellular cytoplasm.

Heart Failure (HF) is characterized by a persistent hyperadrenergic state in which serum catecholamine levels are chronically elevated. The result of this long-term hyperadrenergic state is sustained PKA hyperphosphorylation of RyR2, resulting in 3-4 of 4 Ser2808 in each homotetrameric RyR2 channelLong-term phosphorylation (Marx SO et al Cell, 2000; 101 (4): 365-. In particular, long-term PKA hyperphosphorylation of RyR2 was associated with depletion of the channel-stabilizing subunit calcium channel stabilizing protein 2 from the RyR2 channel macromolecular complex. Depletion of calcium channel stabilizing protein leads to SR Ca²⁺Diastolic "leakage" from the RyR complex contributes to impaired contractility (Marx et al, 2000). This diastolic SR Ca is due to activation of the inward depolarization current²⁺"leak" is also associated with fatal arrhythmias (Lehnart et al, J Clin invest.2008; 118 (6): 2230-. Indeed, HF progression following Myocardial Infarction (MI) was prevented in mice engineered with RyR2 that lack the PKA phosphorylation site (Wehrens XH et al Proc NatlAcad Sci USA.2006; 103 (3): 511-. In addition, long-term PKA hyperphosphorylation of RyR2 in HF is associated with reconstruction of the RyR2 macromolecular complex, which includes phosphatases (Marx et al 2000) PP1 and PP2a (impaired dephosphorylation of Ser 2808) and depletion of cAMP-specific phosphodiesterase type 4 (PDE4D3) from the RyR2 complex. Depletion of PDE4D3 from the RyR2 complex results in a sustained elevation of local cAMP levels (Lehnart SE et al, Cell 2005; 123 (1): 25-35). Thus, stress-relaxing SR Ca²⁺Leakage contributes to HF progression and arrhythmia. In addition, recent reports have demonstrated that constitutive PKA hyperphosphorylated RyR2-S2808D +/+ (aspartate instead of serine 2808) knockout mice that mimic RyR2 show calcium channel stabilizing protein 2 depletion and leaky RyR 2. RyR2-S2808D +/+ mice develop age-dependent cardiomyopathy showing increased oxidation and nitrosylation of RyR2, i.e., reduced SR Ca²⁺Storage content and diastolic SR Ca^{2 +}The leakage increases. Following myocardial infarction, RyR2-S2808D +/+ mice showed increased mortality compared to WT littermates. Treatment with S107, 1, 4-benzothiazepine derivatives that stabilize RyR 2-calchannel stabilizing protein 2 interaction (WO 2007/024717) inhibits RyR 2-mediated diastolic SR Ca²⁺Leakage and reduced HF progression in WT and RyR2-S2808D +/+ mice (Shah et al J Clin invest.2010Dec 1; 120 (12): 4375-87).

Furthermore, RyR2 contains about 33 free sulfhydryl residues, which confer high sensitivity to the redox state of the cell. CysteineOxidation of amino acid favours RyR opening and SR Ca²⁺And (6) leakage. Shann et al, 2010, demonstrated that the oxidative and nitrosylation of RyR2 and the dissociation of the stabilizing subunit, calcium channel stabilizing protein 2, from RyR2 induces SR Ca²⁺And (6) leakage.

Catecholamine-dependent ventricular tachycardia (CPVT) is a genetic disease in individuals with structurally normal hearts. More than 50 different RyR2 mutations were associated with CPVT. CPVT patients experience syncope and Sudden Cardiac Death (SCD) from infancy to adulthood, with a mortality rate of 50% by age 35. Individuals with CPVT have ventricular arrhythmias when performing exercise, but do not develop arrhythmias at rest. CPVT-related RyR2 mutations result in "leaky" RyR2 channels due to reduced calcium channel stabilizing protein 2 subunit binding (Lehnart et al, 2008). Heterozygous mice (RyR2-R2474S mice) generated by the R2474S mutation in RyR2 showed spontaneous generalized tonic-clonic seizures (occurring in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias and SCD. Treatment with S107 enhanced calcium channel stabilizing protein 2 binding to the mutant RyR2-R2474S channel, inhibited the channel from leaking, prevented arrhythmias, and raised seizure threshold (Lehnart et al, 2008).

Lannogenin receptor 1 and skeletal muscle diseases

Skeletal muscle contraction by SR Ca²⁺Is activated by release of RyR 1. Depolarization of the transverse (T) -tubule membrane activates the dihydropyridine receptor baroreceptor (cav1.1), thereby activating the RyR1 channel through direct protein-protein interactions, resulting in SRCa²⁺And (4) storing and releasing. Ca²⁺Binds troponin C, allowing actin-myosin cross-bridging to occur and sarcomere shortening.

In the case of prolonged muscle tone (e.g., during marathon long runs) or diseases such as heart failure, both are characterized by long-term activation of the SNS, with impaired skeletal muscle function, possibly due to altered EC coupling. In particular, Ca during each muscle contraction²⁺Reduced amount of release from SR, abnormal Ca²⁺Release events may occur, and Ca²⁺The re-uptake slowed down (Reiken, S, et al 2003.J.cell biol.160: 919-928). These observations suggest that the deleterious effects of long-term SNS activation on skeletal muscle may be due at least in part to Ca²⁺A signal conduction defect.

The RyR1 macromolecular complex is composed of a tetramer of 560-kDa RyR1 subunits forming the backbone of proteins that regulate channel function PKA and phosphodiesterase 4D3(PDE4D3), protein phosphatase 1(PP1) and calcium channel stabilizing protein 1. The A-kinase dockerin (mAKAP) targets PKA and PDE4D3 to RyR1, while the acanthophilic protein targets PP1 to the channel (Marx et al 2000; Brillants et al, Cell, 1994, 77, 513-523; Bellinger et al J.Clin. invest.2008, 118, 445-53). The catalytic and regulatory subunits of PKA, PP1, and PDE4D3 regulate PKA-mediated phosphorylation of RyR1 at Ser2843 (Ser 2844 in mice). PKA-mediated phosphorylation of RyR1 at Ser2844 has been shown to increase cytoplasmic Ca for this channel²⁺Decreases the binding affinity of calcium channel stabilizing protein 1 to RyR1 and destabilizes the closed state of the channel (Reiken et al, 2003; Marx, S.O. et al, Science, 1998, 281: 818-. It was reported that the concentration of calchannel stabilizing protein 1 in skeletal muscle was about 200nM and PKA phosphorylation of RyR1 reduced the binding affinity of calchannel stabilizing protein 1 to RyR1 from about 100nM to over 600 nM. Thus, under physiological conditions, the decrease in binding affinity of calchannel stabilizing protein 1 to RyR1 due to PKA phosphorylation of RyR1 at Ser2843 is sufficient to substantially reduce the amount of calchannel stabilizing protein 1 present in the RyR1 complex. Long-term PKA hyperphosphorylation of RyR1 at Ser2843 (defined as PKA phosphorylation of 3 or 4 of the 4 PKA Ser2843 sites present in each RyR1 homotetramer) results in "leaky", channels (i.e. channels that are prone to open at rest), which contribute to skeletal muscle dysfunction associated with sustained hyperadrenergic states, such as occurs in individuals with heart failure (Reiken et al, 2003).

Furthermore, it has been reported that modulation of RyR1 by non-phosphorylated post-translational modifications, such as by nitrosylation (S-nitrosylation) of an off-sulfhydryl group upstream of a cysteine residue and channel oxidation increases RyR1 channel activity. S-nitrosylation and oxidation of RyR1 were each shown to reduce calcium channel stabilizing protein 1 binding to RyR 1.

It was previously reported by Bellinger et al (Proc. Natl. Acad. Sci.2008, 105 (6): 2198-Ach 2002) that during extreme exercise in mice and humans, RyR1 progressive PKA-hyperphosphorylation, S-nitrosylation and PDE4D3 and calcium channel stabilizing protein 1 depletion resulted in "leaky" channels which resulted in reduced locomotor ability in mice. Treatment with S107 prevented depletion of the calponin 1 from the RyR1 complex, improved force production and motility, and reduced Ca^2+-Activity of the dependent neutral protease carbaryl and plasma creatinine kinase levels.

Duchenne Muscular Dystrophy (DMD) is one of the major fatal childhood genetic diseases. DMD is X-linked, which affects 1 in 3,500 male newborn infants, and typically leads to death from respiratory or heart failure by the age of-30. Mutations in the dystrophin protein associated with DMD result in a complete deletion of the dystrophin protein, thereby disrupting the linkage between the submucous cytoskeleton and the extracellular matrix. This linkage is necessary to protect and stabilize the muscle contraction induced injury. Currently, there is no cure for DMD and most of the treatments in the clinic are palliative. The emerging interventions in phase I/II clinical trials are exon skipping, tubocurarine inhibition and upregulation of dystrophin-related proteins. However, there are problems with using systemic delivery, supporting exon skipping and up-regulation of dystrophin-related proteins. Furthermore, tubocurarine inactivation, which increased muscle size, did not show muscle strength or function improvement in phase I/II clinical trials. There is a cascade effect of sarcoplasmic instability due to mutations in dystrophin. One of the main effects is cytosolic Ca²⁺Increased concentration of Ca, which results in^2+-Dependent protease (carbaryl) activation. Another effect is inflammation and elevated iNOS activity, which can lead to oxidation/nitrosylation of proteins, lipids and DNA. DMD myopathy is progressive and far exceeds the instability of the sarcolemma. Thus, it is possible to provideThis pathological condition is consistent with myofascial instability that increases susceptibility to further injury. It has recently been demonstrated that transitional oxidation or nitrosylation of RyR1 can disrupt the interaction between the calcium channel stabilizing protein 1 and RyR1 complex, leading to RyR1 leakage and muscle weakness in a muscular dystrophy (mdx) mouse model, and that treatment with S107 improves the muscle function index in this mouse model (Bellinger, a. et al 2009, Nature Medicine, 15: 325-.

Age-related loss of muscle mass and strength (sarcopenia) contributes to disability and increased mortality. Andersson, D.et al (Cell Metab.2011Aug 3; 14 (2): 196-207) report that RyR1 from aged (24-month) mice is oxidized, cysteine-nitrosylated and calcium channel stabilizing protein 1 depleted compared to RyR1 from younger (3-6-month) adult mice. This RyR1 channel complex remodeling results in a "leaky" channel with an increased chance of opening, resulting in leakage of intracellular calcium in skeletal muscle. Treatment of aging mice with S107 stabilized the binding of calcium channel stabilizing protein 1 to RyR1, reduced intracellular calcium leakage, reduced Reactive Oxygen Species (ROS), and enhanced tonic Ca²⁺Release, muscle-specific force and motor ability.

PCT international patent publications WO 2005/094457, WO 2006/101496 and WO2007/024717 disclose 1, 4-benzothiazepine derivatives and their use in the treatment of cardiac, skeletal and cognitive disorders, among others.

PCT international patent publication WO 2008/060332 relates to the use of 1, 4-benzothiazepine derivatives for the treatment and/or prevention of muscle fatigue in individuals suffering from pathological conditions such as muscular dystrophy or individuals suffering from muscle fatigue as a result of sustained, prolonged and/or intense exercise or prolonged stress.

PCT international patent publication WO 2008/021432 relates to the use of 1, 4-benzothiazepine derivatives in the treatment and/or prevention of diseases, disorders and conditions affecting the nervous system.

PCT International patent publication WO 2012/019076 relates to the use of 1, 4-benzothiazepine derivatives in therapy and/orUse in the prevention of myocardial ischemia/reperfusion injury. Fauconnier et al, Proc Natl Acad Sci USA, 2011, 108 (32): 13258-63 reported that caspase-8 activation mediated leakage of RyR leads to myocardial ischemia-reperfusion-post-left ventricular injury, and treatment with S107 inhibited SR Ca²⁺Leakage, reduction of ventricular arrhythmias, infarct area and left ventricular remodeling at 15 days post reperfusion.

PCT international patent publication WO 2012/019071 relates to the use of 1, 4-benzothiazepine derivatives in the treatment and/or prevention of sarcopenia.

PCT international patent publication WO 2012/037105 relates to the use of 1, 4-benzothiazepine derivatives for the treatment and/or prevention of stress-induced neuronal disorders and diseases.

There is a need to identify new compounds for effective treatment of disorders and diseases associated with RyRs, including skeletal muscle and cardiac disorders and diseases. More specifically, there remains a need to identify new agents that can be used to treat RyR-related disorders, for example, by repairing leaks in RyR channels and enhancing calcium channel stabilizing protein binding to PKA-phosphorylated/oxidized/nitrosylated RyRs, and to mutate RyRs that otherwise have reduced affinity for or do not bind to calcium channel stabilizing proteins.

Summary of The Invention

The present invention provides novel 1, 4-benzothiazepine derivatives and pharmaceutically acceptable salts thereof. In some embodiments, the compounds of the present invention are lanolinidine receptor (RyR) calcium channel stabilizers, sometimes referred to as "Rycals^TM". The invention also provides methods of using these compounds in the treatment of disorders and diseases associated with RyRs.

The compounds of the present invention are selected from the 1, 4-benzothiazepine derivatives described in WO 2007/024717. WO2007/024717 describes structurally similar compounds, however, as further described herein, these compounds have been found to be highly unstable and thus their therapeutic use as pharmaceuticals is limited. The problem underlying the present application is therefore to provide alternative 1, 4-benzothiazepine derivatives which not only have pharmacological activity but also advantageous properties, such as a high metabolic stability, and are therefore suitable as medicaments for the treatment of diseases and disorders associated with RyR, such as cardiac, skeletal muscle and Central Nervous System (CNS) disorders. It has surprisingly been found that the compounds of formula (I) are stable and pharmacologically active, thus providing a technical solution to the problem underlying the present invention.

The compounds of the present invention are represented by the structure of formula (I):

wherein

R is COOH;

and pharmaceutically acceptable salts thereof.

The compounds of formula (I) may be present in the form of a salt with a pharmaceutically acceptable acid or base. Such salts are preferably selected from the group consisting of sodium, potassium, magnesium, hemi-fumarate, hydrochloride and hydrobromide salts, with each possibility representing a separate embodiment of the invention. One presently preferred salt is the sodium salt. Another presently preferred salt is the hemifumarate salt.

In some particular embodiments, the compound is selected from the group consisting of compound 1, compound 4, and compound 6, and pharmaceutically acceptable salts thereof. The structures of these compounds are described below.

In a preferred embodiment, the compound is represented by the structure of compound (1):

or a pharmaceutically acceptable salt thereof.

In some embodiments, compound 1 is provided as a parent compound. However, in other embodiments, compound 1 is provided as a salt with a pharmaceutically acceptable acid or base. Preferably, such salts are selected from the group consisting of sodium, potassium, magnesium, hemi-fumarate, hydrochloride and hydrobromide salts, with each possibility representing a separate embodiment of the invention. One presently preferred salt is the sodium salt. Another presently preferred salt is the hemifumarate salt.

The invention also provides methods for synthesizing the compounds of the invention and salts thereof.

The present invention also provides pharmaceutical compositions comprising one or more of the compounds of the present invention and at least one additive or excipient, such as fillers, diluents, binders, disintegrants, buffers, colorants, emulsifiers, taste modifiers, gelling agents, glidants, preservatives, solubilizers, stabilizers, suspending agents, sweeteners, tonicity agents, wetting agents, emulsifiers, dispersing agents, swelling agents, retarding agents, lubricants, absorbents, and viscosity increasing agents. The composition can be made into capsule, granule, powder, solution, sachet, suspension or tablet.

The invention also provides methods of treating or preventing various disorders, diseases and conditions associated with RyRs, such as cardiac, musculoskeletal, cognitive, CNS and neuromuscular disorders and diseases, comprising administering to a subject in need of such treatment an amount of a compound of formula (I) or a salt thereof effective to prevent or treat the disorder or disease associated with RyR. The invention also provides a method of preventing or treating leakage of RyR (including RyR1, RyR2, and RyR3) in a subject, comprising administering to the subject a compound of formula (I) or a salt thereof in an amount effective to prevent or treat leakage of RyR.

In addition, the present invention provides a method of modulating the binding of RyRs to a calcium channel stabilizing protein in a subject comprising administering to the subject an amount of a compound of formula (I) or a salt thereof effective to modulate RyR-binding of the calcium channel stabilizing protein.

The invention also relates to the use of compounds of formula (I) for the preparation of medicaments for the treatment and/or prevention of disorders, diseases and conditions associated with RyRs, such as cardiac, musculoskeletal and cognitive/CNS disorders and diseases. In another embodiment, the invention relates to the use of a compound of formula (I) for the manufacture of a medicament for the prevention and/or treatment of RyR leakage. In another embodiment, the invention relates to the use of a compound of formula (I) for the manufacture of a medicament for modulating the amount of an RyR-bound calcium channel stabilizing protein.

The methods of the invention may be performed on in vitro systems (e.g., cultured cells or tissues) or physically (e.g., in a non-human animal or human).

In some embodiments, there is provided a combination of a compound of the invention with an exon skipping therapy, e.g., an Antisense Oligonucleotide (AOs), in order to enhance exon skipping in an mRNA of interest, e.g., a DMD gene as further described herein. Other features and advantages of the present invention will be apparent from the following detailed description and the accompanying drawings.

Brief Description of Drawings

Figure 1A is an immunoblot using a calcium channel stabilizing protein 2 antibody showing that calcium channel stabilizing protein 2 binds PKA-phosphorylated RyR2 in the absence (-) or presence of 100nM compound 1. (+): the calcium channel stabilizing protein binds non-PKA phosphorylated RyR 2. S36(US 7,544,678) was used as a positive control.

Figure 1B immunoblots with calcium channel stabilizing protein 2 antibodies showing binding of calcium channel stabilizing protein 2 to PKA-phosphorylated RyR2 in the absence (-) or presence 100nM of compound 2, compound 3, or compound 4. (+): the calcium channel stabilizing protein binds non-PKA phosphorylated RyR 2. S36 was used as a positive control.

Figure 1C immunoblots with calcium channel stabilizing protein 1 antibodies showing that calcium channel stabilizing protein 1 binds PKA-phosphorylated RyR1 in the absence (Neg) or in the presence of compound 1 or compound 4 at the indicated concentrations. (Pos): the calcium channel stabilizing protein binds non-PKA phosphorylated RyR 1. S36 was used as a positive control.

FIG. 2A: immunoblotting using a calcium channel stabilizing protein 1 antibody showing the level of calcium channel stabilizing protein 1 in the RyR1 complex of immunoprecipitates of tibial lysates in mice administered with vehicle (50: 50DMSO/PEG), Isoproterenol (ISO) alone, or isoproterenol in osmotic pumps and the indicated concentrations of compound 1. S36 was used as a control at 3.6 mM. FIG. 2B: quantification of% calcium channel stabilizing protein 1 rebinding to RyR 1.

FIG. 3 model of chronic heart failure in rats induced by ischemic-reperfusion (I/R) injury. For the I/R protocol, the Left Anterior Descending (LAD) coronary artery was closed for 1 h.

FIG. 4 shows the administration of compound 1 at 5mg/kg/d (5MK) or 10mg/kg/d (10MK) in drinking water and vehicle (H)₂O) -Left Ventricular (LV) volume and Ejection Fraction (EF) compared in treated rats in treated and mock-operated animals Chronic heart failure induced by ischemia-reperfusion (I/R) injury closed LAD artery 1h treatment started 1 week after reperfusion and continued for 3 months echocardiographic parameters were obtained after 1, 2 or 3 months of treatment 4A: LV end diastolic volume; FIG. 4B: LV end systolic volume; FIG. 4C: EF. FIGS. 4A and 4B § P < 0.001 compared to mock; P < 0.05 compared to vehicle ＊;p is less than 0.001 compared with the solvent. FIG. 4C: p < 0.001 compared to the simulation,p is less than 0.001 compared with the solvent.

FIG. 5 FIGS. 5A-C depict Body Weight (BW) (5A), infarct size (5B), and LV weight (5C), and FIG. 5D depicts administration of Compound 1 at 5mg/kg/D (5MK) and 10 mg/kg/day (10MK) in drinking water and vehicle (H)₂O) -collagen content in treated and mock-operated animals compared to treated rats. Slow induction by ischemia-reperfusion (I/R) injurySexual heart failure. Closing the LAD artery for 1 h; treatment was initiated 1 week after reperfusion and continued for 3 months. Parameters were measured after 3 months of treatment. FIGS. 5A-C: not significant. FIG. 5D:p is less than 0.001 compared with the simulation, and P is less than 0.05 compared with the solvent in ＊.

Fig. 6 invasive hemodynamic: compound 1 at 5mg/kg/d (5MK) or 10 mg/kg/day (10MK) in drinking water with vehicle (H)₂O) -left ventricular systolic pressure (LV SP) (6A), dP/dtmax (6B) and dP/dtmin (6C) in treated and mock-operated animals comparison treated rats Chronic heart failure induced by ischemia-reperfusion (I/R) injury closure of LAD artery 1h, initiation of treatment 1 week after reperfusion and duration of 3 months after treatment 3 months measurement of hemodynamic parameters figure 6A: not significant figure 6B § P < 0.05 compared to mock, P < 0.05 compared to vehicle ＊ figure 6C:p is less than 0.01 compared with the simulation, and P is less than 0.05 compared with the solvent in ＊.

Figure 7 plasma concentration of compound 1(μ M) versus time in days.

FIG. 8 shows the effect of 5mg/kg/d (5MK) of Compound 1 or Compound A in drinking water and vehicle (H)₂O) -EF. in rats treated and treated in mock compared to closed LAD artery for 1h, treatment started 1 week after reperfusion and continued for 3 months, echocardiogram parameters obtained after 1, 2 or 3 months of treatment, § P < 0.001 compared to mock, ＊ P < 0.05 compared to vehicle;p is less than 0.001 compared with the solvent.

FIG. 9 mdx and WT mice with vehicle (H)₂O) -effect of the treated control group on spontaneous physical activity compared to compound 1. Day 1-19 activity in 10 and 50 mg/kg/day (target dose) administered mdx mice administered in drinking water compared to vehicle control group P < 0.001。

Figure 10 specific force-frequency dependence of EDL muscle. (A) Day 1-19 Activity & vehicle (H) in Drinking Water administered Compound 1(5, 10 and 50mg/kg/d (target dose)) treated mdx mice₂O) -treated control (n ═ 5) were compared. For a 50mg/kg/d dose, frequency of 150Hz and above, p is < 0.05. (B) Compound 1(5, 10 and 50mg/kg/d (target dose)) treated WT, C57BL/6 mice in drinking water with vehicle (H)₂O) -treated control (n ═ 4) compared.

FIG. 11 with vehicle (H) administered in drinking water₂O) or Compound 1(50mg/kg/d (target dose) mean body weight (12A) and mean water consumption (12B) of the WT mice treated mdx and WT mice.

Detailed Description

It should be understood that the detailed description and specific examples, while indicating various embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. All publications, patent applications, and other references cited herein are incorporated by reference in their entirety.

The term "Rycals^TM"refers to the ryanodine receptor calcium channel stabilizer, expressed as the compounds of formula (I) or (IA) provided herein, as well as the specific compounds designated by the numerical designations provided herein, and collectively referred to herein as" compounds of the invention ".

Compound (I)

In some embodiments, the compounds of the present invention are represented by the structure of formula (IA):

wherein

R is COOH or its bioisostere, COOR¹Or CN; and is

R¹Is C₁-C₄An alkyl group;

and pharmaceutically acceptable salts thereof.

In some preferred embodiments, R in formula (IA) is carboxylic acid (COOH). In other preferred embodiments, R in formula (IA) is a carboxylic acid bioisostere, such as tetrazole. Alternatively, the carboxylic acid bioisosteres can be acidic heterocycles such as 1, 2, 4-oxadiazol-5 (4H) -one, 1, 2, 4-thiadiazol-5 (4H) -one, 1, 2, 4-oxadiazol-5 (4H) -thione, 1, 3, 4-oxadiazol-2 (3H) -thione, 4-methyl-1H-1, 2, 4-triazole-5 (4H) -thione, 5-fluoroorotic acid, and the like. Additional carboxylic acid bioisosteres are described, for example, in Hamada, y, et al, bioorg.med.chem.lett.2006; 16: 4354-4359; herr, r.j. et al, bioorg.med.chem.2002; 10: 3379-; olesen, p.h., curr. opin. drug discov.devel.2001; 4: 471; patani.g.a. et al, j.chem.rev.1996; 96: 3147; kimura, t, et al bioorg.med.chem.lett.2006; 16: 2380 and 2386; and Kohara, y, et al bioorg.med.chem.lett.1995; 5(17): 1903, and 1908. The contents of each of the above-cited references are incorporated herein by reference.

In a preferred embodiment, the compounds of the present invention are represented by the structure of formula (IA), wherein R is COOH, and pharmaceutically acceptable salts thereof (i.e., compounds of formula (I)).

In other preferred embodiments, R in formula (IA) is in the 4-position of the phenyl ring (i.e., in the 7-position of the benzothiazepine ring). Each possibility represents a separate embodiment of the invention. The compounds of formula (IA) or (I) may be present in the form of a salt with a pharmaceutically acceptable acid or base. Such salts are preferably selected from the group consisting of sodium, potassium, magnesium, hemi-fumarate, hydrochloride and hydrobromide salts, with each possibility representing a separate embodiment of the invention. One presently preferred salt is the sodium salt. Another presently preferred salt is the hemifumarate salt.

In some particular embodiments, the compound is selected from the group consisting of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11, and compound 12, and pharmaceutically acceptable salts thereof. These compounds are represented by the following structures:

chemical hair sense：

The term "alkyl" as used herein refers to a straight or branched chain saturated hydrocarbon ("C") having from 1 to 4 carbon atoms₁-C₄Alkyl "). Representative alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, and tert-butyl. The alkyl group may be unsubstituted or substituted with one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy, haloalkoxy, cycloalkyl, aryl, heterocyclyl, heteroaryl, amido, alkylamido, dialkylamido, nitro, amino, cyano, N₃Oxo, alkylamino, dialkylamino, carboxy, thio, thioalkyl and thioaryl.

The compounds of the present invention may exist in their tautomeric form. All such tautomeric forms are contemplated herein as being part of the present invention.

All stereoisomers of the compounds of the invention (e.g., those present due to asymmetric carbons on different substituents) including enantiomeric and enantiomeric forms are contemplated as being within the scope of the invention. For example, each stereoisomer of a compound of the invention may be substantially free of the other isomers (e.g., as a pure or substantially pure optical isomer having the specified activity) or may be mixed, e.g., as a racemate or as a mixture enriched in one stereoisomer. The chiral centers of the present invention may have the S or R configuration as defined by the IUPAC 1974 recommendation. The racemic forms can be resolved by physical methods such as fractional crystallization, separation or crystallization of diastereomeric derivatives or separation by chiral column chromatography. The optical isomers may be obtained from the racemates by any suitable method, including, but not limited to, conventional methods, such as salt formation with an optically active acid or base, followed by crystallization.

The compounds of the present invention are preferably isolated and purified after their preparation to yield a composition comprising equal to or greater than about 90% of the compound, about 95% of the compound, and even more preferably greater than about 99% of the compound ("substantially pure" compound), by weight, and then used or formulated as described herein. Such "substantially pure" compounds of the invention are also contemplated herein as part of the invention.

Therapeutic uses

The present invention provides compounds that are capable of treating conditions, disorders, and diseases associated with RyRs. More specifically, the present invention provides compounds that can repair leaks in RyR channels, which may be RyR1, RyR2, and/or RyR3 channels. In one embodiment, the compounds of the invention enhance RyR and calcium channel stabilizing proteins (e.g., RyR1 and calcium channel stabilizing protein 1; RyR2 and calcium channel stabilizing protein 2; and RyR3 and calcium channel stabilizing protein 1) binding and/or inhibiting their separation. "conditions, disorders, and diseases associated with RyRs" refers to disorders and diseases that can be treated and/or prevented by modulation of RyRs, and includes, but is not limited to, cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular diseases and disorders, cognitive impairment, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome.

Thus, in one embodiment, the invention relates to a method of treating or preventing a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular diseases and disorders, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or improving cognitive function, comprising the step of administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I) or (IA) as described herein, or a salt thereof, to accomplish such treatment. Presently preferred compounds are compounds of formula (1).

In another embodiment, the invention relates to the use of an effective amount of a compound of formula (I) or (IA) or a salt thereof as described herein for the preparation of a medicament for the treatment or prevention of a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, skeletal muscle disorders and diseases, CNS disorders and diseases, neuromuscular diseases and disorders, cognitive dysfunction, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death and sudden infant death syndrome or for improving cognitive function. Presently preferred compounds are compounds of formula (1).

In another embodiment, the present invention relates to a compound of formula (I) or (IA) or a salt thereof as described herein for the preparation of a medicament for the treatment or prevention of a condition selected from the group consisting of cardiac disorders and diseases, muscular fatigue, skeletal muscle disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular diseases and disorders, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death and sudden infant death syndrome, or for improving cognitive function. Presently preferred compounds are compounds of formula (1).

In one embodiment, the condition, disorder or disease is associated with abnormal function of RyR 1. In another embodiment, the condition, disorder or disease is associated with abnormal function of RyR 2. In another embodiment, the condition, disorder or disease is associated with abnormal function of RyR 3. Each possibility represents a separate embodiment of the invention.

Cardiac disorders and diseases include, but are not limited to, irregular heart beat disorders and diseases, exercise-induced irregular heart beat disorders and diseases, heart failure, congestive heart failure, chronic heart failure, acute heart failure, systolic heart failure, diastolic heart failure, acute compensatory heart failure, myocardial ischemia/reperfusion (I/R) injury (including post-coronary angioplasty or post-thrombolytic I/R injury during Myocardial Infarction (MI)), chronic obstructive pulmonary disease, and hypertension. Irregular heart beat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmias, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmias, atrial and ventricular tachycardias, catecholamine-dependent ventricular tachycardia (CPVT), and exercise-induced variations thereof.

The compounds of the invention are also useful in the treatment of muscle fatigue, which may be due to prolonged exercise or high intensity exercise or may be caused by musculoskeletal disorders. Examples of muscle disorders and diseases include, but are not limited to, skeletal muscle fatigue, central nuclear disease, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, age-related muscle fatigue, sarcopenia, congenital myopathy, skeletal myopathy and/or atrophy, cancer cachexia, myopathy with nuclei and rods, mitochondrial myopathy [ e.g., ji-seiki syndrome, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke) syndrome and MERRF (myoclonic epilepsy with broken red fibers) syndrome ], endocrine myopathy, glycogen storage disease [ e.g., pompe disease, anderson disease and Cori disease ], myoglobinuria [ e.g., McArdle disease, Tarui disease and DiMauro disease ], dermatomyositis, ossification myositis, familial periodic paralysis, polymyositis, inclusion body myositis, neuromyotonia, stiff person syndrome, malignant hyperthermia, hyperrefractory syndrome, and malignant sarcoidosis, General muscle spasms, tics, myasthenia gravis, Spinal Muscular Atrophy (SMA), spinal and bulbar muscular atrophy (SBMA, also known as spinal bulbar muscular atrophy, bulbar atrophy, X-linked bulbar neuropathy (XBSN), X-linked spinal muscular atrophy type 1 (SMAX1) and Kennedy's Disease (KD)), and muscular dystrophies. Preferred skeletal muscle disorders include, but are not limited to, exercise-induced skeletal muscle fatigue, congenital myopathy, muscular dystrophy, age-related muscle fatigue, sarcopenia, central nuclear disease, cancer cachexia, bladder disorders, and incontinence.

Examples of muscular dystrophy include, but are not limited to, Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), limbal marginal band muscular dystrophy (LGMD), Congenital Muscular Dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral dystrophy, myotonic dystrophy, edbi muscular dystrophy and oculopharyngeal muscular dystrophy, with DMD currently preferred.

Congenital muscular dystrophy as used herein refers to muscular dystrophy that occurs at birth. Classifying CMD based on genetic mutation: 1) a gene encoding a structural protein of a skeletal muscle fiber basement membrane or extracellular matrix; 2) a gene encoding a putative or validated glycosyltransferase, which thereby affects the glycosylation of dystrophin glycans, i.e. outer membrane proteins of the basement membrane; and 3) others. Examples of CMD include, but are not limited to, laminin- α 2-deficient CMD (MDC1A), Ullrich CMG (UCMDs 1, 2, and 3), wacker-wabbger syndrome (WWS), ocular muscle brain disease (MEB), Fukuyama CMD (FCMD), CMD + secondary laminin deficiency 1(MDC1B), CMD + secondary laminin deficiency 2(MDC1C), CMD with mental retardation and megagyrus (MDC1D), and ankylosing spine with muscular dystrophy type 1 (RSMD 1).

Cognitive dysfunction may be associated with or include, but is not limited to, memory loss, age-dependent memory loss, post-traumatic stress disorder (PTSD), Attention Deficit Hyperactivity Disorder (ADHD), Autistic Spectrum Disorders (ASD), Generalized Anxiety Disorder (GAD), Obsessive Compulsive Disorder (OCD), schizophrenia, bipolar disorder or major depression.

CNS disorders and diseases include, but are not limited to, Alzheimer's Disease (AD), neuropathy, epilepsy, Parkinson's Disease (PD), and Huntington's Disease (HD).

Neuromuscular disorders and diseases include, but are not limited to, spinocerebellar ataxia (SCA) and amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease).

In some embodiments, the compounds of the invention improve cognitive function, which may be selected from short term memory, long term memory, attention, learning, and any combination thereof.

In some embodiments, the compounds of the invention are used to treat cancer cachexia, i.e., muscle weakness typically associated with cancer, and preferably in metastatic cancer, e.g., bone metastasis. Muscle weakness and muscle atrophy (cachexia) are common paracancerous symptoms in cancer patients. These conditions cause significant fatigue and significantly reduce the quality of life of the patient. The present invention provides methods for treating and preventing muscle weakness in cancer patients based in part on the following findings: in some cancer types, such as prostate and breast cancers with bone metastases, RyR1 is oxidized, which is induced to become "leaky". It was also found that prevention of leakage by administration of Rycal compounds improved muscle function. Exemplary cancers include, but are not limited to, breast cancer, bone cancer, pancreatic cancer, lung cancer, colon cancer, and gastrointestinal cancer.

Exon skipping therapy:

in some embodiments, the compounds of the invention modulate (e.g., enhance) mRNA splicing by enhancing antisense-mediated exon skipping. This modulation of splicing is accomplished in the presence of an Antisense Oligonucleotide (AOs) specific for the spliced sequence of interest. In some embodiments of the invention, the compound of formula (I) or (IA) and AO may act synergistically, wherein the compound of formula (I) or (IA) enhances AO-mediated exon skipping. Thus, in some embodiments, the present invention relates to a pharmaceutical composition for treating or preventing any of the disorders described herein associated with leaky RyR, further comprising the use of an antisense AO specific for a splice sequence in an mRNA sequence for enhancing exon skipping in the mRNA of interest.

One particular embodiment of the compounds of the invention for exon skipping enhancement relates to Duchenne Muscular Dystrophy (DMD). DMD is a fatal X-linked recessive disease characterized by progressive muscle weakness during the life of the patient. DMD results primarily from the deletion of an out-of-frame multi-exon in the DMD gene produced by the removal of dystrophin. Loss of dystrophin expression alone does not explain DMD pathophysiology. Disruption of the dystrophin-glycoprotein complex (DGC) also leads to oxidative stress, mitochondrial Ca²⁺Overload and apoptosis, Ca²⁺Influx of muscle growth and pathological Ca²⁺And (6) conducting signals. There is no therapy to cure DMD and only drug therapy has been shown to be corticosteroids, which can prolong bed-exit activity, but there are significant side effects. Antisense oligonucleotide-mediated exon skipping is a promising therapeutic approach aimed at restoring the DMD reading frame and allowing expression of the intact dystrophin glycoprotein complex. To date, low levels of dystrophin protein have been produced in humans using this approach. Kendall et al (Sci trans Med, 2012, 4(164), p.164ra160) report that some small molecules such as dantrolene and other RyR modulators enhance antisense oligomer-guided exon skipping to increase exon skipping, thereby restoring mRNA reading frame, sarcoplasmic dystrophin and dystrophin glycoprotein complexes in skeletal muscle in an mdx mouse, i.e., DMD mouse model.

Thus, in one embodiment, the invention relates to a method of treating DMD by administering to an individual in need thereof a compound of formula (I) or (IA) of the invention with an Antisense Oligonucleotide (AO) specific for one or more exons of the DMD gene, e.g. the spliced sequences of exons 23, 45, 44, 50, 51, 52 and/or 53 of the DMD gene. Preferred AOs include, but are not limited to, AOs targeting DMD exons 23, 50 and/or 51 of the DMD gene, such as 2 '-O-methyl (2' OMe) phosphorothioate or phosphorodiamidate (phosphorodiamidite) morpholino (PMO) AOs. Examples of such AOs include, but are not limited to, Pro051/GSK2402968, AVI4658/Eteplirsen and PMO E23 morpholino (5'-GGCCAAACCTCGGCTTACCTGAAAT-3').

As used herein, the terms "effective amount", "sufficient amount" or "therapeutically effective amount" of an active agent are used interchangeably, i.e., an amount sufficient to elicit a beneficial or desired effect, including a clinical effect, and as such, an "effective amount" or variations thereof, is dependent upon the context in which it is used. In some embodiments, the response is prophylactic, in other embodiments, therapeutic, and in further embodiments, a combination thereof. The term "effective amount" also includes an amount of a compound of the present invention that is "therapeutically effective" and that avoids or substantially reduces undesirable side effects.

As used herein and as well understood in the art, "treatment" is a method for obtaining a beneficial or desired effect, including a clinical effect. Beneficial or desired effects can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization (i.e., not worsening) of the disease state, prevention of spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and elimination (whether partial or total), whether detectable or undetectable. "treatment" may also refer to prolonging survival compared to expected survival if not receiving treatment.

Pharmaceutical composition

The compounds of the present invention are formulated into pharmaceutical compositions that are administered to a human subject in a biologically compatible form suitable for in vivo administration. According to another aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention together with a pharmaceutically acceptable diluent and/or carrier. The pharmaceutically acceptable carrier is preferably "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

The compounds may be administered alone, but are preferably administered together with one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier used herein may be selected from a variety of organic or inorganic materials which are materials used as pharmaceutical formulations and incorporated as any one or more of fillers, diluents, binders, disintegrants, buffers, colorants, emulsifiers, taste-improving agents, gelling agents, glidants, preservatives, solubilizers, stabilizers, suspending agents, sweeteners, tonicity agents, wetting agents, emulsifiers, dispersants, swelling agents, retarding agents, lubricants, absorbents and viscosity-increasing agents.

The compounds of the invention are administered to human or animal subjects by known methods, including, but not limited to, oral, sublingual, buccal, parenteral (intravenous, intramuscular, or subcutaneous), transdermal or transcutaneous, intranasal, intravaginal, rectal, ocular and respiratory (administration by inhalation). The compounds of the invention can also be administered to a subject by delivery to a muscle of the subject, including, but not limited to, the cardiac or skeletal muscle of the subject. In one embodiment, the compound is administered to the individual by targeted delivery to the myocardium via a catheter inserted into the heart of the individual. In other embodiments, the compound is administered directly to the CNS, for example by intravertebral injection or intraventricular (intraventricular) infusion of the compound directly into cerebrospinal fluid (CSF) or intraventricular, intrathecal or interstitial administration. Oral administration is presently preferred.

Pharmaceutical compositions of the invention for solid oral administration include, inter alia, tablets or lozenges, sublingual tablets, sachets, capsules including gelatin capsules, powders and granules; and those for liquid oral, nasal, buccal or ocular administration include, inter alia, emulsions, solutions, suspensions, drops, syrups and aerosols. The compounds may also be administered together as a suspension or solution through drinking water or food. Examples of pharmaceutically acceptable carriers include, but are not limited to, cellulose derivatives including carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, and microcrystalline cellulose; sugars such as mannitol, sucrose or lactose; gelatin, gum arabic, magnesium stearate, sodium stearyl fumarate, saline, sodium alginate, starch, talc, water, and the like.

Pharmaceutical compositions of the invention for parenteral injection include, inter alia, sterile solutions, which may be aqueous or non-aqueous dispersions, suspensions or emulsions, and also sterile powders for reconstitution into injectable solutions or dispersions. The compounds of the present invention may be combined with a sterile aqueous solution that is isotonic with the blood of the individual. Such formulations are prepared by dissolving the solid active ingredient in water containing a physiologically compatible substance, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions so as to produce an aqueous solution, which is then rendered sterile. The formulations are provided in unit or multi-dose containers, for example sealed ampoules or vials. The formulation is delivered by any injection mode including, but not limited to, suprafascially, intravesicularly, intracranially, intradermally, intramuscularly, intraorbitally, intraperitoneally, intraspinally, intrasternally, intravascularly, intravenously, intraparenchymally, subcutaneously, or sublingually, or by a catheter into the heart of the individual.

Pharmaceutical compositions for rectal or vaginal administration are preferably suppositories, and those for transdermal or transdermal administration include, inter alia, powders, aerosols, creams, ointments, gels and patches.

For transdermal administration, the compounds of the present invention are combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropyl alcohol, ethanol, oleic acid, N-methylpyrrolidine, and the like, which increase the permeability of the skin to the compounds of the present invention and allow the compounds to penetrate through the skin and into the bloodstream. The compound/accelerator composition may be further combined with a polymer, such as ethyl cellulose, hydroxypropyl cellulose, ethylene/vinyl acetate, polyvinyl pyrrolidone, etc., to provide a composition in the form of a gel, which is dissolved in a solvent, evaporated to a desired viscosity, and then applied to a backing material to provide a patch.

The pharmaceutical formulations of the present invention are prepared by methods well known in the pharmaceutical arts including, but not limited to, wet granulation and dry granulation or by direct compression. The choice of carrier will depend on the solubility and chemical nature of the compound, the chosen route of administration and standard pharmaceutical practice.

The above pharmaceutical compositions exemplify the invention, but do not limit the invention in any way.

Any of these compounds can be administered to a subject (or cells contacting a subject) in an amount effective to limit or prevent a decrease in the level of an RyR-bound calcium channel stabilizing protein in the subject, particularly in cells of the subject, according to the methods of the invention. Such amounts are readily determined by one skilled in the art based on known methods, including analysis of titration curves established in vivo and the methods and assays disclosed herein. Suitable amounts of the compound effective to limit or prevent a decrease in the level of RyR-bound calcium channel stabilizing protein in an individual are in the range of about 0.01 mg/kg/day to about 100 mg/kg/day (e.g., 1, 2, 5, 10, 20, 25, 50, or 100 mg/kg/day) and/or in an amount sufficient to result in plasma levels in the range of about 300ng/ml to about 5,000 ng/ml. Alternatively, the amount of the compound of the present invention may range from about 1 mg/kg/day to about 50 mg/kg/day. Alternatively, the compounds of the present invention may be used in amounts of from about 10 mg/kg/day to about 20 mg/kg/day. Also included are amounts that may be administered at about 0.01 mg/kg/day or 0.05 mg/kg/day to about 5 mg/kg/day or about 10 mg/kg/day.

Synthesis method

In another aspect, the invention provides methods for preparing the compounds of the invention and salts thereof. More specifically, the present invention provides a process for the preparation of compounds of formula (I) or (IA) such as compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, compound 9, compound 10, compound 11 and compound 12 or salts thereof. The different synthetic routes to the compounds are described in the examples. The general synthetic Route (ROS) is described in scheme 1 below:

scheme 1

In scheme 1, R^aIs COOR¹Or CN; r¹Is C₁-C₄Alkyl, and L is a leaving group, as examples, halogen, sulfonate (OSO)₂R ', wherein R' is an alkyl or aryl group, such as OMs (mesylate), OTs (tosylate)), and the like. The amine starting material is reacted with an alkylating agent (a benzyl derivative as shown above), preferably in the presence of a base, to give the desired product or its precursor (R ═ R)^a). If desired, such precursors may be further reacted to convert the group R^aConversion to the group R as exemplified in the experimental section below or by any other method known to those skilled in the art. For example, the ester precursor (R) may be hydrolyzed under acidic or basic conditions according to known methods^a＝COOR¹Wherein R is¹Is C₁-C₄Alkyl) to the corresponding carboxylic acid (R ═ COOH). Alternatively, the nitrile precursor (R) may be prepared by reacting sodium azide under suitable conditions^aCN) to tetrazoles (carboxylic acid isosteres) or by hydrolysis to carboxylic acids (R ═ COOH).

The amine starting material may be prepared according to the methods described in WO 2009/111463 or WO2007/024717 or by any other method known to the person skilled in the art. The contents of all of the above references are incorporated herein by reference. The nature of the base is not particularly limited. Preferred bases include, but are not limited to, hydrides (e.g., sodium hydride or potassium hydride) and N, N-diisopropylethylamine. Other suitable bases include, but are not limited to, organic bases, such as tertiary amines selected from acyclic amines (e.g., trimethylamine, triethylamine, dimethylaniline, diisopropylethylamine, and tributylamine), cyclic amines (e.g., N-methylmorpholine), and aromatic amines (dimethylaniline, dimethylaminopyridine, and pyridine).

The reaction may be carried out in the presence or absence of a solvent. If used, the nature of the solvent is not particularly limited, and examples include solvents such as esters (e.g., ethyl acetate), ethers (e.g., THF), chlorinated solvents (e.g., dichloromethane or chloroform), Dimethylformamide (DMF), and other solvents such as acetonitrile or toluene or mixtures of these solvents with each other or with water.

Salts of compounds of formula (I) (wherein R ═ COOH) may be prepared by reacting the parent molecule with a suitable base such as NaOH or KOH to give the corresponding alkali metal salt, e.g. sodium or potassium salt. Alternatively, esters (R ═ COOR) can be prepared by reaction with a suitable base¹) Directly converted into salt.

Salts of the compounds of formula (I) may also be prepared by reacting the parent molecule with a suitable acid, for example HCl, fumaric acid or p-toluenesulfonic acid, to give the corresponding salts, for example the hydrochloride, tosylate or hemi-fumarate salts.

Examples

The following examples are provided as illustrations of some preferred embodiments of the invention.

Example 1: synthesis of

The instrument comprises the following steps:

NMR: bruker AVANCE III 400 or Varian Mercury 300

LC/MS: waters Delta 600 with auto sampler 17Plus, photodiode array detector 2996 and mass detector 3100 mounted, or Shimadzu 210

General procedure for alkylation of 7-methoxy-2, 3, 4, 5-tetrahydrobenzo [ f ] [1, 4] thiazepine ("amine

Amines as pesticides

The amine (structure as shown above) (1mmol) was dissolved in 3ml dichloromethane. To this solution was added alkylating reagent (1mmol) followed by N, N-diisopropylethylamine (0.34ml, 2 mmol). The mixture was stirred at room temperature overnight. The solution was applied directly to the column, eluting with hexane/EtOAc (2: 1, v/v).

Compound 2

3- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzoate:¹HNMR(300MHz，CDCl₃)：7.96(m，2H)，7.46(m，3H)，6.70(dd，J＝8.4Hz，3.0Hz，1H)，6.50(d，J＝2.7Hz，1H)，4.09(s，2H)，3.90(s，3H)，3.72(s，3H)，3.57(s，2H)，3.35(m，2H)，2.72(m，2H)。MS：344(M+1)。

compound 3

4- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzoate:¹HNMR(300MHz，CDCl₃)：7.99(d，J＝8.4Hz，2H)，7.46(d，J＝8.4Hz，1H)，7.37(d，J＝8.7Hz，2H)，6.70(dd，J＝8.4Hz，3.0Hz，1H)，6.50(d，J＝2.7Hz，1H)，4.09(s，2H)，3.90(s，3H)，3.72(s，3H)，3.57(s，2H)，3.35(m，2H)，2.72(m，2H)。MS：344(M+1)。

compound 5

2- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzoate: dissolving with 2M HCl in diethyl etherThe solution converts the compound to the hydrochloride salt.¹HNMR(300MHz，DMSO-d₆)：10.33(br，1H)，8.08(d，J＝7.5Hz，1H)，7.80-7.65(m，3H)，7.51(d，J＝8.1Hz，1H)，7.14(s，1H)，6.99(dd，J＝8.4，2.1Hz，1H)，4.90-4.40(m，br，4H)，3.88(s，3H)，3.78(s，3H)，3.40(m，2H)，3.26(m，1H)，3.11(m，1H)。MS：344(M+1)。

Compound 7

2- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzonitrile:¹HNMR(300MHz，CDCl3)：7.67-7.26(m，5H)，6.73(d，J＝2.7Hz，1H)，6.74(dd，J＝2.7，8.4Hz，1H)，4.14(s，2H)，3.78(s，3H)，3.70(s，2H)，3.36(m，2H)，2.76(m，2H)。MS：311(M+1)。

compound 8

3- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzonitrile:¹HNMR(300MHz，CDCl3)：7.64-7.42(m，5H)，6.74(dd，J＝2.7，8.4Hz，1H)，6.48(d，J＝2.7Hz，1H)，4.08(s，2H)，3.75(s，3H)，3.57(s，2H)，3.36(m，2H)，2.76(m，2H)。MS：311(M+1)。

compound 9

4- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzonitrile:¹HNMR(300MHz，CDCl3)：7.64(d，J＝7.2Hz，2H)，7.42(m，3H)，6.74(dd，J＝2.7，8.4Hz，1H)，6.48(d，J＝2.7Hz，1H)，4.08(s，2H)，3.75(s，3H)，3.58(s，2H)，3.36(m，2H)，2.76(m，2H)。MS：311(M+1)。

hydrolysis of esters (general method)

The methyl ester (3mmol) was dissolved in 30ml THF/methanol/1M NaOH (1: 1, v/v). The mixture was stirred for 8 hours and TLC showed complete disappearance of the ester. 1ml of concentrated HCl was added to adjust the acidic pH. The organic solvent was removed and the solid formed was collected by filtration. The solid was air dried.

Compound 4

3- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzoic acid: the compound was obtained by extraction with EtOAc as solvent.¹HNMR(300MHz，CDCl₃)：8.10(s，1H)，8.04(d，J＝8.4Hz，1H)，7.80(br，1H)，7.46(m，2H)，6.80(m，2H)，4.40(s，2H)，3.90(s，2H)，3.76(s，3H)，3.42(s，2H)，2.86(s，2H)。MS：330(M+1)，328(M-1)。

Compound 1

4- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzoic acid: the compound was obtained by extraction with EtOAc as solvent.¹HNMR(300MHz，CDCl₃)：8.02(d，J＝8.4Hz，2H)，7.46(d，J＝8.4Hz，1H)，7.42(d，J＝8.7Hz，2H)，6.70(dd，J＝8.4Hz，3.0Hz，1H)，6.50(d，J＝3.0Hz，1H)，4.11(s，2H)，3.72(s，3H)，3.62(s，2H)，3.35(m，2H)，2.76(m，2H)。MS：330(M+1)，328(M-1)。

Compound 1, sodium salt:

the sodium salt of compound 1 was prepared from the parent molecule using 1 equivalent of NaOH in EtOH (m.p.: 290 ℃ for the salt).

¹HNMR(DMSO-D6，600MHz)，(ppm)：7.77(2H，m)，7.41(1H，d)，7.13(2H，m)，6.75(1H，dd)，6.63(1H，d)，4.00(2H，s)，3.70(3H，s)，3.49(2H，s)，3.18(2H，m)，2.70(2H，m)。

Compound 1, hemifumarate salt:

1.6g of Compound 1 (neutral form) and 265mg of fumaric acid were introduced into a round-bottomed flask. After addition of 18mL of acetone and 2mL of water, the reaction mixture was refluxed. Partial solubilization (but not complete clarification) was observed, followed by precipitation. The reaction mixture was then refluxed overnight. After cooling, the residual solid was isolated by filtration, washed with 3mL of acetone and dried under vacuum (40 ℃ C./10 mbars) for 4 hours.

¹HNMR(DMSO-D6，600MHz)，(ppm)：12.97(2H，bs)，7.90(2H，m)，7.43(1H，d)，7.40(2H，m)，6.77(1H，dd)，6.64(1H，d)，6.62(1H，s)，4.03(2H，s)，3.70(3H，s)，3.58(2H，s)，3.20(2H，m)，2.72(2H，m)。

Compound 6

2- ((7-methoxy-2, 3-dihydrobenzo [ f)][1，4]Thiazepin-4 (5H) -yl) methyl) benzoic acid: the compound was converted to the hydrochloride salt with 2m hcl in ether.¹HNMR(300MHz，DMSO-d₆)：10.10(br，1H)，8.08(d，J＝7.5Hz，1H)，7.66-7.51(m，4H)，7.17(d，J＝2.1Hz，1H)，6.99(dd，J＝8.4，2.1Hz，1H)，4.80-4.40(m，br，4H)，3.78(s，3H)，3.46(m，2H)，3.13(m，2H)。MS：330(M+1)，328(M-1)。

Synthesis of tetrazole (general method)

The nitrile precursor (3.22mmol), sodium azide (830mg, 12.9mmol) and triethylamine hydrochloride (1.72g, 12.9mmol) were stirred in 40ml of anhydrous DMF at 100 ℃ for 5 days. DMF was removed under high vacuum and the residue was mixed with water. The aqueous solution was extracted with dichloromethane (3 × 100 ml). The pure compound was purified by column chromatography (EtOAc/methanol).

Compound 10

4- (2- (1H-tetrazol-5-yl) benzyl) -7-methoxy-2, 3, 4, 5-tetrahydrobenzo [ f][1，4]Thiazepine:¹HNMR (300MHz, CDCl3 and 1 drop CD3 OD): 8.30(d, J ═ 8.7Hz, 1H), 7.53(m, 2H). 7.14(t, J ═ 7.8Hz, 1H), 7.20(d, J ═ 7.5Hz, 1H), 6.84(dd, J ═ 2.7, 8.4Hz, 1H), 6.69(d, J ═ 2.7Hz, 1H), 4.46(s, 2H), 3.80(s, 2H), 3.75(s, 2H), 3.43(m, 2H), 2.96(m, 2H). MS: 354(M +1), 352 (M-1).

Compound 11

4- (3- (1H-tetrazol-5-yl) benzyl) -7-methoxy-2, 3, 4, 5-tetrahydrobenzo [ f][1，4]Thiazepine:¹HNMR(300MHz，CDCl3)：8.16(s，1H)，7.90(d，J＝7.5Hz，1H)，7.40(d，J＝8.4Hz，1H)，7.20(m，2H)，6.74(dd，J＝2.7，8.4Hz，1H)，6.58(d，J＝2.7Hz，1H)，4.18(s，2H)，3.75(s，5H)，3.36(m，2H)，2.76(m，2H)。MS：354(M+1)，352(M-1)。

compound 12

4- (4- (1H-tetrazol-5-yl) benzyl) -7-methoxy-2, 3, 4, 5-tetrahydrobenzo [ f][1，4]Thiazepine:¹HNMR (300MHz, CDCl3 and 1 drop CD3 OD): 7.99(d, J ═ 7.2Hz, 2H), 7.42(m, 3H), 6.74(dd, J ═ 2.7, 8.4Hz, 1H), 6.53(d, J ═ 2.7Hz, 1H), 4.10(s, 2H), 3.71(s, 3H), 3.58(s, 2H), 3.36(m, 2H), 2.76(m, 2H). MS: 354(M +1), 352 (M-1).

Synthesis of 7-methoxy-2, 3, 4, 5-tetrahydrobenzo [ f ] [1, 4] thiazepine ("amine

2- (4-Methoxyphenylthio) ethylamine (1)

4-Methoxythiophenol (50g, 0.357mol), 2-chloroethylamine monohydrochloride (39.8g, 0.343mol.), K₂CO₃(78.8g, 0.57mol) and diisopropylethylamine (32mL, 0.178mol) were combined in 200mL THF. The mixture was degassed under reduced pressure for 5min and refluxed under argon atmosphere overnight. Water was removed and water (300mL) was added to the flask. The mixture was extracted with dichloromethane (3 × 200 mL). The organic layer was collected, dichloromethane removed, 50mL concentrated HCl added, followed by 200mL water. The solution was extracted with 1: 1 EtOAc/hexanes (3X200 mL). The aqueous layer was adjusted to pH10 with 2M NaOH and extracted with dichloromethane (3 × 200 mL). The combined organic layers were dried over anhydrous sodium sulfate. The solvent was removed to give 61g of the title compound as a colorless liquid in 97% yield.

¹H-NMR(300MHz，CDCl3)：7.35(d，J＝8.7Hz，2H)，6.81(d，J＝8.7Hz，2H)，3.77(s，3H)，2.88-2.80(m，4H)，1.44(s，2H)。

2- (4-Methoxybenzenethio) ethylcarbamic acid benzyl ester (2)

First method

To a flask containing compound 1(8.0g, 43.7mmol), sodium bicarbonate (12.1g, 144mmol), water (100mL), and dichloromethane (200mL) was added dropwise benzyl chloroformate (8.2g, 48.1mmol, diluted with 100mL dichloromethane) at 0 ℃. After addition, the mixture was stirred at r.t. for 5 hr. The organic layer was collected and the aqueous solution was extracted with 100mL of dichloromethane. The combined organic solutions were dried over sodium sulfate. The solvent was removed and the resulting solid was triturated with 200mL THF/hexane (1: 10). The solid was collected and dried to give the desired product (12.9g) in 93% yield.

Alternative method

To a solution of compound 1(10g, 54.6mmol) and triethylamine (15mL, 106mmol) in 200mL of dichloromethane was added benzyl chloroformate (7.24mL, 51.5mmol, diluted with 100mL of dichloromethane) dropwise at 0 ℃. After addition, the solution was stirred at r.t. for 1 hour. The solids were removed by filtration. The solution was extracted with 100mL of 0.1M HCl and 100mL of saturated sodium carbonate and dried over anhydrous sodium sulfate. The solvent was removed and a white solid was obtained which was stirred in 200mL THF/hexane (1: 20) for 3 hours. The solid was collected by filtration to give 14.2g of the title compound in 87% yield.

¹H-NMR(300MHz，CDCl₃)：7.35(m，7H)，6.83(d，J＝8.7Hz，2H)，5.07(m，3H)，3.77(s，3H)，3.10(q，J＝6.3Hz，2H)，2.92(t，J＝6.3Hz，2H)。

7-methoxy-2, 3-dihydrobenzo [ f ] [1, 4] thiazepine-4 (5H) -carboxylic acid benzyl ester (3)

A mixture of compound 2(7.3g, 23mmol), paraformaldehyde (6.9g, 0.23mol) and p-toluenesulfonic acid (1.45g, 7.6mmol) in 250mL of toluene was stirred at 70 ℃ overnight. After cooling to r.t., the solid was filtered off. The solution was extracted with saturated sodium carbonate (100mL), and the organic layer was dried over anhydrous sodium sulfate. The desired product (7.4g) was obtained in 97% yield as a liquid after removal of the solvent.

¹H-NMR(300MHz，CDCl₃)：7.44(d，J＝8.1Hz，0.77H)，7.32(m，5.60H)，7.07(d，J＝2.7Hz，0.33H)，6.68(m，1.30H)，5.04(s，2H)，4.59(ss，2H)，3.96(br，1.80)，3.80(ss，1.23H)，3.55(s，1.97H)，2.76(m，2H)。

7-methoxy-2, 3, 4, 5-tetrahydrobenzo [ f ] [1, 4] thiazepine hydrobromide (amine) (4HBr salt)

First method

A solution of HBr (33% in acetic acid, 10mL) was added to compound 3(4.2g, 12.8 mmol). After the addition, carbon dioxide evolution began to occur, forming a white solid. The mixture was allowed to stand at r.t. for an additional 2 hours. Diethyl ether (150mL) was added to the mixture, which was stirred for 30 min. The solid was collected by filtration and washed with diethyl ether. The solid was dried in vacuo to give 3.40g of the title compound in 91.8% yield.

¹H-NMR(300MHz，DMSO-d₆)：9.02(br，2H)，7.52(d，J＝8.1Hz，1H)，7.27(d，J＝3.3Hz，1H)，6.92(dd，J＝8.4，2.7Hz，1H)，4.41(s，2H)，3.77(s，3H)，3.53(m，2H)，2.96(m，2H)。

Alternative Process (free base 4a)

Compound 3(10g, 30mmol) was combined with 50mL concentrated HCl, 50mL water and 30mL dioxane. The mixture was stirred at 100 ℃ overnight. After cooling to r.t., most of the solvent and HCl were removed under reduced pressure. Water (100mL) was added to the solution and the solid was filtered off. The aqueous solution was extracted with EtOAc/hexanes (1: 1, 3X100mL) and basified by addition of 15g NaOH. The mixture was extracted with dichloromethane (3 × 150 mL). The combined solutions were dried over anhydrous sodium sulfate. The solvent was removed to obtain a liquid, which was solidified after standing at rt. to obtain 6.2g of the objective compound.

¹H-NMR(300MHz，CDCl₃)：7.42(d，J＝8.1Hz，1H)，6.78(d，J＝2.7Hz，H)，6.68(dd，J＝2.7，8.1Hz，1H)，4.08(s，2H)，3.96(br，1.80)，3.76(s，3H)，3.38(m，2H)，2.68(m，2H)。

Example 2: binding of calchannel stabilizing protein 2 to PKA-phosphorylated RyR2

Cardiac SR membranes were prepared as described above (Marx et al, 2000; Kaftan et al, Circ. Res., 1996, 78: 990-97). Microsomes (50. mu.g) were immunoblotted for 1hr at room temperature using an anti-calcium channel stabilizing protein antibody (1: 1,000) (Jayaraman et al, J.biol.chem., 1992, 267: 9474-77) as described (Reiken et al, Circulation, 107: 2459-66, 2003). After incubation with HRP-labeled anti-rabbit IgG (1: 5,000 dilution; transduction laboratories, Lexington, Ky.), developed using ECL developed blots (Amersham Pharmacia, Piscataway, N.J.), on an x-ray film or exposure to secondary antibodies labeled with infrared dyes, using equipment from Li-Corbiosciences (model Odyssey). Unless otherwise stated, the tested concentration of compound was 100 nM. Representative calcium channel stabilizing protein 2 binding assays are shown below.

A.PKA phosphorylation of Cardiac Sarcoplasmic Reticulum (CSR)

The reaction mixture was charged into a 1.5ml microcentrifuge tube. Mu.g of cardiac SR was added to the reaction mixture of kinase buffer, PKA and ATP until the final volume was 100. mu.l (reaction mixture below). ATP was finally added to start the reaction.

Reaction mixture:

20 μ l ═ sample (cardiac SR, 2 or 10 μ g/μ l)

10 μ l ═ 10 × kinase buffer (80mM MgCl₂，100mM EGTA，500mM Tris/PIPES)，pH＝7.0

20 μ l ═ PKA (2 units/ul) (Sigma # P2645)

10μl＝10x ATP(1.0mM)(Sigma A 9187)

40 μ l ═ distilled H₂O

1. The tubes were incubated at 30 ℃ for 30 minutes.

2. The reaction mixture was then transferred to a 0.5ml thick walled glass tube.

3. The glass tube containing the reaction mixture was centrifuged AT 50,000Xg for 10min in a Sorvall centrifuge RCM120EX using a rotor S120AT3 rotor. Centrifugation at 50,000x g for 10min was sufficient to isolate the microsomes.

4. The resulting precipitate was washed 4 times with binding buffer (10mM imidazole, 300mM sucrose, pH 7.4). 100 μ l of 1x binding buffer was added to the tube each time to wash the pellet. The pellet was resuspended by rinsing (nush) up and down using the pipette tip. After the final spin, 50 μ l of binding buffer was added and the pellet from all tubes was collected. The reaction was stored at-20 ℃.

5. Phosphorylation was confirmed by separation of approximately 10. mu.g of CSR by 6% polyacrylamide gel electrophoresis (PAGE) and analysis of immunoblots for total RyR (5029Ab, 1: 3000 dilution; or monoclonal Ab from Affinity Bioreagens, Cat # MA3-916, 1: 2000 dilution) and PKA phosphorylated RyR2(P2809Ab, 1: 10000 dilution).

6. The aliquots were saved at-80C.

B.Calcium channel stabilizing protein rebinding assay

1. PKA-phosphorylated CSR (about 20. mu.g) was incubated with 250nM calcium channel stabilizing protein 2 in 100. mu.l binding buffer (as described above) with or without compound.

2. The reaction was loaded into a 0.5ml thick walled glass tube (Hitachi centrifuge instrument, Catalog # B4105).

3. Calcium channel stabilizing protein 2 was added as the final reagent to the reaction mixture. The reaction was allowed to proceed for 30mins at room temperature.

4. After the reaction, the tubes were centrifuged AT 100,000g for 10min (Sorvall RCM120EX centrifuge with S120AT3 rotor).

5. The resulting pellet was washed 4 times in 1 × binding buffer at 4 ℃. After each wash, the tubes were centrifuged at 50,000g for 10mins at 4 ℃.

6. After the final wash, the supernatant was discarded.

7. Add 20. mu.l sample buffer (2X) [ 6X sample buffer below ], resuspend pellet with tip and/or by simple vortex. The suspension was transferred to a 1.5ml microcentrifuge tube.

8. The tube was heated at 90 ℃ for 4 min.

9. Proteins were separated using 15% SDS/PAGE.

10. Calcium channel stable protein 2 binding was detected using an anti-FKBP (Jayaraman et al, J.biol.chem.1992; 267: 9474-77, 1: 2000) primary antibody and a suitable secondary antibody.

6 Xsample buffer

7.0ml 4x Tris-HCl/SDS，pH6.8

3.0ml glycerol (30% final concentration)

1.0g SDS (10% final concentration)

0.93g DTT (0.6M Final)

1mg bromophenol blue (0.001% final concentration)

Distilled water to a final volume of 10 ml.

1ml aliquots were stored at-70 ℃.

Results：

FIG. 1A depicts an immunoblot using a calcium channel stabilizing protein 2 antibody showing that calcium channel stabilizing protein 2 binds to PKA-phosphorylated RyR2 in the absence (-) or presence of 100nM compound 1. (+): the calcium channel stabilizing protein binds non-PKA phosphorylated RyR 2. S36 (benzothiazepine described in US 7,544,678) was used as a positive control. As shown, compound 1 at a concentration of 100nM prevents dissociation of calchannel stabilizing protein 2 from PKA-phosphorylated RyR2 and/or enhances (re-) binding of calchannel stabilizing protein 2 to PKA-phosphorylated RyR.

As shown in figure 1B, it was also found that the following representative compounds prevent dissociation of ca channel stabilizing protein 2 from PKA-phosphorylated RyR2 and/or enhance (re) binding of ca channel stabilizing protein 2 to PKA-phosphorylated RyR when tested at 100nM in the above described ca channel stabilizing protein 2 re-binding assay: compound 2, compound 3 and compound 4.

Example 3: binding of calchannel stabilizing protein 1 to PKA-phosphorylated RyR1

SR membranes were prepared from skeletal muscle in a manner similar to example 2 and further as described in U.S. patent application publication No. 2004/0224368, the contents of which are incorporated herein by reference. Microsomes (50. mu.g) were immunoblotted with anti-calsium channel stabilizing protein 1 antibody (Zymed) (1: 1,000) as described. The blot was developed and quantified as described in example 2.

Figure 1C depicts an immunoblot using a calc-channel stabilizing protein 1 antibody that shows binding of calc-channel stabilizing protein 1 to PKA-phosphorylated RyR1 in the absence (Neg) or in the presence of compound 1 or compound 4 at the indicated concentrations. (Pos): the calcium channel stabilizing protein binds non-PKA phosphorylated RyR 1. S36 was used as a control. As shown, compound 1 and compound 4 prevented dissociation of calchannel stabilizing protein 1 from PKA-phosphorylated RyR1 and/or enhanced calchannel stabilizing protein 1 (re-) binding to PKA-phosphorylated RyR1 in a dose-dependent manner with estimated EC50 of approximately 100nM and 150nM, respectively.

Example 4: re-association of calc channel stabilizing protein 1 with RyR1 in isoproterenol-treated mice

Isoproterenol, a beta adrenergic receptor agonist, induces heart failure in mice by over-stimulating the beta adrenergic receptor. Concurrent with this is PKA activation, RyR2 phosphorylation on SR and a decrease in calcium channel stabilizing protein 2(FKBP12.6) interaction with RyR 2. A similar cascade of events occurs in skeletal muscle, in which RyR1 is phosphorylated, resulting in reduced binding of calcium channel stabilizing protein 1(FKBP12) to RyR 1.

As described in detail in international publication No. WO2008/064264, the contents of which are incorporated herein by reference, chronic isoproterenol treatment of wild type mice provides a rapid and reliable method for inducing easily quantifiable RyR biochemical changes. These changes include increased RyR phosphorylation and concomitant decrease in calcium channel stabilizing protein binding.

Animals and reagents

C57B1/6 mice were maintained and studied according to an approved protocol synthetic β adrenergic agonist Isoproterenol (ISO) was obtained from Sigma (I65627) and prepared as a 100mg/ml stock solution by adding sucrose (1mM), dithiothreitol (320mM), and 1 protease inhibitor tablet (10X) to 10ml of stock solution (10mM HEPES, 1mM EDTA, 20mM NaF, 2mM Na₃VO₄) The lysis buffer was prepared.

Osmotic pump preparation and surgical implantation

Mice were infused continuously with 10mg/ml isoproterenol (1 μ lhr) for 5 days by a subcutaneously implanted osmotic infusion pump (Alzet miniosmatic pump, Model 2001, durect corporation, Cupertino, CA).

For drug loading, the osmotic pump was mounted vertically and 200 μ l of drug solution was injected into the pump through a syringe (adapter cannula) containing an excess of drug solution (250-. The drug solution is slowly injected down while the syringe is slowly raised until the pump is overfilled. The fluid displaced when the pump was capped flowed out to confirm that the pump was properly filled.

The drug-loaded osmotic pump was implanted subcutaneously by the following procedure. With 1.5-2% isoflurane at O administered at 0.6L/min₂The mice were received under medium anesthesia and their body weights were then measured and recorded. The mice were then placed chest down on styrofoam with their face on the nasal cone. The fur was trimmed on the back of the neck, spreading from behind the ears to the top of the head. The area was gently wiped with 70% alcohol and a small incision was made at the midline on the head/neck. The suture fixture was wiped with alcohol, inserted into the incision, and opened to allow the skin to relax from the underlying tissue. To accommodate the pump, the opening is expanded back into the side back half. Inserting the drug-loaded pump into the opening with its release position away from the incision and allowing for subsidence with minimal tensionIt descends to below the skin. About 5-6 sutures were required to close the incision with 5.0 nylon sutures. And the area was lightly wiped with 70% alcohol. After surgery, mice were placed in individual cages in order to minimize injury and possible activation of the sympathetic nervous system.

Skeletal muscle separation

Mouse skeletal muscle was isolated as follows. The leg muscles were exposed by cutting the skin at the ankle joint and pulling up. Tissue treated with Tyrode buffer (10mM HEPES, 140mM NaCl, 2.68mM KCl, 0.42mM Na)₂HPO₄，1.7mM MgCl₂，11.9mM NaHCO₃5mM glucose, 1.8mM CaCl₂By adding 20mg of CaCl₂To contain no CaCl₂Prepared in 100ml of 1X buffer made of 10X solution) was kept wet. The following muscles were isolated and frozen in liquid nitrogen. Extensor Digitorum Longus (EDL) was isolated by inserting scissors between the lateral tendon and the X formed by EDL and tibialis tendon, cut upward against the knee; cutting the fibular muscle to expose the segmental tendon of the gastrocnemius tendon; inserting forceps in X and muscle to relax EDL tendon; EDL tendon was excised and muscle was pulled up; and finally the relaxed EDL is cut off. The soleus muscle was isolated by the following steps: removing the fibular muscle from the top of the gastrocnemius tendon; exposing the soleus muscle to the underside of the gastrocnemius muscle by cutting and lifting the achilles tendon; the soleus muscle was cut at the top of the muscle behind the knee; and eventually pulls out the soleus muscle and cuts it away from the gastrocnemius muscle. Isolating the tibialis by: the tibialis tendon was excised from the anterior ankle, pulled upward, and excised away from the tibia. The femoral muscle (thigh muscle) is isolated from the leg by cutting the muscle just above the knee and removing the fascicles. The samples were frozen in liquid nitrogen.

RyR1 immunoprecipitation from tissue lysates

By mixing 200-500. mu.g of homogenate with 2. mu.l of anti-RyR 1 antibody (Zymed) in 0.5ml of modified RIPA buffer (50mM Tris-HCl (pH 7.4), 0.9% NaCl, 5.0mM NaF, 1.0mM Na at 4 ℃₃VO₄0.5% Triton-X100 and protease inhibitor)RyR1 was immunoprecipitated at 1.5hr from incubation. The samples were then incubated with protein A agarose beads (Amersham Pharmacia Biotech, Piscatawy, NJ) for 1 hour at 4 ℃ after which the beads were washed 3 times with ice cold RIPA. The samples were heated to 95 ℃ and size fractionated by SDS-PAGE (15% SDS-PAGE for calcium channel stabilizing proteins). Immunoblots were developed using an anti-FKBP antibody (FKBP12/12.6, Jayaraman et al, J.biol.chem.1992; 267: 9474-77) at a dilution of 1: 2,000. 5% milk or TBS-T (20mM Tris-HCl, pH7.5, 0.5M NaCl, 0.05%)20, 0.5% Triton X-100).

Results

An osmotic pump containing isoproterenol with or without test compound was implanted in mice as described above. Mice were perfused osmotically with vehicle alone (DMSO/PEG), Isoproterenol (ISO) alone (0.5mg/kg/hr) or a combination of isoproterenol (0.5mg/kg/hr) and compound 1 at the indicated concentrations for 5 days. On day 6, each mouse was sacrificed and skeletal muscle tissue was isolated and used to analyze calcium channel stabilizing protein 1 binding in RyR1 immunoprecipitates.

The effect of compound 1 on enhancing the binding of calcium channel stabilizing protein 1 to RyR1 in skeletal muscle isolated from isoproterenol-treated mice is depicted in fig. 2A (immunoblot) and 2B (graphical quantification). As shown, compound 1 enhanced the level of calcium channel stabilizing protein 1 binding to RyR1 in skeletal muscle membranes similar to that observed by administration of 3.6mM S36, another benzothiazepine derivative (WO2008/064264), which served as a positive control. Similar results were obtained for compound 4 (data not shown).

Example 5: role of Compound 1 in Long-term post-ischemic Heart failure model in rats

Purpose(s) to

The aim of this study was to test the ability of compound 1 to reduce cardiac dysfunction and attenuate ventricular remodeling in ischemia-reperfusion-induced heart failure models.

Method of producing a composite material

Chronic heart failure was induced in male wistar rats (224-240g, 10-11 weeks old) by ischemia-reperfusion (I/R) injury. For the I/R protocol, the Left Anterior Descending (LAD) coronary artery was closed for 1 h. Drug treatment (5 mg/kg/d or 10mg/kg/d in drinking water) was initiated 1 week after reperfusion and maintained for a study period of 3 months. The potency of compound 1 was assessed according to echocardiography at 1, 2 and 3 months after treatment initiation and according to invasive hemodynamic at3 months compared to vehicle-treated and mock-operated animals. Cardiac samples were also analyzed to assess hypertrophy and collagen content. Blood was collected from each rat at the final study day to evaluate drug plasma concentrations, as shown in figure 3. The study design is shown in figure 3. The experiment was performed in a double blind manner.

Statistical method

For parameters measured over time, comparisons to vehicle and drug treatments were simulated by 2-way ANOVAs with repeated measures analysis. For parameters at sacrifice and morphometry, comparisons to vehicle were simulated by t-test analysis and drug treatment comparisons were analyzed by one-way ANOVA followed by dunnett's test.

Results

Vehicle-treated I/R animals showed increased Left Ventricular (LV) end systole (LVESV) and end diastole (LV EDV) volumes (fig. 4A and B), decreased cardiac function as measured by a decrease in Ejection Fraction (EF) (fig. 4C) and increased interstitial collagen content (fig. 5D) compared to mock-operated animals. From 1 to 3 months, compound 1 administered at 5 and 10mg/kg/D significantly increased EF compared to vehicle, and LVESV and LVEDV decreased (fig. 4A-C), and interstitial collagen content decreased (fig. 5D).

Invasive hemodynamic studies (at 3 months) showed that LV dP/dt max and LV dP/dt min were protected compared to vehicle in animals treated with compound 1 at 5 and 10mg/kg/d (fig. 6B and C), with no statistically significant change in LV systolic blood pressure at the time of treatment (fig. 6A).

No effect on body weight (Bw), infarct size or hypertrophy (LV weight) was observed at the time of treatment (fig. 5A-C). The plasma concentrations of the drugs are shown in figure 7.

The results show that compound 1 at concentrations as low as 5mg/kg/d exerts beneficial effects on systolic and diastolic heart function in the model of heart failure after chronic ischemia in rats.

Compound 1 has more significant and surprising activity than compound a, a structurally related benzothiazepine derivative described in WO 2007/024717. As shown in figure 8, at the end of the study, compound a administered at 5mg/kg/d for 3 months was difficult to improve systolic and diastolic heart function in the chronic post-ischemic heart failure rat model when compared to compound 1. Thus, the beneficial effects of Compound 1, but not Compound A, were observed after 3 months of treatment in the rat CHF model at a dose of 5 mg/kg/d.

Compound A

Example 6: effect of Compound 1 on muscle function in mouse model of muscular dystrophy (mdx)

Purpose(s) to

The aim of this study was to test whether treatment with compound 1 improved muscle function in the dystrophin-deficient mouse model (mdx).

Method of producing a composite material

At the start of the study, 6 weeks and about 20 grams of C57BL/10ScSn-DMD were allowed to stand^mdxMice were mouse-adapted (abbreviated mdx, n ═ 5/group)The wheel was placed in a cage for 6 days, and then the vehicle (H) was randomly assigned₂O) or a target dose of 5mg/kg/d, 10mg/kg/d or 50mg/kg/d administered in ad hoc drinking water (actual doses are: 7.9 mg/kg/d; 12.8 mg/kg/d; and 61.5mg/kg/d, determined as the weekly measured consumption of the drug solution divided by 1 weight, of the sodium salt of compound 1 (based on parent drug weight; sodium salt in the present example hereinafter referred to as "compound 1") in the group treated for 4 weeks. Age-matched C57BL/6 (abbreviated WT, n-4/group) mice were randomly assigned to vehicle (H)₂O) or 50mg/kg/d of the target dose (exact dose: 67.7mg/kg/d) sodium salt of Compound 1.

Voluntary activity, body weight and average water consumption on the wheels were measured during the first 3 weeks. Specific muscle strength was measured at the end of the study 4 weeks after treatment.

Distance traveled over a 24hr period (Km/day) was analyzed as an indicator of improved functional activity (see DMD _ m.2.1.002sop, athttp：//www.treat-nmd.eu/Above). At the end of the study, Extensor Digitorum Longus (EDL) was isolated for muscle strength analysis as described further below. Blood was collected from each mouse by retro-orbital bleeding at the end of the study (after the end of the dark cycle-about 7AM) to evaluate drug plasma concentrations. The experiment was double blind.

Force measurement

At the end of the study, EDL muscles were dissected from hindlimbs for isometric analysis using a 407A muscle testing system from Aurora Scientific (Aurora, Ontario, Canada). Each tendon was tied with 6-0 gauge suture and the EDL muscle between tendons was transferred into O₂/CO₂(95%/5%) foamed Tilode solution (in mM: NaCl 121, KCl 5.0, CaCl₂1.8，MgCl₂，NaH₂PO₄，NaHCO₃24 and glucose 5.5). Using the suture, 1 tendon was tied vertically to a stainless steel hook attached to the force sensor. The other sutured tendon was pressed down into the moving arm of the Aurora system. EDL muscle contraction was stimulated using an electric field between 2 platinum electrodes. In thatAt the beginning of each experiment, muscle length was adjusted to obtain maximum force. Force-frequency dependence was determined by triggering contractions using increasing stimulation frequencies (5-250Hz, 200ms at suprathreshold voltage). Between two stimulations, the muscle was allowed to rest for-3 min. At the end of the force measurement, the length of the EDL muscle (L) simultaneously sutured in the Aurora system was measured₀). The EDL muscle was then removed from the system and weighed after severing the terminal tendon and undoing the suture. EDL muscles were then frozen in liquid nitrogen. By dividing the EDL muscle weight by the EDL muscle length and 1.056mg/m³The cross-sectional area (mm) of the EDL muscle was calculated from the mammalian muscle density constant (Yamada, T., et al, Arthritis and rhematic 60: 3280-3289)²). To determine the specific force (kN/m) of the EDL²) Absolute tonic tension is divided by EDL muscle cross-sectional area.

Statistical method

To determine statistical significance, the student's t-test was used for comparison between the two groups. All collected data are expressed as mean +/-SEM.

Results

Compound 1 was tested for its ability to improve voluntary training in mdx mice. After acclimatization of the mice to the automated wheel cages, the activity of the mice on the automated wheels was monitored with computer 24/7. The collected data was converted into distance of movement per day for 3 weeks. Mdx mice treated with 10 and 50mg/kg/d (target dose) of Compound 1 and vehicle alone (H)₂O) treated mdx mice were significantly longer in distance of movement on the wheel (P < 0.001 from day 1 to day 19). The observed therapeutic effect was observed as early as 2-3 days after the start of treatment and continued throughout the activity monitoring period. No effect of Compound 1 on distance traveled was observed in WT mice treated with 50mg/kg/d of Compound 1 (FIG. 9). Furthermore, the method is simple. Compound 1 treatment increased specific force with muscle mdx dose-dependent as determined from in vitro force in EDL muscle (figure 10). Statistically significant increases in specific muscle strength were shown in mdx mice treated at 150Hz stimulation frequency and above 50mg/kg/d (S) ((S))P is less than 0.05). No effect of compound 1 treatment on specific muscle forces was observed in WT mice.

As shown in figure 11, compound 1 treatment did not affect body weight. No dose-dependent effect on water consumption was observed. Morning blood exposure of compound 1 was (mean ± SEM)3.3 ± 0.4 μ M, 5 mg/kg/d-administered mdx mice; 10.7. + -. 0.9. mu.M, 10 mg/kg/d-administered mdx mice; 52.8. + -. 1.7. mu.M, 50 mg/kg/d-administered mdx mice; and 72.8. + -. 7.0. mu.M, 50 mg/kg/d-administered WT mice. Collectively, these results show that compound 1 treatment at 10mg/kg/d and 50mg/kg/d (target dose) in the mdx mouse Duchenne Muscular Dystrophy (DMD) mouse model improved the voluntary round of training after 3 weeks and improved specific muscle strength after 4 weeks compared to vehicle-treated controls, thereby confirming the use of compound 1 and its analogs claimed herein in treating muscular dystrophy.

Example 7: metabolic stability

Compound 1, a representative Rycol of the invention^TMThe compounds B and C are compared for metabolic stability with the structurally related benzothiazepine derivatives described in WO 2007/024717.

A.Metabolic stability in human liver microsomes

The method comprises the following steps:

solubilization of the compound: stock solutions were made with DMSO and working solutions were made with water containing 1mg/ml BSA.

Prediction of metabolic bioavailability: metabolic bioavailability prediction (MF%) is based on an in vitro metabolic stability assay using liver microsomes with putative total absorption. Briefly, after incubation for 0, 5, 15, 30 and 60min in the presence of NADPH (1mM), incubation with rat and human liver microsomes (0.33mg protein n/ml) (10)^-7M), unchanged drug was quantified by LC-MS-MS. The enzyme reaction was stopped using methanol (v/v) and the protein was precipitated by centrifugation. Expressed as ml/min/g proteinIntrinsic clearance in vitro (Clint _ mic) is the slope of the unchanged drug residual concentration versus incubation time (after LN linearization). Clint in vitro was then amplified to the whole body in vivo (Clint in vivo) using 0.045mg prot/kg liver and 11g of rat and 1.2kg liver weight of human, then in vivo Clint was converted to liver clearance (HepCl) using a well agitated model (HepCl ═ in vivo Clint ＊ HBF/(in vivo Clint + HBF), where HBF (hepatic blood flow) was taken to be 22ml/min rat and 1500ml/min human, then MF%,% was estimated from the extraction ratio using the equation (MF% ═ 1-HepCl/HBF) — the results are shown in table 1:

table 1: stability in human microsomes

Clint _ mic: intrinsic clearance in vitro in ml/min/g protein

MF%: metabolic bioavailability in%

B.Metabolic stability in rat and human hepatocytes

Solubilization of the compound: stock solutions were made with DMSO and working solutions were made with William medium containing 1/10 rat plasma or 1/4 human plasma.

And (3) metabolic stability determination: mixing the compound 10^-7M was incubated with isolated hepatocytes (6E +5 cells/ml, rat hepatocytes; and 4E +5 cells/ml, human hepatocytes) at 37 ℃ in plasma from the same species diluted with Wiliams medium (1/10, rat; and 1/4, human). At sampling times of 0, 10, 20, 30, 60 and 120min, and the enzyme reaction was stopped with methanol (v/v). Proteins were precipitated by centrifugation and supernatants were analyzed by LC/MS/MS. Clint expressed as ml/min/g protein was calculated using the ratio of 0.134mg protein/ml for human 4E +5 cells/ml to 0.201mg protein/ml for rat 6E +5 cells/ml versus liver microsomes. The presence of the reference drug and potential metabolites in each sample was detected by LC/MS/MS in this assay. KnotIf as shown in table 2:

table 2: stability in rat and human hepatocytes

Clint _ mic: intrinsic clearance in vitro in ml/min/g protein

MF%: metabolic bioavailability in%

c.Q: number of cells/ml

C.Metabolic stability in mouse and rat microsomes

Materials and methods

Dilution buffer: 0.1M Tris HCl buffer in pH 7.4 containing 5mM EDTA.

NADPH cofactor solution: to a 50mL falcon tube containing 2.79mL dilution buffer were added 0.429mL of the ADPH-regenerating solution A and 0.079mL of the NADPH-regenerating solution B.

Preparation of microsomes: (1.5mg/mL solution) 50mL falcon tubes containing 3.32mL dilution buffer were warmed at 37 ℃ for 15min (at least 10 min.). 0.178mL of microsomes (24.6mg/mL) was added to the pre-warmed dilution buffer. The protein concentration of the microsomal preparation was 1.25 mg/mL.

Sample (test compound) -original and intermediate stock solutions: a1 mg/mL solution of the test compound in methanol (0.5mg/mL for Compound 1) was prepared. An intermediate solution of 100 μ M of the test compound was prepared from the original stock solution using dilution buffer. A5. mu.M solution was prepared by diluting 100. mu.M of the intermediate solution with dilution buffer.

Experiment:

(this experiment was performed in a 1.5mL eppendorf microcentrifuge tube)

Incubate for 0min.The method comprises the following steps:

a. 100 μ L of pre-warmed microsomes were added.

b. 50 μ L of 5 μ M test compound solution was added.

c. Add 500. mu.L of cold stop solution (ice-cold methanol).

d. Add 100. mu.L of NADPH cofactor solution to eppendorf.

a. Eppendorf was mixed by vortexing.

"t" minute incubation

b. Add 100. mu.L of NADPH cofactor solution to eppendorf.

c. 50 μ L of 5 μ M test compound solution was added.

d. 100 μ L of pre-warmed microsomes were added.

e. Incubate eppendorf't' min on a hot mixer at 37 ℃ 300 rpm.

f. The eppendorf was removed from the hot mixer.

g. Add 500. mu.L of cold stop solution (ice-cold methanol).

h. Eppendorf was mixed by vortexing.

Incubate samples at 4 ℃ for '0' and't' minutes and centrifuge at 15,000rcf for 15min. 500 μ L of the supernatant was removed and subjected to LC/MS analysis (SIM-Selective ion monitoring)

Results are expressed as% remaining test compound ═ 100 (` MS area response for the't' min sample/` MS area response for the '0' min sample). The MS area was used as the average of the repeated injections.

Time points were 0, 15, 30 and 60min for each test compound.

Positive control:

mu.M imipramine-5 min. and 2. mu.M imipramine-15 min. incubations were used as positive controls for rat and mouse liver microsome stability experiments.

The results are shown in table 3:

table 3: stability in mouse and rat microsomes

Surprisingly, as observed in tables 1-3, compound 1 was significantly stable in mouse, rat and human microsomes and rat and human hepatocytes compared to the structural analogs compounds B and C disclosed in WO2007/024717, both of which have been found to have poor in vitro metabolic stability in the test system, making these compounds unsuitable for development as drug candidates. Surprisingly and unexpectedly, replacement of the H or OH moiety in the prior art compounds with the COOH moiety resulted in compound 1, which exhibited a high degree of metabolic stability in all tested systems. The increased metabolic stability of compound 1 compared to its structural analogs is indeed surprising and demonstrates the surprising benefit of this compound over the compounds known in the art.

All publications, references, patents, and patent applications cited herein are incorporated by reference in their entirety to the same extent as if each individual application, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The foregoing description of the specific embodiments will thus fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for use such specific embodiments without undue experimentation and without departing from the general concept, and, therefore, such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of example and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take various alternative forms without departing from the invention.

Claims (33)

1. A compound represented by structural formula (I):

wherein

R is COOH;

and pharmaceutically acceptable salts thereof.

2. The compound represented by the structural formula (I) according to claim 1, wherein the salt is in the form of a salt with a pharmaceutically acceptable acid or base, and pharmaceutically acceptable salts thereof.

3. A compound represented by structural formula (I) according to claim 2, wherein the salt is selected from the group consisting of sodium salt, potassium salt, magnesium salt, hemifumarate salt, hydrochloride salt and hydrobromide salt, and pharmaceutically acceptable salts thereof.

4. A compound represented by structural formula (I) according to claim 3, wherein the salt is a sodium salt or a hemifumarate salt, and pharmaceutically acceptable salts thereof.

5. A compound according to claim 1, selected from:

6. the compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein said compound is represented by structural formula (1):

7. the compound represented by the structural formula (I) according to claim 6, wherein the salt is in the form of a salt with a pharmaceutically acceptable acid or base, and pharmaceutically acceptable salts thereof.

8. The compound represented by structural formula (I) according to claim 7, wherein the salt is selected from the group consisting of sodium salt, potassium salt, magnesium salt, hemifumarate salt, hydrochloride salt and hydrobromide salt, and pharmaceutically acceptable salts thereof.

9. The compound represented by structural formula (I) according to claim 8, wherein the salt is a sodium salt, and pharmaceutically acceptable salts thereof.

10. A compound represented by structural formula (I) and pharmaceutically acceptable salts thereof according to claim 8, wherein the salt is a hemifumarate salt.

11. Use of a compound or salt according to any one of claims 1-10 for the manufacture of a medicament for the treatment or prevention of a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular disorders and diseases, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death and sudden infant death syndrome, or for improving cognitive function.

12. A pharmaceutical composition comprising a compound according to any one of claims 1-10 and one or more pharmaceutically acceptable excipients or carriers.

13. Use of a pharmaceutical composition according to claim 12 for the preparation of a medicament for the treatment or prevention of a condition selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, CNS disorders and diseases, cognitive dysfunction, neuromuscular disorders and diseases, bone disorders and diseases, cancer cachexia, malignant hyperthermia, diabetes, sudden cardiac death and sudden infant death syndrome, or for improving cognitive function.

14. The use according to claim 13, wherein the disorder is associated with dysfunction of ryanodine receptor 1(RyR1), ryanodine receptor type (RyR2), ryanodine receptor type 3 (RyR3), or a combination thereof.

15. Use according to claim 13, wherein the cardiac disorders and diseases are selected from irregular heart beat disorders and diseases; heart failure; myocardial ischemia/reperfusion (I/R) injury; chronic obstructive pulmonary disease; post-thrombolytic (MI) for post-coronary angioplasty I/R injury or treatment of myocardial infarction; and hypertension.

16. Use according to claim 15, wherein the irregular heartbeat disorders and diseases are exercise-induced irregular heartbeat disorders and diseases.

17. The use according to claim 15, wherein the heart failure is selected from the group consisting of congestive heart failure; chronic heart failure; acute heart failure; systolic heart failure; diastolic heart failure; acute compensatory heart failure.

18. Use according to claim 15, wherein the irregular heartbeat disorders and diseases are selected from the group consisting of atrial and ventricular arrhythmias, atrial and ventricular fibrillation, catecholamine dependent ventricular tachycardia (CPVT) and exercise induced irregular heartbeat disorders and diseases of their altered forms.

19. Use according to claim 18, wherein the atrial and ventricular arrhythmias are selected from the group consisting of atrial and ventricular tachyarrhythmias and atrial and ventricular tachycardias.

20. Use according to claim 13, wherein the muscle fatigue is due to a skeletal muscle disease, disorder or condition.

21. Use according to claim 13, wherein the musculoskeletal disorders and diseases are selected from exercise-induced skeletal muscle fatigue; exercise-induced muscle fatigue due to prolonged exercise or high intensity exercise; congenital myopathy; muscular dystrophy; spinal Muscular Atrophy (SMA), Spinal and Bulbar Muscular Atrophy (SBMA), age-related muscle fatigue; sarcopenia, central nuclear disease; cancer cachexia; bladder disorders; and incontinence.

22. Use according to claim 21, wherein the muscular dystrophy is selected from Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD), Limbal Girdle Muscular Dystrophy (LGMD), facioscapulohumeral dystrophy, myotonic dystrophy, Congenital Muscular Dystrophy (CMD), distal muscular dystrophy, idenedial muscular dystrophy and oculopharyngeal muscular dystrophy.

23. The use according to claim 13, wherein the CNS disorders and diseases are selected from Alzheimer's Disease (AD), neuropathy, epilepsy, Parkinson's Disease (PD) and Huntington's Disease (HD); and the neuromuscular disorders and diseases are selected from the group consisting of spinocerebellar ataxia (SCA) and amyotrophic lateral sclerosis (ALS, Lou Gehrig's disease).

24. The use according to claim 13, wherein the cognitive dysfunction is associated with stress or with age, or wherein the improved cognitive function is short term memory, long term memory, attention or learning, or wherein the cognitive dysfunction is associated with a disease or disorder selected from the group consisting of Alzheimer's Disease (AD), Attention Deficit Hyperactivity Disorder (ADHD), Autism Spectrum Disorder (ASD), Generalized Anxiety Disorder (GAD), Obsessive Compulsive Disorder (OCD), Parkinson's Disease (PD), Post Traumatic Stress Disorder (PTSD), schizophrenia, bipolar disorder and major depression.

25. Use according to claim 13, wherein the disorder is cancer cachexia.

26. Use according to claim 25, wherein the cancer cachexia is due to cancer with bone metastasis.

27. The use according to claim 13, wherein the compound is used in a dose sufficient to restore or enhance calcium channel stabilizing protein 2 binding to RyR 2.

28. The use according to claim 13, wherein the compound is used in a dose sufficient to restore or enhance calcium channel stabilizing protein 1 binding to RyR 1.

29. Use according to claim 13, wherein Ca is reduced sufficiently to reduce Ca²⁺The compound is used at doses that leak through the RyR channel.

30. Use according to claim 13, further comprising the application of an Antisense Oligonucleotide (AO) specific for a spliced sequence in an mRNA of interest for enhancing exon skipping in said mRNA of interest.

31. Use according to claim 30, for the treatment of Duchenne Muscular Dystrophy (DMD), wherein said Antisense Oligonucleotide (AO) is specific for a splicing sequence of at least one exon of the DMD gene.

32. Use according to claim 31, wherein said Antisense Oligonucleotide (AO) is specific for a spliced sequence of exons 23, 45, 44, 50, 51, 52 and/or 53 of the DMD gene.

33. A process for the preparation of a compound according to any one of claims 1 to 10, comprising the steps of: reacting a compound of the formula

With a compound of the formula,

wherein R is^aIs COOR¹Or CN; r¹Is C₁-C₄Alkyl, and L is a leaving group, to give a compound of the formula:

and

a radical R^aConversion to the group R gives the compound of formula (I).