# Patent

## Title
Agents for preventing and treating disorders involving modulation of the RyR receptors

## Abstract

The present invention provides novel compounds of Formula I




and salts, hydrates, solvates, complexes, and prodrugs thereof. The present invention further provides methods for synthesizing compounds of Formula I. The invention additionally provides pharmaceutical compositions comprising the compounds of Formula I and methods of using the pharmaceutical compositions of Formula I to treat and prevent disorders and diseases associated with the RyR receptors that regulate calcium channel functioning in cells. Such disorders and diseases include, by way of example only, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof. Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence. Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss.

## Background
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under NIH Grant No. PO1 HL 67849-01. As such, the United States government may have certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to novel compounds and their use to treat and prevent disorders and diseases associated with the RyR receptors that regulate calcium channel functioning in cells. More particularly, the invention discloses compounds that are related to 1,4-benzothiazepines and are useful to treat cardiac and skeletal muscular disorders. The invention also discloses pharmaceutical compositions comprising the compounds and articles of manufacture comprising the pharmaceutical compositions.
BACKGROUND OF THE INVENTION
The sarcoplasmic reticulum (SR) is a structure in cells that functions, among other things, as a specialized intracellular calcium (Ca2+) store. Channels in the SR called ryanodine receptors (RyRs) open and close to regulate the release of Ca2+ from the SR into the intracellular cytoplasm of the cell. Release of Ca2+ into the cytoplasm from the SR increases cytoplasmic Ca2+ concentration. Open probability (Po) of the RyR receptor refers to the likelihood that the RyR channel is open at any given moment, and therefore capable of releasing Ca2+ into the cytoplasm from the SR.
There are three types of ryanodine receptors, all of which are highly-related Ca2+ channels: RyR1, RyR2, and RyR3. RyR1 is found predominantly in skeletal muscle as well as other tissues, RyR2 is found predominantly in the heart as well as other tissues, and RyR3 is found in the brain as well as other tissues. The RyR channels are formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs), specifically FKBP12 (calstabin1) and FKBP12.6 (calstabin2). Calstabin1 binds to RyR1, calstabin2 binds to RyR2, and calstabin1 binds to RyR3. The FKBP proteins (calstabin1 and calstabin2) bind to the RyR channel (one molecule per RyR subunit), stabilize RyR-channel functioning, and facilitate coupled gating between neighboring RyR channels, thereby preventing abnormal activation of the channel during the channel's closed state.
Besides the calstabin binding proteins, protein kinase A (PKA) also binds to the cytoplasmic surface of the RyR receptors. PKA phosphorylation of the RyR receptors causes partial dissociation of calstabins from RyRs. Dissociation of calstabin from RyR causes increased open probability of RyR, and therefore increased Ca2+ release from the SR into the intracellular cytoplasm.
Ca2+ release from the SR in skeletal muscle cells and heart cells is a key physiological mechanism that controls muscle performance, because increased concentration of Ca2+ in the intracellular cytoplasm causes contraction of the muscle.
Excitation-contraction (EC) coupling in skeletal muscles involves electrical depolarization of the plasma membrane in the transverse tubule (T-tubule), which activates voltage-gated L-type Ca2+ channels (LTCCs). LTCCs trigger Ca2+ release from the SR through physical interaction with RyR1. The resulting increase in cytoplasmic Ca2+ concentration induces actin-myosin interaction and muscle contraction. To enable relaxation, intracellular Ca2+ is pumped back into the SR via SR Ca2+-ATPase pumps (SERCAs), which is regulated by phospholamban (PLB) depending on the muscle fiber type.
It has been shown that disease forms that result in sustained activation of the sympathetic nervous system and increased plasma catecholamine levels cause maladaptive activation of intracellular stress pathways resulting in destabilization of the RyR1 channel closed state and intracellular Ca2+ leak. SR Ca2+ leak via RyR1 channels was found to deplete intracellular SR calcium stores, to increase compensatory energy consumption, and to result in significant acceleration of muscle fatigue. The stress-induced muscle defect permanently reduces isolated muscle and in vivo performance particularly in situations of increased demand.
It also has been shown that destabilization of the RyR1 closed state occurs under pathologic conditions of increased sympathetic activation and involves depletion of the stabilizing calstabin1 (FKBP12) channel subunit. Proof-of-principle experiments have shown that PKA activation as an end effector of the sympathetic nervous systems increases RyR1 PKA phosphorylation at Ser-2843 which decreases the binding affinity of calstabin 1 to RyR1 and increases channel open probability.
In cardiac striated muscle, RyR2 is the major Ca2+-release channel required for EC coupling and muscle contraction. During EC coupling, depolarization of the cardiac-muscle cell membrane during phase zero of the action potential activates voltage-gated Ca2+ channels. Ca2+ influx through the open voltage-gated channels in turn initiates Ca2+ release from the SR via RyR2. This process is known as Ca2+-induced Ca2+ release. The RyR2-mediated, Ca2+-induced Ca2+ release then activates the contractile proteins in the cardiac cell, resulting in cardiac muscle contraction.
Phosphorylation of cardiac RyR2 by PKA is an important part of the “fight or flight” response that increases cardiac EC coupling gain by augmenting the amount of Ca2+ released for a given trigger. This signaling pathway provides a mechanism by which activation of the sympathetic nervous system, in response to stress, results in increased cardiac output. PKA phosphorylation of RyR2 increases the open probability of the channel by dissociating calstabin2 (FKBP12.6) from the channel complex. This, in turn, increases the sensitivity of RyR2 to Ca2+-dependent activation.
Despite advances in treatment, heart failure remains an important cause of mortality in Western countries. An important hallmark of heart failure is reduced myocardial contractility. In heart failure, contractile abnormalities result, in part, from alterations in the signaling pathway that allows the cardiac action potential to trigger Ca2+ release via RyR2 channels and muscle contraction. In particular, in failing hearts, the amplitude of the whole-cell Ca2+ transient is decreased and the duration prolonged.
Cardiac arrhythmia, a common feature of heart failure, results in many of the deaths associated with the disease. Atrial fibrillation (AF) is the most common cardiac arrhythmia in humans, and represents a major cause of morbidity and mortality. Structural and electrical remodeling—including shortening of atrial refractoriness, loss of rate-related adaptation of refractoriness, and shortening of the wavelength of re-entrant wavelets—accompany sustained tachycardia. This remodeling is likely important in the development, maintenance and progression of atrial fibrillation. Studies suggest that calcium handling plays a role in electrical remodeling in atrial fibrillation.
Approximately 50% of all patients with heart disease die from fatal cardiac arrhythmias. In some cases, a ventricular arrhythmia in the heart is rapidly fatal—a phenomenon referred to as “sudden cardiac death” (SCD). Fatal ventricular arrhythmias and SCD also occur in young, otherwise-healthy individuals who are not known to have structural heart disease. In fact, ventricular arrhythmia is the most common cause of sudden death in otherwise-healthy individuals.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited disorder in individuals with structurally normal hearts. It is characterized by stress-induced ventricular tachycardia—a lethal arrhythmia that causes SCD. In subjects with CPVT, physical exertion and/or stress induce bidirectional and/or polymorphic ventricular tachycardias that lead to SCD even in the absence of detectable structural heart disease. CPVT is predominantly inherited in an autosomal-dominant fashion. Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest. Studies have identified mutations in the human RyR2 gene, on chromosome 1q42-q43, in individuals with CPVT.
Failing hearts (e.g., in patients with heart failure and in animal models of heart failure) are characterized by a maladaptive response that includes chronic hyperadrenergic stimulation. In heart failure, chronic beta-adrenergic stimulation is associated with the activation of beta-adrenergic receptors in the heart, which, through coupling with G-proteins, activate adenylyl cyclase and thereby increase intracellular cAMP concentration. cAMP activates cAMP-dependent PKA, which has been shown to induce hyperphosphorylation of RyR2. Thus, chronic heart failure is a chronic hyperadrenergic state which results in several pathologic consequences, including PKA hyperphosphorylation of RyR2.
The PKA hyperphosphorylation of RyR2 has been proposed as a factor contributing to depressed contractile function and arrhythmogenesis in heart failure. Consistent with this hypothesis, PKA hyperphosphorylation of RyR2 in failing hearts has been demonstrated, in vivo, both in animal models and in patients with heart failure undergoing cardiac transplantation.
In failing hearts, the hyperphosphorylation of RyR2 by PKA induces the dissociation of FKBP12.6 (calstabin2) from the RyR2 channel. This causes marked changes in the biophysical properties of the RyR2 channel, including increased open probability (Po) due to an increased sensitivity to Ca2+-dependent activation; destabilization of the channel, resulting in subconductance states; and impaired coupled gating of the channels, resulting in defective EC coupling and cardiac dysfunction. Thus, PKA-hyperphosphorylated RyR2 is very sensitive to low-level Ca2+ stimulation, and this manifests itself as a diastolic SR Ca2+ leak through the PKA hyperphosphorylated RyR2 channel.
The maladaptive response to stress in heart failure results in depletion of FKBP12.6 from the channel macromolecular complex. This leads to a shift to the left in the sensitivity of RyR2 to Ca2+-induced Ca2+ release, resulting in channels that are more active at low-to-moderate Ca2+ concentrations. Over time, the increased “leak” through RyR results in resetting of the SR Ca2+ content to a lower level, which in turn reduces EC coupling gain and contributes to impaired systolic contractility.
Additionally, a subpopulation of RyR2 that are particularly “leaky” can release SR Ca2+ during the resting phase of the cardiac cycle, diastole. This results in depolarizations of the cardiomyocyte membrane known as delayed after-depolarizations (DADs), which are known to trigger fatal ventricular cardiac arrhythmias.
In patients with CPVT mutations in their RyR2 and otherwise structurally-normal hearts, a similar phenomenon is at work. Specifically, it is known that exercise and stress induce the release of catecholamines that activate beta-adrenergic receptors in the heart. Activation of the beta-adrenergic receptors leads to PKA hyperphosphorylation of RyR2 channels. Evidence also suggests that the PKA hyperphosphorylation of RyR2 resulting from beta-adrenergic-receptor activation renders mutated RyR2 channels more likely to open in the relaxation phase of the cardiac cycle, increasing the likelihood of arrhythmias.
Cardiac arrhythmias are known to be associated with diastolic SR Ca2+ leaks in patients with CPVT mutations in their RyR2 and otherwise structurally-normal hearts. In these cases, the most common mechanism for induction and maintenance of ventricular tachycardia is abnormal automaticity. One form of abnormal automaticity, known as triggered arrhythmia, is associated with aberrant release of SR Ca2+, which initiates DADs. DADs are abnormal depolarizations in cardiomyocytes that occur after repolarization of a cardiac action potential. The molecular basis for the abnormal SR Ca2+ release that results in DADs has not been fully elucidated. However, DADs are known to be blocked by ryanodine, providing evidence that RyR2 plays a key role in the pathogenesis of this aberrant Ca2+ release.
U.S. Pat. No. 6,489,125 discusses JTV-519 (4-[3-(4-benzylpiperidin-1-yl)propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine monohydrochloride; also known as k201 or ICP-Calstan 100), a 1,4-benzothiazepine, as a new modulator of RyR calcium-ion channels.
Co-pending application U.S. Ser. No. 10/763,498 discusses RyR2 as a target for treating and preventing heart failure and cardiac arrhythmias, including atrial fibrillation and cardiac arrhythmias that cause exercise-induced sudden cardiac death (SCD). RyR2 channels with 7 different CPVT mutations (e.g., S2246L, R2474S, N4104K, R4497C, P2328S, Q4201R, V4653F) were found to have functional defects that resulted in channels that became leaky (i.e., a calcium leak) when stimulated during exercise. The mechanism for the VT in CPVT has been demonstrated to be the same as the mechanism for VT in heart failure.
It has been shown that exercise-induced arrhythmias and sudden death (in patients with CPVT) result from a reduced affinity of FKBP12.6 (calstabin2) for RyR2. Additionally, it has been demonstrated that exercise activates RyR2 as a result of phosphorylation by adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA). Mutant RyR2 channels, which had normal function in planar lipid bilayers under basal conditions, were more sensitive to activation by PKA phosphorylation—exhibiting increased activity (open probability) and prolonged open states, as compared with wild-type channels. In addition, PKA-phosphorylated mutant RyR2 channels were resistant to inhibition by Mg2+, a physiological inhibitor of the channel, and showed reduced binding to FKBP12.6 (aka calstabin2, which stabilizes the channel in the closed state). These findings indicate that, during exercise, when the RyR2 are PKA-phosphorylated, the mutant CPVT channels are more likely to open in the relaxation phase of the cardiac cycle (diastole), increasing the likelihood of arrhythmias triggered by SR Ca2+ leak.
Additionally, co-pending U.S. patent application Ser. No. 09/288,606 discusses a method for regulating contraction of a subject's heart by administering a compound which regulates PKA phosphorylation of an RyR2 receptor and specifically decreases PKA phosphorylation. Co-pending U.S. patent application Ser. No. 10/608,723 also discusses a method for treating and prophylaxis for atrial tachyarrhythmia and exercise and stress-induced arrhythmias by administration of an agent which inhibits PKA phosphorylation of RyR2.
SUMMARY OF THE INVENTION
In view of the foregoing, there is a need to identify new agents effective for treating or preventing disorders and diseases associated with the RyR receptors that regulate calcium channel functioning in cells, including skeletal muscular disorders and diseases and especially cardiac disorders and diseases. More particularly, a need remains to identify new compounds that can be used to treat RyR associated disorders by, for example, repairing the leak in RyR channels, and enhancing binding of FKBP proteins (calstabin1 and calstabin2) to PKA-phosphorylated RyR, and to mutant RyR that otherwise have reduced affinity for, or do not bind to, FKBP12 and FKBP12.6. Embodiments of the invention solve some or all of these needs.
Accordingly, the present invention generally provides compounds that may be classified as 1,4-benzothiazepines and sometimes are referred to herein as “RyCals.”
The present invention further provides compounds of Formula I:




wherein,

n is 0, 1, or 2;

R is located at one or more positions on the benzene ring;

each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R1 is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R2 is selected from the group consisting of —C═O(R5), —C═S(R6), —SO2R7, —POR8R9, —(CH2)m-R10, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R3 is selected from the group consisting of H, CO2Y, CONY, acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl; and wherein Y is selected from the group consisting of H, alkyl, aryl, cycloalkyl, and heterocyclyl;

R4 is selected from the group consisting of H, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R5 is selected from the group consisting of—NR16, NHNHR16, NHOH, —OR15, CONH2NHR16, CO2R15, CONR16, CH2X, acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R6 is selected from the group consisting of —OR15, NHNR16, NHOH, —NR16, CH2X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R7 is selected from the group consisting of —OR15, —NR16, NHNHR16, NHOH, CH2X, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R8 and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R10 is selected from the group consisting of NH2, OH, —SO2R11, —NHSO2R11, C═O(R12), NHC═O(R12), —OC═O(R12), and —POR13R14;

R11, R12, R13, and R14 independently are selected from the group consisting of H, OH, NH2, NHNH2, NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, —N—, —O—, —S—, —CN, —N3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

X is selected from the group consisting of halogen, CN, CO2R15, CONR16, —NR16, —OR15, —SO2R7, and —POR8R9; and

R15 and R16 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, —N—, —O—, —S—, —CN, —N3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

and salts, hydrates, solvates, complexes, and prodrugs thereof. In one embodiment, the compound is selected from the group consisting of S1, S2, S3, S4, S5, S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, and S100.

The present invention also provides methods for the synthesis of compounds of Formula I:




wherein R, R1, R2, R3, R4, and n are defined as above.

The present invention further provides a method of treating or preventing various disorders and diseases in a subject that are associated with RyR receptors, such as muscular and cardiac disorders, comprising administering to the subject an amount of a compound effective to prevent or treat a disorder or disease associated with the RyR receptors, wherein the compound is of Formula I:




wherein R, R1, R2, R3, R4, and n are defined as above; and salts, hydrates, solvates, complexes and pro-drugs thereof.

Also provided is a method of preventing or treating a leak in a RyR2 receptor in a subject, including administering to the subject an amount of a compound effective to prevent or treat a leak in the RyR2 receptor, wherein the compound is of Formula I:




wherein R, R1, R2, R3, R4, and n are as defined above; and salts, hydrates, solvates, complexes and pro-drugs thereof. The subject is, for example, an in vitro system (e.g., cultured cells or tissues) or in vivo system (e.g., animal or human).

In addition, the present invention provides a method of modulating the binding of RyR and FKBP in a subject, including administering to the subject an amount of a compound effective to modulate the level of RyR-bound FKBP, wherein the compound is of Formula I:




wherein R, R1, R2, R3, R4, and n are as defined above; and salts, hydrates, solvates, complexes and pro-drugs thereof. The subject is, for example, an in vitro system (e.g., cultured cells or tissues) or in vivo system (e.g., animal or human).

The present invention also provides articles of manufacture for treating and preventing disorders and diseases associated with the RyR receptors, such as muscular and cardiac disorders, in a subject. The articles of manufacture comprise a pharmaceutical composition of one or more of the compounds of Formula I. The articles of manufacture are packaged with indications for various disorders that the pharmaceutical compositions are capable of treating and/or preventing.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the 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.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1, embodiments A, B, C, and D are, respectively, (A) immunoblots of PKA phosphorylated RyR2 in the presence of FKBP12.6 and increasing JTV-519 concentrations; (B) immunoblots of PKA phosphorylated RyR2 in the presence of 0.5 nM S36; (C) a graph of current through plasma membrane, voltage dependent L-type Ca2+ channels which are completely blocked by nifedipine but not by S36 in isolated mouse cardiomyocytes; and (D) a graph of the voltage-dependence of L-type Ca2+ current in channels in the presence of JTV-519 and S36.
FIG. 2, embodiments A, B, C, and D demonstrate the prevention of exercise-induced ventricular arrhythmias by JTV-519 in haploinsufficient calstabin (FKBP12.6)+/− mice. Embodiment A are representative telemetric electrocardiograms (ECGs) of an untreated calstabin2 (FKBP12.6)+/− mouse (left), a JTV-519-treated calstabin2 (FKBP12.6)+/− mouse (middle), and a calstabin2 (FKBP12.6)−/− mouse (right). Embodiment B are telemetry recordings of a sustained polymorphic ventricular tachycardia (sVT) in (upper) an untreated haploinsufficient calstabin2 (FKBP12.6)+/− mouse and (lower) a JTV-519-treated calstabin2 (FKBP12.6)+/− mouse, each subjected to exercise testing immediately followed by injection with 0.5 mg epinephrine per kilogram of body weight. Embodiment C are graphs showing the numbers of mice with cardiac death (left), sustained VTs (middle), and nonsustained VTs (right) in experimental groups of mice subjected to exercise testing and injection with 0.5 mg/kg epinephrine. Embodiment D are graphs comparing the dose dependence of pharmacological effects of JTV-519 and S36 in regard to sudden cardiac death (left), sustained VTs (middle), and nonsustained VTs (right).
FIG. 3 is a graph showing fractional shortening (FS) of the left ventricle assessed by M-mode echocardiography 2 weeks post-myocardial infarction in placebo versus S36-treated mice. S36 treated mice show a significant improvement in FS in the 100 nM and 200 nM groups as compared to placebo.
FIG. 4 is a graph showing heart weight to body weight (HW/BW) ratios and pressure-volume loops quantifications (dP/dt) one week post-myocardial infarction of placebo and S36-treated mice. S36 treatment results in a beneficial reduction of the HW/BW ratio and increased velocity of pressure development in S36 as compared to placebo treated mice.
FIG. 5 is a graph summarizing EC50 values of JTV-519 and a series of Rycal compounds indicating several compounds with a higher biologic activity as evidenced by significantly lower EC50 values compared to JTV-519.
FIG. 6, embodiments A, B, and C are, respectively, (A) single-channel current traces of unphosphorylated RyR2-P2328S and unphosphorylated RyR2-WT treated with S36; (B) single-channel current traces of phosphorylated RyR2-P2328S and unphosphorylated RyR2-P2328S treated with S36; and (C) immunoblot analysis of calstabin-2 binding of RyR2-P2328S in the presence or absence of PKA and S36.
FIG. 7, embodiments A and B, are, respectively, (A) an immunoblot of RyR2 immunoprecipitated with an antibody against RyR2, and immunoblots of RyR2 PKA phosphorylation at Ser-2809 and calstabin2; and (B) a bar graph quantifying the relative amount of PKA phosphorylated RyR2 at Ser-2808 (corresponding to human Ser-2809) bound to RyR2 in wild-type (contol) and calstabin2-deficient (FKBP12.6−/−) mice.
FIG. 8, embodiments A, B, and C, are, respectively, bar graphs of (A) quantitative in vivo M-mode echocardiograms comparing ejection fraction (EF) before and following sham operation or permanent left anterior descending (LAD) coronary artery ligation in wild-type and RyR2-S2808A knockin mice; (B) in vivo pressure-volume loop quantification of maximal pressure change over time (dP/dt) in wild-type and RyR2-S2808A knockin mice after sham operation or permanent left anterior descending coronary artery (LAD) ligation; and (C) quantitative M-mode echocardiographic assessment of end-systolic diameter (ESD) in wildtype and RyR2-S2808A knockin mice after sham operation or permanent left anterior descending coronary artery (LAD) ligation.
FIG. 9, embodiments A, B, C, and D demonstrate the effect of JTV-519 on calstabin2 affinity to RyR2 in haploinsufficient calstabin2 (FKBP12.6)+/− mice after exercise. Embodiment A are immunoblots of equivalent amounts of RyR2 immunoprecipitated with an antibody against RyR2 (top). Embodiment B are bar graphs showing the amount of PKA phosphorylation of RyR2 at Ser-2809 and the amount of calstabin2 bound to RyR2 from control animals and animals after exercise immediately followed by injection with 0.5 mg/kg epinephrine. Embodiment C are single-channel tracings of RyR2 channels isolated from hearts of haploinsufficient calstabin2+/− and calstabin2−/− deficient mice immediately following exercise testing and injection of 0.5 mg epinephrine per kg of body weight, both untreated (top) and after treatment (middle and bottom) with JTV-519. Average open probality (Po), open time (To) and average closed time (Tc) are as indicated; and closed state is indicated by ‘c’. The dotted lines indicate subconductance levels for partial RyR2 openings. Embodiment D is a bar graph summarizing average open probabilities of single RyR2 channels from haploinsufficient calstabin2± and calstabin2−/− deficient mice after exercise with and without JTV-519 treatment. “*” indicates significance level P<0.05. Numbers in the bars indicate the number of single channels measured.
FIG. 10, embodiments A, B, C, D, E, and F demonstrate normalized RyR2 channel gating and increased calstabin2 binding to RyR2 channels after treatment with JTV-519. Embodiment A are immunoblots of immunoprecipitated wild-type RyR2 (RyR2-WT) channels phosphorylated by PKA in the absence or presence of the inhibitor peptide PKI5-24 and incubated with calstabin2 (250 nM) at the indicated JTV-519 concentrations; the immunoblots show the amount of RyR2 (top) and the amount of calstabin2 (bottom) associated with immunoprecipitated RyR2 after incubation with or without the indicated concentrations of JTV-519. Embodiment B are immunoblots of RyR2-S2809D, which mimics constitutive PKA phosphorylation of RyR2, analyzed as in Embodiment A. Embodiment C are binding curves of 35S-radiolabeled calstabin2 to unphosphorylated or PKA phosphorylated RyR2 or to RyR2-S2809D in the presence or absence of JTV-519, documenting differences in the calstabin2 binding affinity for RyR2. Embodiments D, E, and F are single channel tracings (left) and amplitude histograms (right) of PKA-phosphorylated RyR2s (embodiments E and F) or unphosphorylated (embodiment D in presence of PKA inhibitor PKI4) incubated with calstabin2 (250 nM) with (embodiment F) or without (embodiments D and E) JTV-519 (1 μM). Single-channel tracings are shown at 150 nM [Ca2+] which mimics the diastolic or resting phase in the heart; channel openings are upward, the dash indicates the full level of channel opening (4 pA), and “c” indicates the closed state of the channels. The amplitude histograms have amplitude represented on the x axis, and events on the y axis indicates the number of channel openings.
FIG. 11, embodiments A, B, C, D, E, and F demonstrate that RyR1 channel function is increased and normalized in mdx mice treated with JTV-519. Embodiments A and B are, respectively, a single channel current trace and an amplitude histogram of RyR1 from soleus muscle of a control (wild-type) mouse under resting conditions. Embodiments C and D are, respectively, a single channel current trace and an amplitude histogram of RyR1 from soleus muscle of an mdx mouse. Embodiment E and F are, respectively, a single channel current trace and an amplitude histogram of RyR1 from soleus muscle of an mdx mouse treated with JTV-519.
FIG. 12, embodiments A and B, are, respectively, immunoblots of RyR1, RyR1-pSer2843, and RyR1-associated calstabin1 in mdx mice and wild-type mice; and bar graphs of the relative amounts of RyR1-pSer2843 and calstabin1 in mdx and wild-type mice.
FIG. 13, embodiments A, B, and C, demonstrate that a SR Ca2+ leak is detectable in the skeletal muscles of animals with heart failure. Embodiments A and B are fluorescence line scan images of Ca2+ sparks in myofibers from, respectively, sham and postmyocardial infarction (PMI) rats. Embodiment C are a bar graphs summarizing the amplitude, rise time, FDHM, and FWHM of the Ca2+ sparks for the sham (open symbols) and PMI (closed symbols) rats.
FIG. 14, embodiments A and B, demonstrate that treatment of wild-type mice with JTV-519 improves soleus muscle fatigue times. Embodiment A are maximal tetanic force fatigue time tracings for wild-type and calstabin2−/− mice, treated with JTV-519 or placebo as indicated. Embodiment B are bar graphs summarizing the mean time to fatigue for wild-type and calstabin2−/− mice, treated with JTV-519 or placebo as indicated.
FIG. 15, embodiments A and B, demonstrate that the beneficial effects of JTV-519 treatment on skeletal muscle function depends on calstabin1 and not calstabin2 binding to RyR1. Embodiment A are bar graphs of PKA phosphorylation of RyR1 at Ser-2844 in mice, which corresponds to Ser-2843 in humans. Embodiment B are bar graphs of the amount of calstabin 1 bound to RyR1 from wild-type and calstabin2−/− mice treated with JTV-519 or placebo.
FIG. 16, embodiments A, B, C, and D demonstrate that JTV-519 normalizes abnormal or leaky RyR1 channel function in vivo. Embodiments A and C are single channel current traces of wild-type mice with heart failure treated with placebo (A) and JTV-519 (C). Embodiments B and D are amplitude histograms for wild-type mice with heart failure treated with placebo (B) and JTV-519 (D).
FIG. 17, embodiments A and B, demonstrate that JTV-519 increases calstabin binding to PKA phosphorylated RyR. Embodiment A are immunoblots of calstabin 1 incubated and associated with RyR1 and calstabin2 incubated and associated with RyR2 at increasing concentrations of JTV-519. Embodiment B are graphs summarizing the proportion of calstabin 1 and calstabin2 bound to RyR1 and RyR2, as indicated.
FIG. 18, embodiments A, B, C, and D, demonstrate that PKA phosphorylation of Ser-2843 increases the open probability and gating kinetics of RyR1 channels. Embodiment A are single channel current traces and corresponding histogram of wild-type RyR1. Embodiment B are single channel current traces and corresponding histogram of wild-type RyR1 that is PKA phosphorylated. Embodiment C are are single channel current traces and corresponding histogram of RyR1-Ser-2843A. Embodiment D are single channel current traces and corresponding histogram of RyR1-Ser-2843D.
FIG. 19, embodiments A and B, demonstrate the PKA hyperphosphorylation and calstabin1 deficiency of RyR1 channels after sustained exercise. Embodiment A are immunoblots of RyR1, RyR1-pSer2844, RyR1-pSer2849, and calstabin1 for control and swim mice following an exercise regime. Embodiment B is a bar graph summarizing the relative amounts of the indicated compounds following the exercise regime.
FIG. 20, embodiments A and B, demonstrate that RyR1 PKA phosphorylation increases after exposure to increasing durations of sustained exercise. Embodiment A are immunoblots of RyR1 and RyR1-pSer2844 following increasing durations of sustained exercise. Embodiment B is a graph showing the relative PKA phosphorlation of RyR1 for varying durations of exercise.
FIG. 21, embodiments A, B, and C, demonstrate that RyR1 PKA phosphorylation increases with muscle fatigue. Embodiments A and B are, respectively, fatigue time tracings and a bar graph showing mean fatigue times for rat soleus muscle of heart failure and control subjects. Embodiment C is a graph of PKA phosphorylation versus fatigue time.
FIG. 22 are trichrome and hematoxylin-eosin stains of cross-sections of the mouse M. extensor digitorum longus, and demonstrates myofiber degeneration consistent with dystrophic remodeling following sustained exercise.



DETAILED DESCRIPTION OF THE INVENTION
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents and equivalents thereof known to those skilled in the art, and reference to “the FKBP12.6 polypeptide” is a reference to one or more FKBP12.6 polypeptides (also known as calstabin2) and equivalents thereof known to those skilled in the art, and so forth. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
The present invention provides novel compounds that are capable of treating and preventing disorders and diseases associated with the RyR receptors that regulate calcium channel functioning in cells. More particularly, the present invention provides novel compounds that are capable of treating or preventing a leak in RyR channels. “Disorders and diseases associated with the RyR receptors” means disorders and diseases that can be treated and/or prevented by modulating the RyR receptors that regulate calcium channel functioning in cells. “Disorders and diseases associated with the RyR receptors” include, without limitation, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof. Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence. Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss.
Compounds
Provided herein are compounds of Formula I:




wherein,

n is 0, 1, or 2;

R is located at one or more positions on the benzene ring;

each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R1 is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R2 is selected from the group consisting of —C═O(R5), —C═S(R6), —SO2R7, —POR8R9, —(CH2)m-R10, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R3 is selected from the group consisting of H, CO2Y, CONY, acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl; and wherein Y is selected from the group consisting of H, alkyl, aryl, cycloalkyl, and heterocyclyl;

R4 is selected from the group consisting of H, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R5 is selected from the group consisting of —NR16, NHNHR16, NHOH, —OR15, CONH2NHR16, CO2R15, CONR16, CH2X, acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R6 is selected from the group consisting of —OR15, NHNR16, NHOH, —NR16, CH2X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R7 is selected from the group consisting of —OR15, —NR16, NHNHR16, NHOH, CH2X, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R8 and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R10 is selected from the group consisting of NH2, OH, —SO2R11, —NHSO2R11, C═O(R12), NHC═O(R12), —OC═O(R12), and —POR13R14;

R11, R12, R13, and R14 independently are selected from the group consisting of H, OH, NH2, NHNH2, NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

X is selected from the group consisting of halogen, CN, CO2R15, CONR16, —NR16, —OR15, —SO2R2R7, and —POR8R9; and

R15 and R16 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, —N—, —O—, —S—, —CN, —N3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

and salts, hydrates, solvates, complexes, and prodrugs thereof.

The compounds of Formula I treat and prevent disorders and diseases associated with the RyR receptors. Examples of such compounds include, without limitation, S1, S2, S3, S4, S5, S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S65, S66, S67, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, and S100, which have the following structures:














































In an embodiment of the present invention, if R2 is C═O(R5) or SO2R7, then R is at positions 2, 3, or 5 on the benzene ring.
In another embodiment of the invention, if R2 is C═O(R5) or SO2R7, then each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
In another embodiment of the invention, if R2 is C═O(R5) or SO2R7, then there are at least two R groups attached to the benzene ring. Furthermore, there are at least two R groups attached to the benzene ring, and both R groups are attached at positions 2, 3, or 5 on the benzene ring. Still furthermore, each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
In another embodiment of the invention, if R2 is C═O(R5), then R5 is selected from the group consisting of —NR16, NHNHR16, NHOH, —OR15, CONH2NHR16, CONR16, CH2X, acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
In another embodiment, the present invention provides compounds of Formula II:




wherein R═OR′, SR′, NR′, alkyl, or halide and R′=alkyl, aryl, or H, and wherein R can be at position 2, 3, 4, or 5. Formula II is discussed also in co-pending application Ser. No. 10/680,988, the disclosure of which is incorporated herein in its entirety by reference.

Routes of Activity

The compounds of Formula I reduce the open probability of RyR by increasing the affinity of FKBP12 (calstabin1) and FKBP12.6 (calstabin2) for, respectively PKA-phosphorylated RyR1 and PKA-phosphorylated RyR2. Moreover, the compounds of Formula I normalize gating of mutant RyR channels, including CPVT-associated mutant RyR2 channels, by increasing FKBP12 (calstabin1) and FKBP12.6 (calstabin2) binding affinity. Therefore, the compounds of Formula I prevent disorders and conditions involving modulation of the RyR receptors, particularly the RyR1 and RyR2 receptors. Examples of such disorders and conditions include, without limitation, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof. Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence. Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss. The compounds of Formula I treat these disorders and conditions by increasing FKBP12 (calstabin1)-RyR1 binding affinity and increasing FKBP12.6 (calstabin2)-RyR2 binding affinity.
In accordance with the foregoing, the present invention provides a method for limiting or preventing a decrease in the level of RyR-bound FKBP (calstabin) in cells of a subject. As used herein, “RyR” includes RyR1, RyR2, and RyR3. Additionally, FKBP includes both FKBP12 (calstabin1) and FKBP12.6 (calstabin2). “RyR-bound FKBP” therefore refers to RyR1-bound FKBP12 (calstabin1), RyR2-bound FKBP12.6 (calstabin2), and RyR3-bound FKBP12 (calstabin1).
As used herein, “RyR” also includes an “RyR protein” and an “RyR analogue.” An “RyR analogue” is a functional variant of the RyR protein, having RyR biological activity, that has 60% or greater amino-acid-sequence homology with the RyR protein. The RyR of the present invention are unphosphorylated, phosphorylated (e.g., by PKA), or hyperphosphorylated (e.g., by PKA). As further used herein, the term “RyR biological activity” refers to the activity of a protein or peptide that demonstrates an ability to associate physically with, or bind with, FKBP12 (calstabin1) in the case of RyR1 and RyR3, and FKBP12.6 (calstabin2) in the case of RyR2 (i.e., binding of approximately two fold or, approximately five fold, above the background binding of a negative control), under the conditions of the assays described herein.
As used herein, “FKBP” includes both an “FKBP protein” and an “FKBP analogue,” whether it be FKBP12 (calstabin1) or FKBP12.6 (calstabin2). Unless otherwise indicated herein, “protein” shall include a protein, protein domain, polypeptide, or peptide, and any fragment thereof. An “FKBP analogue” is a functional variant of the FKBP protein, having FKBP biological activity, that has 60% or greater amino-acid-sequence homology with the FKBP protein, whether it be FKBP12 (calstabin1) or FKBP12.6 (calstabin2). As further used herein, the term “FKBP biological activity” refers to the activity of a protein or peptide that demonstrates an ability to associate physically with, or bind with, unphosphorylated or non-hyperphosphorylated RyR2 (i.e., binding of approximately two fold, or approximately five fold, above the background binding of a negative control), under the conditions of the assays described herein.
FKBP binds to the RyR channel, one molecule per RyR subunit. Accordingly, as used herein, the term “RyR-bound FKBP” includes a molecule of an FKBP12 (calstabin1) protein that is bound to an RyR1 protein subunit or a tetramer of FKBP12 that is in association with a tetramer of RyR1, a molecule of FKBP12.6 (calstabin2) protein that is bound to an RyR2 protein subunit or a tetramer of FKBP12.6 that is in association with a tetramer of RyR2, and a molecule of an FKBP12 (calstabin1) protein that is bound to an RyR3 protein subunit or a tetramer of FKBP12 that is in association with a tetramer of RyR3. Therefore, “RyR-bound FKBP” refers to “RyR1-bound FKBP12,” “RyR2-bound FKBP12.6,” and “RyR3-bound FKBP12.”
In accordance with the method of the present invention, a “decrease” or “disorder” in the level of RyR-bound FKBP in cells of a subject refers to a detectable decrease, diminution or reduction in the level of RyR-bound FKBP in cells of the subject. Such a decrease is limited or prevented in cells of a subject when the decrease is in any way halted, hindered, impeded, obstructed or reduced by the administration of compounds of Formula I, such that the level of RyR-bound FKBP in cells of the subject is higher than it would otherwise be in the absence of the administered compound.
The level of RyR-bound FKBP in a subject is detected by standard assays and techniques, including those readily determined from the known art (e.g., immunological techniques, hybridization analysis, immunoprecipitation, Western-blot analysis, fluorescence imaging techniques and/or radiation detection, etc.), as well as any assays and detection methods disclosed herein. For example, protein is isolated and purified from cells of a subject using standard methods known in the art, including, without limitation, extraction from the cells (e.g., with a detergent that solubilizes the protein) where necessary, followed by affinity purification on a column, chromatography (e.g., FTLC and HPLC), immunoprecipitation (with an antibody), and precipitation (e.g., with isopropanol and a reagent such as Trizol). Isolation and purification of the protein is followed by electrophoresis (e.g., on an SDS-polyacrylamide gel). A decrease in the level of RyR-bound FKBP in a subject, or the limiting or prevention thereof, is determined by comparing the amount of RyR-bound FKBP detected prior to the administration of JTV-519 or a compound having Formula I (in accordance with methods described below) with the amount detected a suitable time after administration of the compound.
A decrease in the level of RyR-bound FKBP in cells of a subject is limited or prevented, for example, by inhibiting dissociation of FKBP and RyR in cells of the subject; by increasing binding between FKBP and RyR in cells of the subject; or by stabilizing the RyR-FKBP complex in cells of a subject. As used herein, the term “inhibiting dissociation” includes blocking, decreasing, inhibiting, limiting or preventing the physical dissociation or separation of an FKBP subunit from an RyR molecule in cells of the subject, and blocking, decreasing, inhibiting, limiting or preventing the physical dissociation or separation of an RyR molecule from an FKBP subunit in cells of the subject. As further used herein, the term “increasing binding” includes enhancing, increasing, or improving the ability of phosphorylated RyR to associate physically with FKBP (e.g., binding of approximately two fold or, approximately five fold, above the background binding of a negative control) in cells of the subject and enhancing, increasing or improving the ability of FKBP to associate physically with phosphorylated RyR (e.g., binding of approximately two fold, or, approximately five fold, above the background binding of a negative control) in cells of the subject. Additionally, a decrease in the level of RyR-bound FKBP in cells of a subject is limited or prevented by directly decreasing the level of phosphorylated RyR in cells of the subject or by indirectly decreasing the level of phosphorylated RyR in the cells (e.g., by targeting an enzyme (such as PKA) or another endogenous molecule that regulates or modulates the functions or levels of phosphorylated RyR in the cells). In one embodiment, the level of phosphorylated RyR in the cells is decreased by at least 10% in the method of the present invention. In another embodiment, the level of phosphorylated RyR is decreased by at least 20%.
The subject of the present invention are in vitro and in vivo systems, including, without limitation, isolated or cultured cells or tissues, non-cell in vitro assay systems and an animal (e.g., an amphibian, a bird, a fish, a mammal, a marsupial, a human, a domestic animal (such as a cat, dog, monkey, mouse or rat) or a commercial animal (such as a cow or pig)).
The cells of a subject include striated muscle cells. A striated muscle is a muscle in which the repeating units (sarcomeres) of the contractile myofibrils are arranged in registry throughout the cell, resulting in transverse or oblique striations that are observed at the level of a light microscope. Examples of striated muscle cells include, without limitation, voluntary (skeletal) muscle cells and cardiac muscle cells. In one embodiment, the cell used in the method of the present invention is a human cardiac muscle cell. As used herein, the term “cardiac muscle cell” includes cardiac muscle fibers, such as those found in the myocardium of the heart. Cardiac muscle fibers are composed of chains of contiguous heart-muscle cells, or cardiomyocytes, joined end to end at intercalated disks. These disks possess two kinds of cell junctions: expanded desmosomes extending along their transverse portions, and gap junctions, the largest of which lie along their longitudinal portions.
A decrease in the level of RyR-bound FKBP is limited or prevented in cells of a subject by administering the compounds of Formula I to the subject; this would also permit contact between cells of the subject and the compounds of Formula I. The compounds of Formula I are modulators of calcium-ion channels. In addition to regulating Ca2+ levels in myocardial cells, the compounds of Formula I modulate the Na+ current and the inward-rectifier K+ current in cells, such as guinea pig ventricular cells, and inhibits the delayed-rectifier K+ current in cells, such as guinea pig atrial cells.
Pharmaceutical Composition
The compounds of the invention are formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo. According to another aspect, the present invention provides a pharmaceutical composition comprising compounds of Formula I in admixture with a pharmaceutically acceptable diluent and/or carrier. The pharmaceutically-acceptable carrier must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The pharmaceutically-acceptable carrier employed herein is selected from various organic or inorganic materials that are used as materials for pharmaceutical formulations and which are incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles and viscosity-increasing agents. If necessary, pharmaceutical additives, such as antioxidants, aromatics, colorants, flavor-improving agents, preservatives, and sweeteners, are also added. Examples of acceptable pharmaceutical carriers include carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others.
The pharmaceutical formulations of the present invention are prepared by methods well-known in the pharmaceutical arts. For example, the compounds of Formula I are brought into association with a carrier and/or diluent, as a suspension or solution. Optionally, one or more accessory ingredients (e.g., buffers, flavoring agents, surface active agents, and the like) also are added. The choice of carrier is determined by the solubility and chemical nature of the compounds, chosen route of administration and standard pharmaceutical practice.
The compounds of Formula I are administered to a subject by contacting target cells (e.g., cardiac muscle cells) in vivo in the subject with the compounds. The compounds of Formula I are contacted with (e.g., introduced into) cells of the subject using known techniques utilized for the introduction and administration of proteins, nucleic acids and other drugs. Examples of methods for contacting the cells with (i.e., treating the cells with) the compounds of Formula I include, without limitation, absorption, electroporation, immersion, injection, introduction, liposome delivery, transfection, transfusion, vectors and other drug-delivery vehicles and methods. When the target cells are localized to a particular portion of a subject, it is desirable to introduce the compounds for Formula I directly to the cells, by injection or by some other means (e.g., by introducing the compounds into the blood or another body fluid). The target cells are contained in tissue of a subject and are detected by standard detection methods readily determined from the known art, examples of which include, without limitation, immunological techniques (e.g., immunohistochemical staining), fluorescence imaging techniques, and microscopic techniques.
Additionally, the compounds of Formula I of the present invention are administered to a human or animal subject by known procedures including, without limitation, oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation or intranasally, vaginally, rectally, and intramuscularly. The compounds of Formula I are administered parenterally, by epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous or sublingual injection, or by way of catheter. In one embodiment, the agent is adiminstered to the subject by way of delivery to the subject's muscles including, but not limited to, the subject's cardiac muscles. In an embodiment, the agent is administered to the subject by way of targeted delivery to cardiac muscle cells via a catheter inserted into the subject's heart.
For oral administration, a formulation of the compounds of Formula I are presented as capsules, tablets, powders, granules or as a suspension or solution. The formulation has conventional additives, such as lactose, mannitol, corn starch or potato starch. The formulation also is presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins. Additionally, the formulation is presented with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose. The formulation also is presented with dibasic calcium phosphate anhydrous or sodium starch glycolate. Finally, the formulation is presented with lubricants, such as talc or magnesium stearate.
For parenteral administration (i.e., administration by injection through a route other than the alimentary canal), the compounds of Formula I are combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation is prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation is presented in unit or multi-dose containers, such as sealed ampoules or vials. The formulation is delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymatous, subcutaneous, or sublingual or by way of catheter into the subject's heart.
For transdermal administration, the compounds of Formula I are combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increase the permeability of the skin to the compounds of Formula I and permit the compounds to penetrate through the skin and into the bloodstream. The compounds of Formula I/enhancer composition also are further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which are dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch.
In some embodiments, the composition is in unit dose form such as a tablet, capsule or single-dose vial. Suitable unit doses, i.e., therapeutically effective amounts, can be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will, of course, vary depending on the desired clinical endpoint. The present invention also provides articles of manufacture for treating and preventing disorders, such as cardiac disorders, in a subject. The articles of manufacture comprise a pharmaceutical composition of one or more of the compounds of Formula I as described herein. The articles of manufacture are packaged with indications for various disorders that the pharmaceutical compositions are capable of treating and/or preventing. For example, the articles of manufacture comprise a unit dose of a compound disclosed herein that is capable of treating or preventing a muscular disorder, and an indication that the unit dose is capable of treating or preventing a certain disorder, for example an arrhythmia.
In accordance with a method of the present invention, the compounds of Formula I are administered to the subject (or are contacted with cells of the subject) in an amount effective to limit or prevent a decrease in the level of RyR-bound FKBP in the subject, particularly in cells of the subject. This amount is readily determined by the skilled artisan, based upon known procedures, including analysis of titration curves established in vivo and methods and assays disclosed herein. A suitable amount of the compounds of Formula I effective to limit or prevent a decrease in the level of RyR-bound FKBP in the subject ranges from about 5 mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml. In an embodiment, the amount of compounds from Formula I ranges from about 10 mg/kg/day to about 20 mg/kg/day.
Uses
The present invention provides a new range of therapeutic treatments for patients with various disorders involving modulation of the RyR receptors, particularly skeletal muscular disorders (RyR1), cardiac (RyR2) disorders, and cognitive (RyR3) disorders.
In one embodiment of the present invention, the subject has not yet developed a disorder, such as cardiac disorders (e.g., exercise-induced cardiac arrhythmia). In another embodiment of the present invention, the subject is in need of treatment for a disorder, including a cardiac disorder.
Various disorders that the compounds of Formula I treat or prevent include, but are not limited to, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof. Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence. Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss. One skilled in the art will recognize still other diseases, including but not limited to muscular and cardiac disorders, that the compounds of Formula I are be useful to treat, in accordance with the information provided herein.
The amount of compounds of Formula I effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject is an amount effective to prevent exercise-induced cardiac arrhythmia in the subject. Cardiac arrhythmia is a disturbance of the electrical activity of the heart that manifests as an abnormality in heart rate or heart rhythm. As used herein, an amount of compounds of Formula I “effective to prevent exercise-induced cardiac arrhythmia” includes an amount of compounds of Formula I effective to prevent the development of the clinical impairment or symptoms of the exercise-induced cardiac arrhythmia (e.g., palpitations, fainting, ventricular fibrillation, ventricular tachycardia and sudden cardiac death). The amount of compounds of Formula I effective to prevent exercise-induced cardiac arrhythmia in a subject will vary depending upon the particular factors of each case, including the type of exercise-induced cardiac arrhythmia, the subject's weight, the severity of the subject's condition, and the mode of administration of the compounds of Formula I. This amount is readily determined by the skilled artisan, based upon known procedures, including clinical trials, and methods disclosed herein. In an embodiment, the amount of the compounds of Formula I effective to prevent the exercise-induced cardiac arrhythmia is an amount effective to prevent exercise-induced sudden cardiac death in the subject. In another embodiment, the compounds of Formula I prevent exercise-induced cardiac arrhythmia and exercise-induced sudden cardiac death in the subject.
Because of its ability to stabilize RyR-bound FKBP and maintain and restore balance in the context of dynamic PKA phosphorylation and dephosphorylation of RyR, the compounds of Formula I are also useful in treating a subject who has already experienced clinical symptoms of these various disorders. For example, if the symptoms of the disorder are observed in the subject early enough, the compounds of Formula I are effective in limiting or preventing a further decrease in the level of RyR-bound FKBP in the subject.
Additionally, the subject of the present invention is a candidate for exercise-induced cardiac disorders, such as exercise-induced cardiac arrhythmia. Exercise-induced cardiac arrhythmia is a heart condition (e.g., a ventricular fibrillation or ventricular tachycardia, including any that leads to sudden cardiac death) that develops during/after a subject has undergone physical exercise. A “candidate” for an exercise-induced cardiac disorder is a subject who is known to be, or is believed to be, or is suspected of being, at risk for developing a cardiac disorder during/after physical exercise. Examples of candidates for exercise-induced cardiac arrhythmia include, without limitation, an animal/person known to have catecholaminergic polymorphic ventricular tachycardia (CPVT); an animal/person suspected of having CPVT; and an animal/person who is known to be, or is believed to be, or is suspected of being at risk for developing cardiac arrhythmia during/after physical exercise, and who is about to exercise, is currently exercising or has just completed exercise. As discussed above, CPVT is an inherited disorder in individuals with structurally-normal hearts. It is characterized by stress-induced ventricular tachycardia—a lethal arrhythmia that causes sudden cardiac death. In subjects with CPVT, physical exertion and/or stress induce bidirectional and/or polymorphic ventricular tachycardias that lead to sudden cardiac death (SCD) in the absence of detectable structural heart disease. Individuals with CPVT have ventricular arrhythmias when subjected to exercise, but do not develop arrhythmias at rest.
Accordingly, in still another embodiment of the present invention, the subject has been exercising, or is currently exercising, and has developed an exercise-induced disorder. In this case, the amount of the compounds of Formula I effective to limit or prevent a decrease in the level of RyR-bound FKBP in the subject is an amount of compound effective to treat the exercise-induced disorder in the subject. As used herein, an amount of compounds of Formula I “effective to treat an exercise-induced disorder” includes an amount of compound of Formula I effective to alleviate or ameliorate the clinical impairment or symptoms of the exercise-induced disorder (e.g., in the case of cardiac arrhythmia, palpitations, fainting, ventricular fibrillation, ventricular tachycardia, and sudden cardiac death). The amount of compounds of Formula I effective to treat an exercise-induced disorder in a subject will vary depending upon the particular factors of each case, including the type of exercise-induced disorder, the subject's weight, the severity of the subject's condition, and the mode of administration of the compounds of Formula I. This amount is readily determined by the skilled artisan, based upon known procedures, including clinical trials, and methods disclosed herein. In an embodiment, the compounds of Formula I treat exercise-induced disorders in the subject.
The present invention further provides a method for treating exercise-induced disorders in a subject. The method comprises administering the compounds of Formula I to the subject in an amount effective to treat the exercise-induced disorder in the subject. A suitable amount of the compounds of Formula I effective to treat, for example, exercise-induced cardiac arrhythmia in the subject ranges from about 5 mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml. The present invention also provides a method for preventing an exercise-induced disorder in a subject. The method comprises administering the compounds of Formula I to the subject in an amount effective to prevent the exercise-induced disorder in the subject. A suitable amount of the compounds of Formula I effective to prevent the exercise-induced disorder in the subject ranges from about 5 mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml. Additionally, the present invention provides a method for preventing exercise-induced disorders in a subject. The method comprises administering the compounds of Formula I to the subject in an amount effective to prevent an exercise-induced disorder in the subject. A suitable amount of the compounds of Formula I effective to prevent an exercise-induced disorder in the subject ranges from about 5 mg/kg/day to about 20 mg/kg/day, and/or is an amount sufficient to achieve plasma levels ranging from about 300 ng/ml to about 1000 ng/ml.
Additionally, the compounds prevent irregular heartbeat disorders in subjects with heterozygous defects in the FKBP12.6 gene.
The compounds of Formula I can be used alone, in combination with each other, or in combination with other agents that have cardiovascular activity including, but not limited to, diuretics, anticoagulants, antiplatelet agents, antiarrhythmics, inotropic agents, chronotropic agents, α and β blockers, angiotensin inhibitors and vasodilators. Further, such combinations of the compounds of the present invention and other cardiovascular agents are administered separately or in conjunction. In addition, the administration of one element of the combination is prior to, concurrent to or subsequent to the administration of other agent(s).
In various embodiments of the above-described methods, the exercise-induced cardiac arrhythmia in the subject is associated with VT. In some embodiments, the VT is CPVT. In other embodiments of these methods, the subject is a candidate for exercise-induced cardiac arrhythmia, including candidates for exercise-induced sudden cardiac death.
In view of the foregoing methods, the present invention also provides use of the compounds of Formula I in a method for limiting or preventing a decrease in the level of RyR-bound FKBP in a subject who is a candidate for a disorder. The present invention also provides use of the compounds of Formula I in a method for treating or preventing a muscular disorder in a subject. Furthermore, the present invention provides use of the compounds of Formula I in a method for preventing treating or preventing exercise-induced muscular disorders in a subject.
Accordingly, the present invention further provides a method for assaying the effects of the compounds of Formula I in preventing disorders and diseases associated with the RyR receptors. The method comprises the steps of: (a) obtaining or generating a culture of cells containing RyR; (b) contacting the cells with one or more of the compounds of Formula I; (c) exposing the cells to one or more conditions known to increase phosphorylation of RyR in cells; and (d) determining if the one or more compounds from Formula I limits or prevents a decrease in the level of RyR-bound FKBP in the cells. As used herein, a cell “containing RyR” is a cell in which RyR, including RyR1, RyR2, and RyR3, or a derivative or homologue thereof, is naturally expressed or naturally occurs. Conditions known to increase phosphorylation of RyR in cells include, without limitation, PKA.
In the method of the present invention, cells are contacted with one or more of the compounds of Formula I by any of the standard methods of effecting contact between drugs/agents and cells, including any modes of introduction and administration described herein. The level of RyR-bound FKBP in the cell is measured or detected by known procedures, including any of the methods, molecular procedures and assays known to one of skill in the art or described herein. In one embodiment of the present invention, the one or more compounds of Formula I limits or prevents a decrease in the level of RyR-bound FKBP in the cells.
RyR, including RyR1, RyR2, and RyR3, has been implicated in a number of biological events in cells. For example, it has been shown that RyR2 channels play an important role in EC coupling and contractility in cardiac muscle cells. Therefore, it is clear that preventive drugs designed to limit or prevent a decrease in the level of RyR-bound FKBP in cells, particularly RyR2-bound FKPB12.6 in cardiac muscle cells, are useful in the regulation of a number of RyR-associated biological events, including EC coupling and contractility. Thus, the one or more compounds of Formula I are evaluated for effect on EC coupling and contractility in cells, particularly cardiac muscle cells, and therefore, usefulness for preventing exercise-induced sudden cardiac death.
Accordingly, the method of the present invention further comprises the steps of contacting one or more compounds of Formula I with a culture of cells containing RyR; and determining if the one or more compounds has an effect on an RyR-associated biological event in the cells. As used herein, an “RyR-associated biological event” includes a biochemical or physiological process in which RyR levels or activity have been implicated. As disclosed herein, examples of RyR-associated biological events include, without limitation, EC coupling and contractility in cardiac muscle cells. According to this method of the present invention, the one or more compounds of Formula I are contacted with one or more cells (such as cardiac muscle cells) in vitro. For example, a culture of the cells is incubated with a preparation containing the one or more compounds of Formula I. The compounds' effect on a RyR-associated biological event then is assessed by any biological assays or methods known in the art, including immunoblotting, single-channel recordings and any others disclosed herein.
The present invention is further directed to one or more compounds of Formula I identified by the above-described identification method, as well as a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier and/or diluent. The compounds are useful for preventing exercise-induced sudden cardiac death in a subject, and for treating or preventing other RyR-associated conditions. As used herein, an “RyR-associated condition” is a condition, disease, or disorder in which RyR level or activity has been implicated, and includes an RyR-associated biological event. The RyR-associated condition is treated or prevented in the subject by administering to the subject an amount of the compound effective to treat or prevent the RyR-associated condition in the subject. This amount is readily determined by one skilled in the art. In one embodiment, the present invention provides a method for preventing exercise-induced sudden cardiac death in a subject, by administering the one or more compounds of Formula I to the subject in an amount effective to prevent the exercise-induced sudden cardiac death in the subject.
The present invention also provides an in vivo method for assaying the effectiveness of the compounds of Formula I in preventing disorders and diseases associated with the RyR recpetors. The method comprises the steps of: (a) obtaining or generating an animal containing RyR; (b) administering one or more of the compounds of Formula I to the animal; (c) exposing the animal to one or more conditions known to increase phosphorylation of RyR in cells; and (d) determining the extent the compound limits or prevents a decrease in the level of RyR-bound FKBP in the animal. The method further comprises the steps of: (e) administering one or more of the compounds of Formula I to an animal containing RyR; and (f) determining the extent of the effect of the compound on a RyR-associated biological event in the animal. Also provided is a pharmaceutical composition comprising this compound; and a method for preventing exercise-induced sudden cardiac death in a subject, by administering this compound to the subject in an amount effective to prevent the exercise-induced sudden cardiac death in the subject.
It has been demonstrated that compounds which block PKA activation would be expected to reduce the activation of the RyR channel, resulting in less release of calcium into the cell. Compounds that bind to the RyR channel at the FKBP binding site, but do not come off the channel when the channel is phosphorylated by PKA, would also be expected to decrease the activity of the channel in response to PKA activation or other triggers that activate the RyR channel. Such compounds would also result in less calcium release into the cell.
By way of example, the diagnostic assays screen for the release of calcium into cells via the RyR channel, using calcium-sensitive fluorescent dyes (e.g., Fluo-3, Fura-2, and the like). Cells are loaded with the fluorescent dye of choice, then stimulated with RyR activators to determine the reduction of the calcium-dependent fluorescent signal (Brillantes, et al., Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell, 77:513-23, 1994; Gillo, et al., Calcium entry during induced differentiation in murine erythroleukemia cells. Blood, 81:783-92, 1993; Jayaraman, et al., Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science, 272:1492-94, 1996). Calcium-dependent fluorescent signals are monitored with a photomultiplier tube, and analyzed with appropriate software. This assay can easily be automated to screen the compounds of Formula I using multiwell dishes.
To demonstrate that the compounds of Formula I inhibit the PKA-dependent activation of RyR-mediated intracellular calcium release, an assay involves the expression of recombinant RyR channels in a heterologous expression system, such as Sf9, HEK293, or CHO cells. RyR could also be co-expressed with beta-adrenergic receptors. This would permit assessment of the effect of compounds of Formula I on RyR activation, in response to addition of beta-adrenergic receptor agonists.
The level of PKA phosphorylation of RyR2 which correlates with the degree of heart failure also is assayed and then used to determine the efficacy of the one or more compounds of Formula I to block the PKA phosphorylation of the RyR2 channel. Such an assay is based on the use of antibodies that are specific for the RyR2 protein. For example, the RyR2-channel protein is immunoprecipitated and then back-phosphorylated with PKA and [gamma32P]-ATP. The amount of radioactive [32P] label that is transferred to the RyR2 protein then is measured using a phosphorimager (Marx, et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000).
Another assay of the compounds of Formula I involves use of a phosphoepitope-specific antibody that detects RyR1 that is PKA phosphorylated on Ser 2843 or RyR2 that is PKA phosphorylated on Ser 2809. Immunoblotting with such an antibody can be used to assess efficacy of these compounds for therapy for heart failure and cardiac arrhythmias. Additionally, RyR2 S2809A and RyR2 S2809D knock-in mice are used to assess efficacy of therapy for heart failure and cardiac arrhythmias. Such mice further provide evidence that PKA hyperphosphorylation of RyR2 is a contributing factor in heart failure and cardiac arrhythmias by showing that the RyR2 S2809A mutation inhibits heart failure and arrhythmias, and that the RyR2 S2809D mutation worsens heart failure and arrhythmias.
Therefore, in a specific embodiment, the present invention provides a method of treating heart failure, atrial fibrillation or exercise-induced cardiac arrhythmia, comprising administering to an animal in need thereof, a therapeutically effective amount of a compound selected from the compounds of Formula I.
Intracellular Ca2+ leak is proposed as a principal mediator of depressed muscle performance and dystrophic muscle remodeling. Muscular dystrophies are heterogeneous hereditary diseases characterized by weakness and progressive muscle wasting. Of all forms of muscular dystrophies involving the dystrophin-associated protein complex (referred to as dystrophinopathies), Duchenne muscular dystrophy (DMD) is one of the most frequent genetic diseases (X-linked; 1 in 3,500 boys) with death usually occurring before age 30 by respiratory and/or cardiac failure in high numbers of patients. Becker muscular dystrophy (BMD) represents a milder form of the disease associated with a reduction in the amount or expression of a truncated form of the dystrophin protein whereas Duchenne patients have been characterized by complete absence or very low levels of dystrophin. Duchenne and Becker's muscular dystrophy (DMD/BMD) are caused by mutations in the gene encoding the 427-kDa cytoskeletal protein dystrophin. However, with increasing age in BMD cardiac symptoms are more common than in DMD patients and do not correlate with skeletal muscle symptoms. Since genetic screening will not eliminative DMD due to a high incidence of sporadic cases, an effective therapy is highly desirable. DMD/BMD have been consistently associated with disturbed intracellular calcium metabolism. Because alterations of intracellular Ca2+ concentrations in DMD myofibers are believed to represent a central pathogenic mechanism, development of a therapeutic intervention that prevents intracellular Ca2+ abnormalities as a cause of skeletal muscle degeneration is highly desirable.
It is well established that lack of dystrophin expression is the primary genetic defect in DMD and BMD. However, the key mechanism leading to progressive muscle damage is largely unknown. It has been suggested that elevations of intracellular Ca2+ concentrations ([Ca2+]i) under resting conditions directly contributed to toxic muscle cell (myofiber) damage and concurrent activation of Ca2+-dependent proteases. Since calpain activity is increased in necrotic muscle fibers of mdx mice and calpain dysfunction contributes to limb-girdle muscular dystrophy, preventing activation of calcium-dependent proteases by inhibiting intracellular Ca2+ elevations represents a strategy to prevent muscle wasting in DMD. Significant differences in [Ca2+]i between normal and dystrophic muscles have been reported in myotubes and animal models including the dystrophin-deficient mdx mouse. Intracellular Ca2+ elevations are prevented by administration of a pharmaceutical composition comprising a compound of Formula I.
The present invention also provides a method of diagnosis of a disease or disorder in a subject, said method comprising: obtaining a cell or tissue sample from the subject; obtaining DNA from the cell or tissue; comparing the DNA from the cell or tissue with a control DNA encoding RyR to determine whether a mutation is present in the DNA from the cell or tissue, the presence of a mutation indicating a disease or disorder. In one embodiment, the mutation is a RyR2 mutation on chromosome 1q42-q43. In another embodiment, the mutation is one or more CPTV mutations. In another embodiment, the mutation may be a mutation which is present in the DNA encoding RyR2 of a SIDS subject. The diagnostic method is used to detect the presence of a disease or disorder in an adult, a child or a fetus. The disease and disorders include, but are not limited to, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof. Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence. Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss.
The present invention further provides a method of diagnosis of disorders and diseases in a subject, said method comprising: obtaining cells or tissue sample from the subject; incubating the cells or tissue sample with the compound of Formula I under conditions which increase phosphorylation of RyR in cells; determining (a) whether RyR bound to calstabin (i.e. RyR1 bound to calstabin1, RyR2 bound to calstabin2, or RyR3 bound to calstabin1) is increased in the cells or tissue compared to RyR bound to calstabin in control cells or tissues said control cells or tissues lacking mutant RyR calcium channels, or (b) whether a decrease in release of calcium occurs in RyR channels compared to a lack of decrease in release of calcium in the control cells; an increase in RyR-bound calstabin in (a) indicating a disorder or disease in the subject or a decrease in release of calcium in RyR channels in (b) compared to the control cells indicating a cardiac disease or disorder in the subject. The diagnostic method is used to detect the presence of a disease or disorder in an adult, a child or a fetus. The disease and disorders include, but are not limited to, cardiac disorders and diseases, skeletal muscular disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome. Cardiac disorder and diseases include, but are not limited to, irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof. Skeletal muscular disorder and diseases include, but are not limited to, skeletal muscle fatigue, exercise-induced skeletal muscle fatigue, muscular dystrophy, bladder disorders, and incontinence. Cognitive disorders and diseases include, but are not limited to, Alzheimer's Disease, forms of memory loss, and age-dependent memory loss.
The present invention further provides a method of diagnosis of a cardiac disorder or disease in a subject, said method comprising: obtaining cardiac cells or tissue sample from the subject; incubating the cardiac cells or tissue sample with the compound of Formula I under conditions which increase phosphorylation of RyR2 in cells; determining (a) whether RyR2 bound to calstabin2 is increased in the cells or tissue compared to RyR2 bound to calstabin2 in control cells or tissues said control cells or tissues lacking mutant RyR2 calcium channels, or (b) whether a decrease in release of calcium occurs in RyR2 channels compared to a lack of decrease in release of calcium in the control cells; an increase in RyR2-bound calstabin2 in (a) indicating a disorder or disease in the subject or a decrease in release of calcium in RyR2 channels in (b) compared to the control cells indicating a cardiac disease or disorder in the subject. The provided method is used to diagnose CPTV. The provided method also is used to diagnose sudden infant death syndrome (SIDS). The provided method additionally is used to diagnose cardiac irregular heartbeat disorders and diseases; exercise-induced irregular heartbeat disorders and diseases; sudden cardiac death; exercise-induced sudden cardiac death; congestive heart failure; chronic obstructive pulmonary disease; and high blood pressure. Irregular heartbeat disorders and diseases include and exercise-induced irregular heartbeat disorders and diseases include, but are not limited to, atrial and ventricular arrhythmia; atrial and ventricular fibrillation; atrial and ventricular tachyarrhythmia; atrial and ventricular tachycardia; catecholaminergic polymorphic ventricular tachycardia (CPVT); and exercise-induced variants thereof.
In addition to the above-mentioned therapeutic uses, the compounds of the invention are also useful in diagnostic assays, screening assays and as research tools.
Methods of Synthesizing Same
The present invention, provides, in a further aspect, processes for the preparation of a compound of Formula I, and salts, solvates, hydrates, complexes, and pro-drugs thereof, and pharmaceutically acceptable salts of such pro-drugs. More particularly, the present invention provides processes for the preparation of compounds selected from the group consisting of S1, S2, S3, S4, S5, S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, and S100, and salts, solvates, hydrates, complexes, and pro-drugs thereof, and pharmaceutically acceptable salts of such pro-drugs. The various synthetic routes to the compounds are described herein.
Some of the following syntheses utilize solvents. In one embodiment, the solvent is an organic solvent. In another embodiment, the organic solvent is methylene chloride (CH2Cl2), chloroform (CCl4) or methanol (CH3OH). Some of the following syntheses also utilize a base catalyst. In one embodiment, the base catalyst is an amine compound. In another embodiment, the base catalyst is an alkylamine such as triethylamine (TEA). In still another embodiment, the base catalyst is pyridine. Some of the following synthesis also utilize basic solutions. In one embodiment, the basic solution is sodium bicarbonate or calcium carbonate. In another embodiment, the basic solution is saturated sodium bicarbonate or saturated calcium carbonate. Some of the following syntheses use acidic solutions. In one embodiment, the acidic solution is a sulfuric acid solution, a hydrochloric acid solution, or a nitric acid solution. In one embodiment, the solution is 1N HCl. One of skill in the art will appreciate still other solvents, organic solvents, base catalysts, basic solutions, and acidic solutions that are used in the embodiments, according to the description herein. The solvents, organic solvents, reactants, catalysts, wash solutions, and so forth are added at appropriate temperatures (e.g. room temperature or about 20° C.-25° C., 0° C., etc.).
Some of the following syntheses requiring purification of the reaction mixture to yield a final product. Purification of the reaction mixture involves one or more processes such as removal of any solvent, crystallization of the product, chromatographic separation of the product (including HPLC, silica gel chromatography, column chromatography, and so forth), washing with basic solution, washing with acidic solution, re-dissolving the product in another solvent, and so forth. One of skill in the art will appreciate still other processes that are used in the embodiments, according to the description herein.
The reactions are carried out as long as needed (e.g., one hour, several hours, overnight, 24 hours, etc.) to obtain the desired or optimal yields of the desired compounds. Often, the reaction mixtures are stirred. The reactions are carried out at appropriate temperatures (e.g. room temperature or about 20° C.-25° C., 0° C., 100° C., etc.).
Synthon S26 is prepared according to methods described in U.S. patent application Ser. No. 10/680,988.
S3, S4, S5, and S54 are prepared from S26. S26 is reacted with RSO2Cl, wherein R is CH2═CH— (S3), Me— (S4), p-Me—C6H4— (S5), or NH-2-Py (S54), to form a product. The product is purified, for example by column chromatography, to yield S3, S4, S5, or S54. In one embodiment, the reaction occurs in a solvent, such as an organic solvent like CH2Cl2, so that a reaction mixture is formed, and the solvent is removed from the reaction mixture before or during purification of the product. If necessary, a base catalyst, such as triethylamine, is used in the synthesis. Also, basic (e.g., saturated sodium bicarbonate) and acidic washes (e.g., 1N HCl) are used if needed to purifying the reaction mixture and/or product, and are accompanied by drying, for example over sodium sulfate, if needed. Column chromatography, for example, is used to purifying the residue to isolate the desired product.
S1 and S2 are prepared from S3 by reaction with HNR1R2, where R is




(S1) or NBu2 (S2). The product is purified, for example by column chromatography, to yield S1 or S2. In one embodiment, the reaction occurs in a solvent, such as an organic solvent like CH2Cl2, so that a reaction mixture is formed, and the solvent is removed from the reaction mixture before or during purification of the product. Column chromatography, for example, is used to purifying the residue to isolate the desired product.

S7, S9, S27 and S40 are prepared from S26 by reaction with an alcohol of formula RCOX, where X is Cl or NHS and R is ICH2— (S7), Ph- (S9), CH2═CH— (S27), or 4-N3-2-OH—C6H5 (S40). In one embodiment, the reaction occurs in a solvent, such as an organic solvent like CH2Cl2, so that a reaction mixture is formed, and the solvent is removed from the reaction mixture before or during purification of the product. If necessary, a base catalyst, such as triethylamine, is used in the synthesis. Also, basic (e.g., saturated sodium bicarbonate) and acidic washes (e.g., 1N HCl) are used if needed to purifying the reaction mixture and/or product, and are accompanied by drying, if needed. In another embodiment, S40 is formed by reaction with an alcohol of formula RCOX, where R is 4-N3-2-OH—C6H5 and X is NHS. Column chromatography, for example, is used to purifying the residue to isolate the desired product.
S11 and S12 are prepared from S26 by reaction with a compound of formula C6H4—NCX, wherein X is O (S11) or S (S12). In one embodiment, the reaction occurs in a solvent, such as an organic solvent like CH2Cl2, so that a reaction mixture is formed, and the solvent is removed from the reaction mixture before or during purification of the product. If necessary, a base catalyst, such as triethylamine or pyridine, is used in the synthesis. In another embodiment, a base catalyst such as pyridine is used as the solvent in which the reaction takes place, and additional solvent, such as ethyle acetate or another appropriate organic solvent, is added after the reaction occurs. Also, basic (e.g., saturated sodium bicarbonate) and acidic washes (e.g., 1N HCl) are used if needed to purifying the reaction mixture and/or product, and are accompanied by drying, if needed. Column chromatography, for example, is used to purifying the residue to isolate the desired product.
The isomers S13 and S14 are prepared from S26 by reaction with phenyl methoxyphosphonyl chloride (Ph(MeO)P(O)Cl). In one embodiment, the reaction occurs in a solvent, such as an organic solvent, such as methylene chloride. If necessary, a base catalyst such as triethylamine may be used, for example, by adding it to a reaction mixture formed by mixing the reactants in a solvent. Also, the reaction mixture is washed with basic solution, for example saturated sodium bicarbonate, if necessary. The isomers are separated and purified, for example, using silica gel chromatography.
S19 and 22 are prepared from S26 by reaction with a compound of formula ClOC—X—COCl, where X is CH2—CH2 (S19) or




In one embodiment, the reaction occurs in the presence of a solvent, such as an organic solvent, such as methylene chloride. If necessary, a base catalyst such as triethylamine is added to the reaction mixture formed by mixing the reactants in a solvent. Also, base (e.g., saturated sodium bicarbonate), acid (e.g., 1N HCl), and water washes are used to remove unwanted compounds from the reaction mixture, if needed.

S20 and S23 are prepared from an intermediate compound of formula




where R is CH2═CH— (S20) or





The intermediate compound is treated with H2O2. If necessary, sodium thiosulfate also is used to treat the intermediate. In one embodiment, the reaction occurs in the presence of a solvent, such as an organic solvent, such as methanol (CH3OH), forming a reaction mixture. The solvent is removed from the reaction mixture after the reaction takes place and, if desired, the residue is redissolved in another solvent, such as another organic solvent, such as ethyl acetate. The reaction mixture is washed with basic solution (e.g., saturated sodium carbonate) if desired to remove unwanted compounds from the reaction mixture. The reaction mixture is dried (e.g., using sodium sulfate) if it is washed with basic solution. The final residue is purified, for example by column chromatography, to obtain the final product.

S57 is prepared from S26 and methyl chlorooxoacetate. In an embodiment, the reaction occurs in the presence of a solvent, such as an organic solvent, such as methylene chloride. A base catalyst such as pyridine is used as necessary to facilitate or hasten the reaction. The reaction mixture formed by mixing the reactants and a solvent is washed with basic solution (e.g., saturated sodium bicarbonate), acidic solution (e.g. HCl), and water. Purification such as silic gel chromatography yields S57.
S36 is prepared from S57 by reaction with sodium hydroxide. In one embodiment, the reaction takes place in a solvent, such as an organic solvent, such as methanol. The solvent is removed from the reaction mixture formed by mixing the reactants and the solvent, thereby forming a residue. The residue is dissolved in water and washed with another organic solvent, such as ether, to remove unwanted hydrophobic compounds. The aqueous phase from the basic washes is acidified and the product is extracted therefrom using an organic solvent, such as methylene chloride. Further purification is used if necessary.
S38 is prepared in a manner similar to S36, except a compound of formula




is used as the starting material in the synthesis.

S44 is prepared by treating S36 with thionyl chloride to form crude S36-Cl. Excess thionyl chloride, if any, is removed from the reaction mixture. The crude S36-Cl then is dissolved in a solvent, such as an organic solvent like methylene chloride, and reacted with mono-protected (e.g., mono-Boc protected) cystamine. A base catalyst such as pyridine is used if desired, and the reaction mixture is quenched with a basic solution (e.g., saturated sodium bicarbonate). The reaction mixture formed by mixing the cystamine and S36-Cl reactants is purified. The protecting groups (e.g., Boc) are removed using an appropriate acid or base wash (e.g., trifluoroacetic acid in an organic solvent in the case of the Boc protecting group). The final product then is purified, for example, using chromatography techniques.
S57 and S59 are prepared from S36-Cl, which is reacted with methanol (S57) or ethylamine (S59).
S43 and S45 are prepared from S36-cystamine, which is prepared as disclosed herein. S-36 cystamine is reacted with an NHS activated ester of an appropriate azido compound to yield S43 and S45. The reaction takes place in a solvent, such as an organic solvent.
S37 is prepared from S26 by reaction with 4-nitrophenyl chloroformate (NO2C6H5OCOCl). The reaction takes place in a solvent and, if desired, a base catalyst such as triethylamine may be used. The reaction mixture formed by mixing the reactants and a solvent is washed with water to remove unwanted hydrophilic compounds. The solvent is removed from the reaction mixture to form a residue, which is purified (e.g., using chromatography techniques) to yield S37.
S6, S46-53, S64, S66, and S67 are prepared from S37 by reaction with an amine of formula RNH2, wherein NR is NH2 (S46), NEt2 (S48), NHCH2Ph (S49), NHOH (S51),




The reaction takes place in the presence of a solvent, such as an organic solvent, such as DMF. In one embodiment, only one equivalent of amine is used in the reaction. Purification is accomplished, for example, by SiO2 column chromatography.

S6, S46-53, S64, S66, and S67 also are prepared from S26, via the S26-phosgene intermediate. The S26-phosgene intermediate is formed by reacting S26 with triphosgene. Thereafter, the S26-phosgene is reacted with an amine of formula RNH2, where NR is NH2




The reaction takes place in the presence of a solvent, such as an organic solvent. In one embodiment, only one equivalent of amine is used in the reaction. Purification is accomplished, for example, by SiO2 column chromatography.

S55, S56, S58, and S60-63 are prepared from S27 by reaction with HNR1R2, where




The reaction occurs in a solvent, such as an organic solvent, such as chloroform, thereby forming a reaction mixture. The solvent is removed from the reaction mixture to form a residue, which is purified, for example, by silica gel column chromatography to yield the final product.

S69-75 are prepared from S68, via the S68-phosgene intermediate. S68 is treated with triphosgene to form the intermediate, which in turn is treated with an amine RNH2, where R is




The reaction occurs in a solvent, such as an organic solvent, such as chloroform, thereby forming a reaction mixture. The solvent is removed from the reaction mixture to form a residue, which is purified, for example, by silica gel column chromatography to yield the final product.

S76 is prepared from S68 by reaction with methyl chlorooxoacetate. In an embodiment, the reaction occurs in the presence of a solvent, such as an organic solvent, such as methylene chloride. A base catalyst such as pyridine is used as necessary to facilitate or hasten the reaction. The reaction mixture formed by mixing the reactants and a solvent is washed with basic solution (e.g., saturated sodium bicarbonate), acidic solution (e.g. HCl), and water. Purification such as silic gel chromatography yields S76.
S77 is prepared from S76 by reaction with sodium hydroxide. In one embodiment, the reaction takes place in a solvent, such as an organic solvent, such as methanol. The solvent is removed from the reaction mixture formed by mixing the reactants and the solvent, thereby forming a residue. The residue is dissolved in water and washed with another organic solvent, such as ether, to remove unwanted hydrophobic compounds. The aqueous phase from the basic washes is acidified and the product is extracted therefrom using an organic solvent, such as methylene chloride. Further purification is used if necessary.
S78-S81 are prepared by treating S77 with thionyl chloride to form crude S77-Cl. Excess thionyl chloride, if any, is removed from the reaction mixture. The crude S77-Cl then is dissolved in a solvent, such as an organic solvent like methylene chloride, and reacted with HX, where X is NHEt (S78), NHPh (S79), NH2 (S80), and NHCH2-pyridine (S81). The solvent is removed, and the residue is purified.
S82 is prepared from S68. S68 is reacted with CH2CHSO2Cl in a manner analogous to the production of S3. The product then is treated with HNR1R2 in a manner analogous to the production of S1 and S2, except that NR1R2 is




S83 is prepared from S68. S68 is reacted with RCOCl, wherein R is




in a manner analogous to the production of S7, S9, and S40.

S84 is prepared from S68 by reaction with benzyl bromide. In an embodiment, the reaction takes place in a solvent, such as an organic solvent like methylene chloride. A base catalyst such as triethylamine is added as necessary to catalyze the reaction. The reaction mixture formed by mixing the reactants and the solvent is purified to yield S84.
S85 is prepared from S26. S26 is reacted with di-tert-butyl dicarbonate in a solvent, for example an organic solvent like methylene chloride. A base catalyst such as triethylamine also is used, if necessary. The reaction mixture formed by mixing the reactants and the solvent is washed with saturated sodium bicarbonate solution and the aqueous layer is extracted with an organic solvent. The combined organic layers are dried and concentrated provide S85.
S86 is prepared from S85 in a solvent, for example an organic solvent. S85 is treated with BBr3 to form a reaction mixture. If necessary, a base catalyst, such as triethylamine, is used in the reaction. The reaction is quenched (e.g., in the case of triethylamine, with methanol) and concentrated. Purification, for example by column chromatography, yields S86.
S87 is prepared by reacting S86 with trifluoromethylsulfonyl anhydride. The reaction is carried out in a solvent, such as an organic solvent. A base catalyst such as triethylamine is added if necessary. In the case of triethylamine, the reaction mixture formed by mixing the reactants and the solvent is quenched with water, after which the aqueous layer is extracted with an appropriate organic solvent. If desired, the organic layers are dried (e.g., using magnesium sulfate), and the organic layers are concentrated. Purification of the concentrated organic layers yields S87.
S88 is prepared from S87 by reaction of morpholine, tris(dibenzylideneacetone)dipalladium(0), 2-(di-tert-butylphosphino)-biphenyl, and potassium phosphate. The reaction mixture is diluted with a solvent, such as methylene chloride or another appropriate organic solvent, and washed with water. The aqueous layers formed by washing with water is extracted with an organic solvent, such as methylene chloride. The organic layers then are dried (e.g., over magnesium sulfate) and concentrated. The residue is purified, for example by silica gel flash chromatography, to yield S88.
S89 is prepared from S87 by reaction with benzenethiol and i-Pr2NEt in a solvent, such as CH3CN or another appropriate organic solvent. After reaction, an organic solvent such as ethyl acetate is added to the reaction mixture. If necessary, the reaction mixture is washed with one or more of acidic (e.g., HCl), basic (e.g. NaOH), and water solutions. After drying (e.g. with Na2SO4), the solution is concentrated. Purification, for example by chromatography, yields S89. In an alternative, refluxing S87 with benezethiol in an appropriate solvent such as dioxane with a catalyst such as i-Pr2NEt/Pd2(dba)3/xantphos yields S89.
S90 is prepared from S87 reacted with a base, phenylboronic acid, and a catalyst. In an embodiment, the base is K2CO3 and the catalyst is (Pd(Ph3P)4). In one embodiment, the reaction occurs in a solvent, such as an organic solvent, such as dioxane. The reaction mixture formed by mixing the reactants and the solvent is diluted with a solvent (e.g. methylene chloride), and washed with water to remove unwanted hydrophilic compounds. Concentration and purification of the residue yields S90.
S92 is prepared from S87 reacted with zinc cyanide. In an embodiment, the reaction occurs in a solvent, such as an organic solvent like DMF. A catalyst such as Pd(Ph3P)4 also is used to facilitate and hasten the reaction. The reaction mixture formed by mixing the reactants and the solvent, if necessary, is diluted with water and an acidic solution and extacted with an organic solvent. The organic extracts then are washed using a salt solution, dred, filtered, and concentrated. Purification of the residue proceeds, for example, by silica gel column chromatography.
S94 is prepared for S86 by reaction with acetic anhydride. In an embodiment, the reaction takes place in a solvent, such as an organic solvent like methylene chloride. Triethylamine or another base catalyst is added as necessary. Washing with water, followed by drying (e.g., using sodium sulfate), is used as desired. Purification of the residue yields S94.
S95 is prepared from S94 by reaction with anhydrous AlCl3, in a solvent if desired. The solvent is an organic solvent like benzene. The reaction mixture is refluxed and cooled on ice. Extraction with an organic solvent, concentration, and purification of the residue yields S95.
S96 is prepared from S86 by iodination. For example, S86 is added to a solvent, such as an organic solvent like methanol, with excess NaI and Chloramine-T. The reaction mixture is quenched with Na2S2O3 solution. Concentration and purification of the residue yields S96 as a mixture of mono-iodinated and di-iodinated products.
S97 is prepared from S86 by reaction with an nitric acid. S86 is protected (e.g., using the Boc protecting groups) and added to concentrated sulfuric acid. Nitric acid is added to the reaction mixture. The reaction mixture is cooled and neutralized (e.g., using Na2CO3) to quench the reaction. Organic extraction and subsequent concentration is used to isolate the product. Purification yields S97.
S98 is prepared by hydrogenation of S97. For example, S97 is added to a solution, such as an organic solution like methanol. H2 gas is bubbled through the solution and Pd/C catalyst or another applicable catalyst is added. Filtration to remove the catalyst and purification yields S97.
S100 is prepared from S98. S98 is dissolved in acid solution, such as aqueous HCl. To this a solution of sodium nitrite, and then NaN3 in water, are added. The reaction mixture is extracted using an organic solvent. If needed, the extract is washed with a basic solution (e.g., saturated sodium bicarbonate) and water. Organic layers from the washing are dried using, for example, anhydrous sodium sulfate, and concentrated to form a residue. The residue is purified to yield S100. To prepare S99, NaN3 is substituted with NaBF4 in a similar manner.
The compounds of the present invention are prepared in different forms, such as salts, hydrates, solvates, complexes, pro-drugs or salts of pro-drugs and the invention includes all variant forms of the compounds.
The term “compound(s) of the invention” as used herein means a compound of Formula I and salts, hydrates, prodrugs and solvates thereof.
A “pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, or pharmaceutically acceptable salts, hydrates or pro-drugs thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
A “pro-drug” refers to an agent which is converted into the parent drug in vivo. Pro-drugs are often useful because, in some situations, they are easier to administer than the parent drug. They are bioavailable, for instance, by oral administration whereas the parent drug is not. The pro-drug also has improved solubility in pharmaceutical compositions over the parent drug. For example, the compound carries protective groups which are split off by hydrolysis in body fluids, e.g., in the bloodstream, thus releasing active compound or is oxidized or reduced in body fluids to release the compound.
A compound of the present invention also can be formulated as a pharmaceutically acceptable salt, e.g., acid addition salt, and complexes thereof. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of the agent without preventing its physiological effect. Examples of useful alterations in physical properties include, but are not limited to, lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.
The term “pharmaceutically acceptable salt” means an acid addition salt which is suitable for or compatible with the treatment of a patient or a subject such as a human patient or an animal such as a dog.
The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compounds represented by Formula I or any of their intermediates. Illustrative inorganic acids which form suitable acid addition salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable acid addition salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed and such salts exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of compounds of Formula I are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g., oxalates, are used, for example, in the isolation of compounds of Formula I for laboratory use or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The compounds of Formula I of the present invention form hydrates or solvates, which are included in the scope of the claims. When the compounds of Formula I of the present invention exist as regioisomers, configurational isomers, conformers or diasteroisomeric forms all such forms and various mixtures thereof are included in the scope of Formula I. It is possible to isolate individual isomers using known separation and purification methods, if desired. For example, when a compound of Formula I of the present invention is a racemate, the racemate can be separated into the (S)-compound and (R)-compound by optical resolution. Individual optical isomers and mixtures thereof are included in the scope of Formula I.
The term “solvate” as used herein means a compound of Formula I, or a pharmaceutically acceptable salt of a compound of Formula I, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate.”
The term an “effective amount,” “sufficient amount” or “therapeutically effective amount” of an agent as used herein is that amount sufficient to effect beneficial or desired results, including clinical results and, as such, an “effective amount” depends upon the context in which it is being applied. The response is preventative and/or therapeutic. The term “effective amount” also includes that amount of the compound of Formula I which is “therapeutically effective” and which avoids or substantially attenuates undesirable side effects.
As used herein and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
The terms “animal,” “subject” and “patient” as used herein include all members of the animal kingdom including, but not limited to, mammals, animals (e.g., cats, dogs, horses, etc.) and humans.
The present invention further provides a composition, comprising radio labeled compounds of Formula I. Labeling of the compounds of Formula I is accomplished using one of a variety of different radioactive labels known in the art. The radioactive label of the present invention is, for example, a radioisotope. The radioisotope is any isotope that emits detectable radiation including, without limitation, 35S, 125I, 3H, or 14C. Radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from the radioisotope is detected using gamma imaging techniques, particularly scintigraphic imaging.
By way of example, radio-labeled compounds of Formula I are prepared as follows. A compound of Formula I is demethylated at the phenyl ring using BBr3. The resulting phenol compound then is re-methylated with a radio-labeled methylating agent (such as 3H-dimethyl sulfate) in the presence of a base (such as NaH) to provide 3H-labeled compounds.
The present invention further provides novel compounds that may be classified as 1,4-benzothiazepines, including, by way of example and without limitation, S1, S2, S3, S4, S5, S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S65, S66, S67, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, and S100.
These and any other novel compounds of the present invention are associated with a pharmaceutically acceptable carrier, as described above, so as to form a pharmaceutical composition.
In accordance with the method of the present invention, the decrease in the level of RyR-bound FKBP is limited or prevented in the subject by decreasing the level of phosphorylated RyR in the subject. In one embodiment, the amount of the agent effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject is an amount of the agent effective to treat or prevent heart failure, atrial fibrillation and/or exercise-induced cardiac arrhythmia in the subject. In another embodiment, the amount of the agent effective to limit or prevent a decrease in the level of RyR2-bound FKBP12.6 in the subject is an amount of the agent effective to prevent exercise-induced sudden cardiac death in the subject.
In view of the foregoing, the present invention further provides a method for treating or preventing exercise-induced cardiac arrhythmia in a subject, comprising administering to the subject a novel 1,4-benzothiazepine compound, as disclosed herein, in an amount effective to treat or prevent exercise-induced cardiac arrhythmia in the subject. Similarly, the present invention provides a method for preventing exercise-induced sudden cardiac death in a subject, comprising administering to the subject a novel 1,4-benzothiazepine compound, as disclosed herein, in an amount effective to prevent exercise-induced sudden cardiac death in the subject. Additionally, the present invention provides a method for treating or preventing atrial fibrillation or heart failure in a subject, comprising administering to the subject a compound, as disclosed herein, in an amount effective to treat or prevent the atrial fibrillation or heart failure in the subject. In each of these methods, the compound is selected from the group of compounds consisting of compounds of the formula:




wherein,

n is 0, 1, or 2;

R is located at one or more positions on the benzene ring;

each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R1 is selected from the group consisting of H, oxo, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R2 is selected from the group consisting of —C═O(R5), —C═S(R6), —SO2R7, —POR8R9, —(CH2)m-R10, alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl; wherein each alkyl, aryl, heteroaryl, cycloalkyl, cycloalkylalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R3 is selected from the group consisting of H, CO2Y, CONY, acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each acyl, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl; and wherein Y is selected from the group consisting of H, alkyl, aryl, cycloalkyl, and heterocyclyl;

R4 is selected from the group consisting of H, alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl; wherein each alkyl, alkenyl, aryl, cycloalkyl, and heterocyclyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R5 is selected from the group consisting of —NR16, NHNHR16, NHOH, —OR15, CONH2NHR16, CO2R15, CONR16, CH2X, acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R6 is selected from the group consisting of —OR15, NHNR16, NHOH, —NR16, CH2X, acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R7 is selected from the group consisting of —OR15, —NR16, NHNHR16, NHOH, CH2X, alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each alkyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R8 and R9 independently are selected from the group consisting of OH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl;

R10 is selected from the group consisting of NH2, OH, —SO2R11, —NHSO2R11, C═O(R12), NRC═O(R12), —OC═O(R12), and —POR13R14;

R11, R12, R13, and R14 independently are selected from the group consisting of H, OH, NH2, NHNH2, NHOH, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, —N—, —O—, —S—, —CN, —N3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

X is selected from the group consisting of halogen, CN, CO2R15, CONR16, —NR16, —OR15, —SO2R7, and —POR8R9; and

R15 and R16 independently are selected from the group consisting of H, acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, alkenyl, alkoxyl, alkyl, alkylamino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, —N—, —O—, —S—, —CN, —N3, nitro, oxo, acyl, alkenyl, alkoxyl, alkyl, alkylamino, amino, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, and hydroxyl;

and salts, hydrates, solvates, complexes, and prodrugs thereof.

Examples of such compounds include, without limitation, S1, S2, S3, S4, S5, S6, S7, S9, S11, S12, S13, S14, S19, S20, S22, S23, S36, S37, S38, S40, S43, S44, S45, S46, S47, S48, S49, S50, S51, S52, S53, S54, S55, S56, S57, S58, S59, S60, S61, S62, S63, S64, S66, S67, S69, S70, S71, S72, S73, S74, S75, S76, S77, S78, S79, S80, S81, S82, S83, S84, S85, S86, S87, S88, S89, S90, S91, S92, S93, S94, S95, S96, S97, S98, S99, and S100.
In an embodiment of the present invention, if R2 is C═O(R5) or SO2R7, then R is at positions 2, 3, or 5 on the benzene ring.
In another embodiment of the invention, if R2 is C═O(R5) or SO2R7, then each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
In another embodiment of the invention, if R2 is C═O(R5) or SO2R7, then there are at least two R groups attached to the benzene ring. Furthermore, there are at least two R groups attached to the benzene ring, and both R groups are attached at positions 2, 3, or 5 on the benzene ring. Still further, each R is independently selected from the group consisting of H, halogen, —OH, —NH2, —NO2, —CN, —N3, —SO3H, acyl, alkyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino; wherein each acyl, alkyl, alkoxyl, alkylamino, cycloalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, (hetero-)aryl, (hetero-)arylthio, and (hetero-)arylamino may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, —SH, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
In another embodiment of the invention, if R2 is C═O(R5), then R5 is selected from the group consisting of —NR16, NHNHR16, NHOH, —OR15, CONH2NHR16, CONR16, CH2X, acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl; wherein each acyl, aryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl may be substituted with one or more radicals independently selected from the group consisting of halogen, N, O, —S—, —CN, —N3, nitro, oxo, acyl, alkyl, alkoxyl, alkylamino, alkenyl, aryl, (hetero-)cycloalkyl, and (hetero-)cyclyl.
Efficacy Demonstrations
As demonstrated by FIG. 1, embodiments A, B, C, and D, S36 is more potent at increasing the binding of FKBP12.6 and RyR2 than JTV-519 and does not block the L-type Ca2+ channel (ICa,L) or HERG K+ channel (IKr). In embodiment A, PKA phosphorylated RyR2 is generated as follows: cardiac SR membrane preparations (5 μl, 50 μg) are added to a total of 100 μl of kinase buffer (8 mM MgCl2, 10 mM EGTA, 50 mM Tris-PIPES, pH 6.8) containing 100 μM MgATP and 40 units of PKA, and incubated at room temperature. Samples are centrifuged at 95,000 g for 10 min and the pellets are washed three times in 0.2 ml imidazole buffer. The final pellets are pooled and resuspended in imidazole buffer (final concentration 10 μg/μl). To test for the FKBP12.6 rebinding efficiency of JTV-519, PKA phosphorylated cardiac SR (50 mg) is incubated for 30 minutes at room temperature with the test compounds and 250 nM FKBP12.6 in 10 mM imidizol buffer, pH 7.0. Samples then are centrifuged at 100,000 g for 10 minutes and pellets washed 3 times with imidizol buffer. After washing, proteins are size-fractionated on 15% PAGE. Immunoblots are developed using an anti-FKBP antibody (1:3,000 dilution). The amount of rebinding is quantified using densitometry of Western blots and is compared to the amount of FKBP associated with RyR in non-phosphorylated SR. EC50's for the compounds are determined by generating FKBP binding data using concentrations of compounds ranging from 0.5-1000 nM. In embodiment B, currents through L-type Ca2+ channels in isolated mouse cardiomyocytes are recorded using whole-cell patch clamp recording conditions with Ba2+ as the charge carrier. The extracellular solution contains (in mM): N-methyl-D-glucamine, 125; BaCl2, 20; CsCl, 5; MgCl2, 1; HEPES, 10; glucose, 5; pH 7.4 (HCl). The intracellular solution contains (in mM): CsCl, 60; CaCl2, 1; EGTA, 11; MgCl2, 1; K2ATP, 5; HEPES, 10; aspartic acid, 50; pH 7.4 (CsOH). Under these conditions, it is expected that the measured current was carried by Ba2+ primarily through L-type calcium channels which is referred to as ICa,L. Drugs are applied by a local solution changer and reach the cell membrane within 1 s. The effects of nifedipine and S36 are tested with 20 ms long voltage-clamp steps to +10 or +20 mV (peak of current-voltage relation for each individual cell) from holding potentials of −80 mV or −40 mV. In embodiment C, the voltage-dependence of L-type Ca2+ current blocked by JTV-519 (1 μM) and S36 (1 μM) are measured and presented.
As demonstrated by FIG. 2, embodiments A, B, C, and D, S36 prevents exercise-induced sudden cardiac death at lower plasma levels compared with JTV-519. In embodiment A are shown representative ECGs of an untreated FKBP12.6± mouse and JTV519-treated FKBP12.6+/− and FKBP12.6−/− mice. Mice are treated with 0.5 mg JTV-519/per kilogram of body weight per hour for 7 days with an implanted osmotic mini-pump. JTV-519 has no effect on resting heart rate or other ECG parameters such as heart rate (HR). In embodiment B are shown sustained polymorphic ventricular tachycardia recorded by telemetry in an untreated FKBP12.6+/− mouse (upper tracing) subjected to exercise testing, immediately followed by injection with 0.5 mg epinephrine per kilogram of body weight. Representative telemetry ECG recording of a JTV-519-treated FKBP12.6+/− mouse following the same protocol is shown in the bottom tracing. In embodiment C are shown numbers of mice with cardiac death (left), sustained VTs (>10 beats, middle), and nonsustained VTs (3 to 10 arrhythmogenic beats, right) in experimental groups of mice subjected to exercise testing and injection with 0.5 mg/kg epinephrine. In embodiment D, the dose-dependence of pharmacological effects of JTV-519 and S36 is shown. Plasma levels of 1 μM JTV519 prevent cardiac arrhythmias and sudden cardiac death in FKBP12.6+/− mice. Plasma levels of 1 μM and 0.02 μM S36 also prevent cardiac arrhythmias and sudden cardiac death in FKBP12.6+/− mice.
As demonstrated by FIG. 3, S36 prevents the development of acute heart failure post-myocardial infarction. Placebo-treated or S36 (100 nM or 200 nM plasma concentrations)-treated mice are subjected to permanent ligation of the left anterior descending coronary artery resulting in myocardial infarction. S36 significantly improves fractional shortening assessed by M-mode echocardiography 2 weeks post-myocardial infarction, compared with placebo.
As demonstrated by FIG. 4, S36 improves cardiac function in chronic heart failure post-myocardial infarction. Wild-type mice are subjected to permanent ligation of the left anterior descending coronary artery resulting in myocardial infarction. Seven days following myocardial infartion, mice are treated with S36 (plasma concentration 200 nM) or placebo: Heart weight to body weight (HW/BW) ratios and pressure-volume loops quantifications (dP/dt, slope of the maximum derivative of change in systolic pressure over time) show reverse remodeling and improved cardiac contractility in S36-treated mice compared with placebo.
FIG. 5 is a summary graph of EC50 values of JTV-519 and compounds disclosed herein. The FKBP12.6 rebinding assay described above is used to determine the amount of FKBP12.6 binding to PKA-phosphorylated RyR2 at various concentrations (0.5-1000 nM) of the compounds shown. EC50 values are calculated using Michaelis-Menten curve fitting.
As demonstrated by FIG. 6, embodiments A, B, and C, S36 normalizes CPVT-associated RyR2-P2328S channel function and structure. In embodiment A are shown representative single-channel current traces of unphosphorylated RyR2-P2328S and PKA-phosphorylated RyR2-WT treated with S36 showing no influence of JTV-519 on baseline channel function. However, in PKA phosphorylated RyR2-P2328S, as shown in embodiment B, S36 normalizes the single channel closed state to levels approaching those seen in embodiment A, reducing the open probability from 14.4% to 0.3% following administration of 0.1 μmol/L S36. Insets in embodiments A and B show channel openings >1 pA at a higher resolution. Embodiment C shows immunoblot analysis of calstabin-2 binding of RyR2-P2328S in the presence or absence of PKA and 0.1 mol/L S36, as indicated. RyR2-P2328S is immunoprecipitated and in vitro PKA was phosphorylated as described above.
As demonstrated by FIG. 7, embodiments A and B, treatment with JTV-519 reduces PKA phosphorylation of RyR2 in mice with heart failure. Equivalent amounts of RyR2 are immunoprecipitated with an antibody against RyR2 (top blot). Representative immunoblots (embodiment A) and bar graphs (embodiment B) show the amount of PKA phosphorylated RyR2 at Ser-2808 bound to RyR2 in wild-type and calstabin2(FKBP12.6)−/− mice. Treatment with JTV-519 (0.5 mg/kg/h) for 28 days post-myocardial infarction reduces PKA-phosphorylation of RyR2, presumably due to reverse cardiac remodeling, in wildtype but not calstabin-2 (FKBP12.6)−/− mice.
As demonstrated by FIG. 8, embodiments A and B, mice in which cardiac RyR2 cannot be PKA phosphorylated (RyR2-S2808A knockin mice) have improved cardiac function following myocardial infarction. Shown in embodiment A is the quantification of M-mode echocardiograms showing improved ejection fraction in RyR2-S2808A knockin mice compared with wildtype 28 days following permanent coronary artery ligation. Shown in embodiments B and C are pressure-volume loop quantifications showing (embodiment B) improved cardiac contractility and decreased cardiac dilation (embodiment C) in RyR2-S2808A knockin mice compared with wildtype following myocardial infarction.
FIG. 9, embodiments A, B, C, and D demonstrate the effect of JTV-519 on calstabin2 affinity to RyR2 in haploinsufficient calstabin2 (FKBP12.6)+/− mice after exercise. The average probability that RyR2 channels will be open in calstabin2+/− mice subjected to exercise were significantly increased compared to those of channels from exercised wild type (control; calstabin2+/+) mice, which are predominately closed under conditions that simulate diastole in the heart. As shown in embodiments C and D, treatment of exercised calstabin2+/− mice with JTV-519 significantly reduced the channel open probability (Po) compared with that of channels from exercised mice that were not treated, consistent with increased amounts of calstabin2 in the RyR2 channel complex. Therefore, JTV-519 increases the binding affinity of calstabin2 to RyR2. In contrast, JTV-519 treatment of exercised Calstabin2−/− deficient mice did not result in channels with a low Po, indicating that the presence of calstabin2 is necessary for JTV-519 effects which are documented as binding of calstabin2 to RyR2.
FIG. 10, embodiments A, B, C, D, E, and F demonstrate, respectively, normalized RyR2 channel gating and increased calstabin2 binding to RyR2 channels after treatment with JTV-519. The immunoblots in embodiments A and B show the amounts of RyR2 and calstabin2 associated with immunoprecipitated RyR2 after incubation with the indicated concentrations of JTV-519 for, respectively, wild-type RyR2 (RyR2-WT) channels and RyR2-S2809D. The binding curves in embodiment C demonstrate that JTV-519 significantly increases the affinity of calstabin2 for PKA-phosphorylated RyR2 channels. The results also show that depletion of calstabin2 from the RyR2 macromolecular complex which is associated with increased RyR2 open probability, ventricular tachycardias, and sudden cardiac death in haploinsufficient calstabin2+/− mice is reversed by treatment with JTV-519. Therefore, the JTV-519 and related compounds prevent disorders and conditions associated with the RyR2 receptors.
As shown in FIG. 11, embodiments A, B, C, D, and E RyR1 channel functioning is increased and normalized in mdx (dystrophin deficient) mice treated with JTV-519. In FIG. 11, channel openings are represented as upward deflections; ‘c’ indicates the closed state; and 4 pA current open amplitude is indicated by dash. Upper traces represent 5 secs and lower traces 500 ms; dotted lines indicate subconductance states.
Embodiment A of FIG. 11 shows a single channel current trace of RyR1 from soleus muscle of control (wild-type) mouse under resting conditions (cytoplasmic Ca2+ 150 nM). As seen, RyR1 is predominantly closed. Embodiment C of FIG. 11 shows that RyR1 channel function in mdx mouse shows significantly increased open probability, increased average open, and decreased average closed dwell times, To and Tc, respectively. Increased Po in mdx mice is consistent with intracellular Ca2+ leak. The amplitude histograms in embodiments B, D, and F show multiple subconductance states consistent with calstabin1 (FKBP12) depletion in RyR1 from mdx soleus muscle. Embodiment E of FIG. 11 shows an mdx mouse treated with 1.0 μM JTV-519. As seen, the RyR1 channels JTV-519-treated mouse demonstrates normal activity which is not significantly different from untreated wild-type trace, thereby indicating that JTV-519 can normalize RyR1 channel function in mdx mice.
The data of FIG. 11 are consistent with intracellular SR Ca2+ leak via RyR1 channels as the cause of increased cytosolic Ca2+ leak in skeletal muscles from mdx (dystrophin deficient) mice.
FIG. 12, embodiments A and B, demonstrate that mdx skeletal muscle has normal levels of RyR1 PKA phosphorylation, but depleted levels of calstabin1. The immunoblots in embodiment A show that mdx type mice have depeleted levels of calstabin 1 compared to a control (wild-type) mouse. The summary bar graphs of embodiment B show that the mdx mouse, nevertheless, has an equivalent level of PKA-phosphorylation. Therefore, it is concluded that calstabin1 depeletion is a defect that is consistent with the intracellular Ca2+ leak observed in skeletal muscle cells from mdx mice and myofibers from human mutation carriers. Intracellular SR Ca2+ leak is likely to contribute to myofiber death and wasting of muscle mass by toxic intracellular Ca2+ overload and activation of proteases.
FIG. 13, embodiments A, B, and C, demonstrates that SR Ca2+ leak at the subcellular level in skeletal muscles of animals with heart failure is detectable. Life quality and prognosis in heart failure (HF) patients is severely decreased due to skeletal muscle dysfunction (e.g., shortness of breath due to diaphragmatic weakness, and exercise intolerance due to limb skeletal muscle fatigue) in addition to depressed cardiac function. Dysregulation of intracellular SR Ca2+ release is a pathogenic mechanism underlying skeletal muscle dysfunction in HF. HF in animals causes significantly accelerates intrinsic skeletal muscle fatigue.
Embodiments A and B of FIG. 13 are ΔF/F fluorescence line scan images of representative examples of Ca2+ sparks in myofibers from sham and postmyocardial infarction (PMI) rats and corresponding Ca2+ spark time course. Embodiment C shows the relative distribution of the spatio-temporal properties of the Ca2+ sparks. Charts indicate 25, 50, 75 percentiles, the horizontal lines indicate the range from 1-99% of the distribution. Sham, open symbols (n=137, three animals); postmyocardial infarction (PMI), gray symbols (n=82, two animals). *, P<0.05. FDHM, full duration at 50% peak amplitude; FWHM, full width at 50% peak amplitude.
FIG. 14, embodiments A and B, demonstrate that treatment of wild-type mice with JTV-519 improved soleus muscle fatigue times compared with placebo. Soleus muscles from JTV519-treated wild-type mice or calstabin2−/− mice with heart failure from myocardial infarction are more resistant to fatigue (P<0.05) compared to placebo-treated mice. After completion of treatment, soleus muscle was dissected and mounted in a tissue bath to assess isolated skeletal muscle function. Representative 50% of maximal tetanic force fatigue time tracings are shown for wild-type and calstabin2−/− mice, treated with JTV519 or placebo, in embodiment A. In embodiment B are shown bar graph summarizing mean time to fatigue.
In summary, JTV-519 treatment improved skeletal muscle fatigability in heart failure animals in vivo. Interestingly, in calstabin2−/− knockout mice, fatigue times were also significantly improved in JTV519 treated mice, suggesting that the beneficial effects on isolated skeletal muscle function depend on calstabin1 and not calstabin2 binding to RyR1. Indeed, calstabin1 appears to be the only isoform of functional significance expressed in skeletal muscles.
FIG. 15, embodiments A and B, demonstrates that in a mouse model of post-myocardial infarction heart failure, RyR1 in soleus muscle is also PKA-hyperphosphorylated. Both in wild-type and calstabin2−/− mice, JTV-519 increased binding of calstabin1 to RyR1 in soleus muscle, suggesting that JTV-519 improves skeletal muscle fatigability by normalizing calstabin1 rebinding to the channel complex. Equivalent amounts of RyR1 were immunoprecipitated with an antibody against RyR1. Embodiment A are bar graphs show the amount of PKA phosphorylation of RyR1 at Ser-2844 in mice (corresponding to human Ser-2843). The significant decrease of RyR1 PKA phosphorylation in JTV519 treated wild-type animals is likely resulting from beneficial cardiac effects and secondary reduction of sympathetic nervous activity. Embodiment B are bar graphs showing the amount of calstabin1 bound to RyR1 from wild-type or calstabin2−/− mice treated with JTV519 or placebo. Mice were treated with JTV519 by implantable osmotic minipumps using a dose of 0.5 mg/kg/day. In summary, JTV-519 treatment resulted in a highly significant increase of calstabin1 in the RyR1 complex in soleus muscles in vivo.
FIG. 16, embodiments A, B, C, and D demonstrates that calstabin1 rebinding to RyR1 by JTV519 normalizes abnormal or leaky RyR1 channel function in vivo. In embodiments A and C are shown RyR1 single-channel tracings at 150 nM cytoplasmic Ca2+ representing resting conditions in skeletal muscle for wild-type mice treated with placebo and JTV-519. JTV-519 treatment of mice with heart failure and increased muscle fatigue normalized RyR1 channel gating in skeletal muscle in vivo. Channel openings are upward, the dash indicates the full level of channel opening (4 pA), the dotted lines indicate subconductance levels, and ‘c’ indicates the closed state of the channels. For the amplitude histograms in embodiment B and D, amplitude is represented on the x axis, and events indicates the number of channel openings. Po, To and Tc values correspond to the representative traces. Treatment as indicated on top of traces. Inset shows higher resolution of open states.
In summary, the data shows that in vivo JTV519 treatment normalizes skeletal muscle function and RyR1 channel dysfunction consistent with preventing intracellular SR Ca2+ leak as a cause of increased skeletal muscle fatigue.
FIG. 17 demonstrate that JTV-519 also increases the binding affinity of calstabin1 for RyR1 in skeletal muscle in vivo. This probably explains why JTV519 treated mice with heart failure have increased levels of calstabin1 bound to RyR1 in soleus muscle. In embodiment A, equivalent amounts of skeletal RyR1 or cardiac RyR2 were immunoprecipitated, PKA phosphorylated and incubated with calstabin1 or calstabin2 at increasing concentrations of JTV519, respectively. Sediment represents only calstabin bound to RyR. Immunoblotting showed that ≧50 nM of JTV519 increased the binding affinity of calstabin for RyR. The graphs of embodiment B further demonstrate that PKA phosphorylation of RyR1 reduced the affinity of calstabin1 for RyR1 (open circles), whereas treatment with JTV519 (filled circles) restored the binding affinity of calstabin1 for RyR1 to that of non-PKA phosphorylated RyR1 (open squares).
FIG. 18, embodiment A, B, C, and D demonstrates that Ser-2843 is the unique PKA phosphorylation site in skeletal RyR1 channels. (A) Representative single channel traces of wild-type RyR1, (B) effect of exogenous PKA phosphorylation of RyR1 (wt RyR1-P), (C) PKA does not affect RyR1-S2843A which contains a non-functional PKA phosphorylation site. Since PKA does not increase RyR1-S2843A activity, Ser-2843 appears to constitute the unique PKA phosphorylation site in RyR1 channels in skeletal muscle. Accordingly, (D) constitutively phosphorylated RyR1-S2843D mimics exogenous PKA phosphorylation shown in (B) confirming that Ser-2843 is the unique PKA phosphorylation site in skeletal RyR1 channels. RyR1 single channel recordings in planar lipid bilayers show activity of the channels at 150 nM [Ca2+]cis, (cytosolic side) with 1 mM ATP. Recordings were at 0 mV, closed state of the channels as indicated by ‘c’, and channel openings are upward deflections. All point amplitude histograms are shown on the right. Open probability (Po) and mean closed (Tc) and open (To) dwell times are indicated above each channel tracing.
FIG. 19, embodiments A and B, demonstrates the depletion of stabilizing calstabin1 and PKA hyperphosphorylation of RyR1 channels from sustained exercise. Aerobic exercise can be defined as a form of physical exercise that increases the heart rate and enhances oxygen intake to improve performance. Examples of aerobic exercise are running, cycling, and swimming. During the study of FIG. 19, mice were challenged by aerobic exercise (forced swimming) for 90 mins twice daily. The animals were accustomed to swimming in preliminary training sessions: day-3 twice 30 mins, day-2 twice 45 mins, day-1 twice 60 mins, day 0 and following twice 90 mins. Mice were then exercised for 1, 7, or 21 additional, consecutive days for 90 mins twice daily. Between swimming sessions separated by a 4 hour rest period the mice are kept warm and given food and water. An adjustable-current water pool was used to exercise mice by swimming. An acrylic pool (90 cm long×45 cm wide×45 cm deep) filled with water to a depth of 25 cm was used. A current in the pool was generated with a pump. The current speed during the swimming session was at a constant speed of 1 l/min flow rate. The water temperature was maintained at 34° C. with an electric heater. Age- and weight-matched mice were used to exclude differences in buoyancy from body fat.
Using forced swimming as an efficient protocol to increase skeletal muscle aerobic capacity in mice, the composition and phosphorylation status of the skeletal RyR1 channel complex have been investigated. Unexpectedly, after 3 weeks of 90 mins swimming twice daily, C57B16 wild-type mice showed significantly increased RyR1 phosphorylation by PKA while Ca2+-calmodulin kinase II (CaMKII) phosphorylation was not changed indicating specificity of the stress pathway RyR1 protein expression was stable, however, RyR1 channels were depleted of the stabilizing subunit calstabin1 (FKBP12). It has been shown that RyR1 hyperphosphorylation and calstabin1 depletion are consistent with leaky RyR1 channels that cause intracellular SR Ca2+ leak.
RyR1 channels are PKA hyperphosphorylated and depleted of the stabilizing calstabin1 subunit after 3 weeks of 90 mins swimming twice daily. As seen in Embodiment A, the immunoprecipitated RyR1 macromolecular channel complex shows increased PKA phosphorylation at Ser-2844 (corresponding to human RyR1-Ser-2843) whereas CaMKII phosphorylation at Ser-2849 (corresponding to human RyR1-Ser-2848) is unchanged. Concomitant with increased RyR1-Ser-2844 PKA hyperphosphorylation, calstabin1 is depleted from the channel complex. As seen in embodiment B, normalization of phosphorylation and calstabin1 content to four subunits of the tetrameric channel complex shows a significant in increase in PKA phosphorylation and depletion of the stabilizing calstabin1 subunit. Control, non-exercised mice; swim, mice exercised 90 mins twice daily for 3 weeks (preliminary data). P<0.05.
FIG. 20, embodiments A and B demonstrate that PKA phosphorylation increases for increasing durations of sustained exercise. To investigate the influence of the duration of sustained exercise on the RyR1 Ca2+ release channel defect, mice were exposed to swimming for 1, 7, or 21 days followed by immediate sacrifice. Longer exposure to sustained exercise results in a significant increase of RyR1 PKA hyperphosphorylation beginning at 7 days and saturating at 21 days.
In FIG. 20, embodiment A, the immunoprecipitated RyR1 channel complex shows significantly and above physiologic levels increased PKA phosphorylation at Ser-2844 (corresponding to human RyR1-Ser-2843) after 7 days of swimming exercise. In FIG. 20, embodiment B, normalization of RyR2-Ser-2844 phosphorylation within the tetrameric channel complex documents a significant increase in PKA phosphorylation. *, P<0.05; **, P<0.005.
In summary, the data of FIG. 20 shows that sustained exercise results in significantly increased RyR1 phosphorylation by protein kinase A (PKA) which contributes to depletion of the stabilizing calstabin1 subunit from the channel complex as the cause of a gain-of-function defect.
FIG. 21 provides data showing that showing that chronically increased sympathetic stimulation of skeletal muscles, results in RyR1-dependent intracellular Ca2+ leak and significantly increased muscle fatigue. What is the functional consequence of chronically increased RyR1 PKA hyperphosphorylation? As shown in FIG. 21 for mice and rats with heart failure from myocardial infarction, chronic RyR1 PKA hyperphosphorylation results in increased muscle fatigue.
In embodiment A, it can be seen that heart failure skeletal muscle fatigues earlier than control. Rat soleus muscle (n=5 control, n=8 HF) was mounted in a tissue bath to assess contractile function. Representative fatigue time tracing is shown for control and HF skeletal muscles. Bar graph shows mean (±S.D.) time to 40% fatigue. *, P<0.05. In embodiment B, it can be seen that heart failure skeletal muscle achieved maximal tetanic force more slowly than control skeletal muscles. Tetanic force was induced by high-frequency field stimulation. Bar graph shows tetanic 50% contraction time. **, P<0.01. Embodiment C demonstrates the correlation between time to fatigue and RyR1 PKA phosphorylation (r=0.88) in rat skeletal muscle from sham and heart failure animals. Muscle function and RyR1 PKA phosphorylation were assessed using contralateral soleus muscles from each animal.
In summary, FIG. 21 provides data showing that sustained exercise causes RyR1 PKA hyperphosphorylation and calstabin1 depletion, and FIG. 21 shows that the identical defect occurs in disease forms with increased sympathetic activity causing intracellular SR Ca2+ leak and significantly accelerated skeletal muscle fatigue.
An additional problem during sustained exercise and stress is skeletal muscle degeneration further contributing to decreased skeletal muscle performance. To assess structural changes during sustained exercise, histologic changes in the fast-twitch muscles of mice exposed to 3 weeks of exercise by swimming have been characterized. Results are shown in FIG. 22. Cross-sections of the mouse M. extensor digitorum longus (EDL) showed histologic changes consistent with myofiber degeneration from intracellular Ca2+ overload from defective RyR1 channels. Therefore sustained exercise for 90 mins twice daily triggers a dystrophic phenotype in EDL muscles of normal C57B16 mice.
Trichrome stain shows packed myofibers of similar cross-sectional dimension in non-exercised control (WT) mice (left). Three weeks swimming results in myofiber degeneration and interstitial collagen deposits with irregular fiber sizes. Hematoxylin-eosin (H&E) stain indicates nuclear changes and myofiber death. These changes are consistent with dystrophic remodeling.
The present invention is described in the following Examples, which are set forth to aid in the understanding of the invention and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
High-Throughput Screening Method
In addition to the compounds disclosed herein, other compounds can be discovered that are capable of modulating calcium ion channel activity, in particular those channels related to the RyR series of caclium ion channels. Provided herein is a highly-efficient assay for high-throughput screening of other compounds that are capable of modulating calcium ion channel activity.
By way of example, and as shown in Example 5 below, a highly-efficient assay for high-throughput screening for small molecules is developed by immobilizing FKBP, either FKBP12 or FKBP12.6 (e.g., wild-type FKBP12.6 or a fusion protein, such as GST-FKBP12.6) onto a 96-well plate coated with glutathione, using standard procedures. PKA-phosphorylated ryanodine receptor (RyR), specifically RyR1 or RyR3 in the case of FKBP12 and RyR2 in the case of FKBP12.6, is loaded onto the FKBP-coated plate, and incubated with compounds at various concentrations (10-100 nM) for 30 min. Thereafter, the plate is washed to remove the unbound RyR, and then incubated with anti-RyR antibody (e.g., for 30 min). The plate is washed again to remove unbound anti-RyR antibody, and then treated with florescent-labeled secondary antibody. The plate is read by an automatic fluorescent plate reader for binding activity.
Alternatively, RyR is PKA-phosphorylated in the presence of 32P-ATP. Radioactive PKA-phosphorylated RyR is loaded onto an FKBP-coated, 96-well plate, in the presence of JTV-519 analogues and other compounds at various concentrations (10-100 nM) for 30 min. The plate is washed to remove the unbound radiolabeled RyR, and then read by an automatic plate reader. PKA-phosphorylated RyR also is coated to the plate, and incubated with 32S-labeled FKBP in the presence of the compounds.
EXAMPLES
Example 1
RyR2 PKA Phosphorylation and FKBP12.6 Binding
Cardiac SR membranes are prepared, as previously described (Marx, et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000; Kaftan, et al., Effects of rapamycin on ryanodine receptor/Ca(2+)-release channels from cardiac muscle. Circ. Res., 78:990-97, 1996). 35S-labelled FKBP12.6 was generated using the TNTTM Quick Coupled Transcription/Translation system from Promega (Madison, Wis.). [3H] ryanodine binding is used to quantify RyR2 levels. 100 μg of microsomes are diluted in 100 μl of 10-mM imidazole buffer (pH 6.8), incubated with 250-nM (final concentration) [35S]-FKBP12.6 at 37° C. for 60 min, then quenched with 500 μl of ice-cold imidazole buffer. Samples are centrifuged at 100,000 g for 10 min and washed three times in imidazole buffer. The amount of bound [35S]-FKBP12.6 is determined by liquid scintillation counting of the pellet.
Example 2
Immunoblots
Immunoblotting of microsomes (50 μg) is performed as described, with anti-FKBP12/12.6 (1:1,000), anti-RyR-5029 (1:3,000) (Jayaraman, et al., FK506 binding protein associated with the calcium release channel (ryanodine receptor). J. Biol. Chem., 267:9474-77, 1992), or anti-phosphoRyR2-P2809 (1:5,000) for 1 h at room temperature (Reiken, et al., Beta-blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation, 107:2459-66, 2003). The P2809-phosphoepitope-specific anti-RyR2 antibody is an affinity-purified polyclonal rabbit antibody, custom-made by Zymed Laboratories (San Francisco, Calif.) using the peptide, CRTRRI-(pS)-QTSQ, which corresponds to RyR2 PKA-phosphorylated at Ser2809. After incubation with HRP-labeled anti-rabbit IgG (1:5,000 dilution; Transduction Laboratories, Lexington, Ky.), the blots are developed using ECL (Amersham Pharmacia, Piscataway, N.J.).
Example 3
Single-Channel Recordings
Single-channel recordings of native RyR2 from mouse hearts, or recombinant RyR2, are acquired under voltage-clamp conditions at 0 mV, as previously described (Marx, et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell, 101:365-76, 2000). Symmetric solutions used for channel recordings are: trans compartment—HEPES, 250 mmol/L; Ba(OH)2, 53 mmol/L (in some experiments, Ba(OH)2 is replaced by Ca(OH)2); pH 7.35; and cis compartment—HEPES, 250 mmol/L; Tris-base, 125 mmol/L; EGTA, 1.0 mmol/L; and CaCl2, 0.5 mmol/L; pH 7.35. Unless otherwise indicated, single-channels recordings are made in the presence of 150-nM [Ca2+] and 1.0-mM [Mg2+] in the cis compartment. Ryanodine (5 mM) is applied to the cis compartment to confirm identity of all channels. Data is analyzed from digitized current recordings using Fetchan software (Axon Instruments, Union City, Calif.). All data is expressed as mean±SE. The unpaired Student's t-testis used for statistical comparison of mean values between experiments. A value of p<0.05 is considered statistically significant.
Example 4
Novel Compounds and Methods for Their Synthesis




Synthon S26 was prepared according to methods described in U.S. patent application Ser. No. 10/680,988.
Synthesis of S3 (Scheme 1): To a stirred solution of vinylsulfonic acid (22 mg, 0.2 mmol) in anhydrous CH2Cl2 (5 ml) is added thionyl chloride (2M in CH2Cl2, 0.1 ml, 0.2 mmol) The reaction mixture is stirred at room temperature overnight and evaporated under vacuum. The residue is dissolved in CH2Cl2 (5 ml). To this solution, a solution of S26 (20 mg, 0.1 mmol) in CH2Cl2 (3 ml) is added drop-wise at 0° C. The reaction mixture is stirred at 0° C. for one hour and at room temperature for another hour and washed with saturated sodium bicarbonate and 1N HCl. After removal of the solvent, the product S3 is purified by SiO2 column chromatography as a colorless oil (18 mg, 65%).
Synthesis of S4 (Scheme 1): To a stirred solution of S26 (20 mg, 0.1 mmol) in CH2Cl2 (5 ml) is added methylsulfonyl chloride (26 mg, 0.2 mmol) and triethylamine (30 mg, 0.3 mmol) at 0° C. The resulting mixture is stirred at 0° C. for one hour and at room temperature overnight. The organic phase is washed with aqueous saturated sodium bicarbonate and dried over sodium sulfate. After filtration and evaporation of the organic solvents, the product S4 is purified by SiO2 column chromatography (25 mg oil, yield: 90%). Similarly, S5 and S54 are synthesized in 95% and 91% yields respectively.




Synthesis of S1 and S2 (Scheme 2): To a solution of S3 (28 mg, 0.1 mmol) in chloroform (5 ml) is added 4-benzylpiperidine (18 mg, 0.1 mmol). The resulting mixture is stirred at room temperature for 1 day. After removal of organic solvent, the residue is purified on silica gel column. Product S1 is obtained as a colorless oil (34 mg, yield 75%). S2 is synthesized similarly from S3 and dibutylamine in 78% yield.




Synthesis of S7, S9, S27 and S40 (Scheme 3): To a stirred solution of iodoacetic acid (37 mg, 0.2 mmol) in CH2Cl2 (10 ml) is added thionyl chloride (2 M solution in CH2Cl2, 0.1 ml, 0.2 mmol). The resulting mixture is stirred at 0° C. for one hour and at room temperature overnight. After removal of solvent, the crude acid chloride is added to a stirred solution of S26 (20 mg, 0.1 mmol) and triethylamine (30 mg, 0.3 mmol) in CH2Cl2 (10 ml) at 0° C. The mixture is stirred at 0° C. for one hour and at room temperature overnight. The organic phase is washed with saturated sodium bicarbonate and 1N HCl. The crude product is purified by column chromatography to give S7 as a colorless oil (34 mg, yield, 95%). Similarly, S9 is synthesized in 95% yield; synthon S27 is synthesized in 96% yield; and S40 is synthesized in 91% yield using N-hydroxysuccinimidyl 4-azidosalicylic acid (NHS-ASA).




Synthesis of S11 and S12 (Scheme 4): To a solution of S26 (20 mg, 0.1 mmol) in pyridine (1 ml) is added phenyl isocyanate (18 mg, 0.15 mmol). The resulting mixture is stirred at room temperature for 24 hours. Then ethyl acetate (10 ml) is added and the organic phase is washed with 1N HCl and saturated sodium bicarbonate. The product S11 is purified by SiO2 column chromatography as a white solid (27 mg, yield: 86%). Similarly, S12 is synthesized from S26 and phenyl isothiocyanate in 85% yield.




Synthesis of S13 and S14 (Scheme 5): To S26 (20 mg, 0.1 mmol) in CH2Cl2 (5 ml) is added triethylamine (30 mg, 0.3 mmol) and phenyl methoxyphosphonyl chloride (38 mg, 0.2 mmol) at 0° C. After stirring for 2 hours at room temperature, the reaction mixture is washed with saturated sodium bicarbonate. Isomers are separated and purified by silica gel column to yield S13 (14 mg, yield: 40%) and S14 (16 mg, yield: 45%).




Synthesis of S19 (Scheme 6): To a stirred solution of S26 (20 mg, 0.1 mmol) and triethylamine (30 mg, 0.3 mmol) in CH2Cl2 (5 ml) is added 1,4-butyldiacid chloride (8 mg, 0.05 mmol) at 0° C. The resulting mixture is stirred at 0° C. for one hour and at room temperature overnight. The organic phase is washed with saturated sodium bicarbonate and 1N HCl and water. After removal of solvent, product S19 is purified by column chromatography (oil, 19 mg, 80% yield). Similarly S22 is prepared from 2,6 pyridyl dicarboxylic acid dichloride.




Synthesis of S20 and S23 (Scheme 7): S27 (25 mg, 0.1 mmol) in MeOH (5 ml) is treated with H2O2 (30%, 0.5 ml) at room temperature for 1 day. After treatment with sodium thiosulfate solution, methanol is removed by evaporation. The resulting residue is dissolved in ethyl acetate (10 ml) and washed with saturated sodium carbonate. After drying over sodium sulfate, solvent is evaporated to provide a crude product which is purified by silica gel column chromatography to yield S20 as colorless oil (16 mg, 60% yield). Similarly, S23 is synthesized from S10.




Synthesis of S36 and S57 (Scheme 8): To a stirred solution of S26 (0.85 g, 4.4 mmol) and pyridine (0.70 g, 8.8 mmol) in CH2Cl2 (50 ml) at 0° C. is added drop-wise methyl chlorooxoacetate (0.81 g, 6.6 mmol). The reaction mixture is stirred at 0° C. for 2 hours then washed with saturated sodium bicarbonate, 1N HCl, and water. Silica gel column chromatography provides S57 as a white solid (1.1 g, 90% yield). S57 (1.1 g, 3.9 mmol) is dissolved in methanol (10 ml) and then a solution of sodium hydroxide (0.3 g, 7.5 mmol) in water (10 ml) is added. The reaction mixture is stirred at room temperature for one hour. After solvent is removed, the residue is dissolved in water (10 ml) and washed with ether (2×10 ml). The aqueous phase is acidified with 1N HCl to pH=2. The product is extracted with CH2Cl2 (2×10 ml). Removal of solvent yields product S36 as a white solid (1.0 g, yield 100%). The product can be further purified by recrystalization. S38 is similarly synthesized (see Structure List).
Synthesis of S43, S44, S45 and S59 (Scheme 8): S36 (150 mg, 0.56 mmol) is treated w thionyl chloride (5 ml) at room temperature overnight. After removal of the excess thionyl chloride, the crude product S36-Cl is dissolved in CH2Cl2 (10 ml) and, to this solution, mono-Boc protected cystamine and pyridine (0.2 ml, 196 mg, 2.48 mmol) are added at 0° C. The reaction mixture is stirred at 0° C. for one hour and at room temperature overnight and quenched with saturated sodium bicarbonate. The organic phase is separated and the solvent is removed to give intermediate S36-cystamine which is purified by SiO2 column chromatography in 80% yield. Deprotection of the Boc-group is achieved with trifluoroacetic acid in CH2Cl2, and the deprotected S36-cystamine is used for the synthesis of S43 and S45 by reaction with NHS-activated ester of azido compounds. Yield is 75% for S43 and 80% for S45.
S44 is synthesized as a by-product of the following reaction: S36 (50 mg, 0.19 mmol) is treated with thionyl chloride (2 ml) at room temperature overnight. After removal of the excess of thionyl chloride, the crude product is dissolved in CH2Cl2 (5 ml). To this solution, cystamine (134 mg, 0.88 mmol) and pyridine (98 mg, 1.23 mmol) in CH2Cl2 (10 ml) is added and the reaction mixture is stirred at room temperature overnight. S44 is purified by column as a white solid (20 mg, 16%). Similarly, S57 and S59 are synthesized by reaction of S36-Cl with methanol or ethylamine (Scheme 8).




Synthesis of urea-based analogs S6, S46-53, S64, S66, S67 (Scheme 9). S26 (195 mg, 1.0 mmol) in CH2Cl2 (20 ml) is added 4-nitrophenyl chloroformate (220 mg, 1.1 mmol) and triethylamine (120 mg, 1.2 mmol) at 0° C. The reaction mixture is stirred for 2 hours at room temperature and washed with water. Removal of the solvents, followed by purification using column chromatography provides compound S37 (330 mg, 91%). Reaction of S37 (36 mg, 0.1 mmole) with one equivalent of amine in DMF (3 ml) overnight provides urea-based compounds in >60% yield after purification by SiO2 column chromatography. Alternatively, the urea-based compounds can be synthesized through a versatile and more reactive intermediate S26-phosgene shown in Scheme 9.




Synthesis of S55, S56, S58, S60-S63 (Scheme 10): The reaction mixture of S27 (25 mg, 0.1 mmol) and 4-(4-aminobenzyl)piperidine (19 mg, 0.1 mmol) in chloroform (5 ml) is stirred at room temperature for 2 days. After removal of solvent, the product S60 is purified by silica gel column chromatography as a white solid (36 mg, yield 90%). S55, S56, S58, and S61-S63 similarly are synthesized according to method described above.
Experimental for New Compounds




Analogs S69-S75, having no methoxyl groups on the benzene ring (R═H in Formula I), are synthesized as shown in Scheme 11 in a similar manner to that employed in the synthesis of S46-S53 (see Scheme 9). The synthesis starts with commercially available S68 and involves a versatile intermediate, S68-phosgene, to provide S69-S75 in 60-95% yield.




By analogy to the syntheses of S36, S43-S45, S57, and S59 (Scheme 8), S76-S81 are synthesized from commercially available S68 as shown in Scheme 12 in 70-95% yield. Using S68 as starting material, the compounds S82, S83, and S84, as shown in Scheme 13, also are synthesized similarly to the compounds which have a methoxy group on the benzene ring (R=4-OCH3 in Formula I).




Synthesis of S85-S93 is accomplished as shown in Scheme 14. The following are examples of the synthesis.
Synthesis of S85: A solution of S26 (10 mmol), di-tert-butyl dicarbonate (11 mmol), and triethylamine (12 mmol) in dichloromethane (100 ml) is stirred at room temperature for 5 hours. The reaction mixture is washed with saturated sodium bicarbonate solution (10 ml) and the aqueous layer is extracted with dichloromethane (2×15 ml). The combined organic layers are dried over magnesium sulfate and concentrated under vacuum to provide S85 as colorless oil (2.90 g, 98% yield).
Synthesis of S86: To a solution of S85 (2.36 g, 8 mmol) in dichloromethane (100 ml) at −78° C. is added BBr3 (1.0 M solution in dichloromethane) (18 ml, 18 mmol) drop-wise. The solution is warmed to room temperature and the reaction mixture is quenched with methanol (100 ml) and concentrated under vacuum. The product S86 is purified by column chromatography.
Synthesis of S87: To a solution of S86 (6 mmol) in dichloromethane (40 ml) at 0° C. is added triethylamine (7 mmol) followed by trifluoromethylsulfonyl anhydride (7 mmol). The solution is stirred at room temperature for 30 minutes, and the reaction mixture is quenched with water (10 ml). The aqueous layer is extracted with dichloromethane (2×15 ml), and the combined organic layers are dried over magnesium sulfate and concentrated under vacuum. The crude product is purified by silica gel flash chromatography to provide S87 in 75% yield.
Synthesis of S88: A mixture of S87 (1 mmol), morpholine (8 ml), tris(dibenzylideneacetone)dipalladium(0) (5 mol %), 2-(di-tert-butylphosphino)-biphenyl (20 mol %), and potassium phosphate (1.2 mmol) is heated at 80° C. in a sealed tube for 12 hours. The reaction mixture is cooled to room temperature, diluted with dichloromethane (50 ml), and washed with water (10 ml). The aqueous layer is extracted with dichloromethane (2×15 ml), and the combined organic layers are dried over magnesium sulfate and concentrated under vacuum. The crude product is purified by silica gel flash chromatography to give S88 in 81% yield.
Synthesis of S89: A solution of S87 (1 mmol), benzenethiol (2 mmol) and i-Pr2NEt (2 mmol) in CH3CN (20 ml) is heated at 80° C. for 18 hours. After cooling, ethyl acetate (30 ml) is added and then washed with 1N HCl, water, and then 1N NaOH. After drying with Na2SO4, the solution was concentrated. The product S89 was purified by chromatography in 59% yield. Alternatively, S89 is synthesized by refluxing of S87 with benzenethiol in dioxane for 10 hours using i-Pr2NEt/Pd2(dba)3/xantphos as catalyst.
Synthesis of S90: To a solution of S87 (1.0 mmol) in dioxane (10 mL) are added K2CO3 (2 mmol), phenylboronic acid (1 mmol), and (Pd(Ph3P)4 (0.11 mmol), and the mixture is stirred at 90° C. for 16 hours. The reaction mixture is cooled to 25° C., diluted with CH2Cl2 (30 mL) washed with water (10 mL), and the organic phase is evaporated to dryness under vacuum. Purification by column chromatography gives S90 in 40% yield.
Synthesis of S92: To a solution of S87 (1.0 mmol) in DMF (5 mL) are added zinc cyanide (1 mmol) and Pd(Ph3P)4 (0.11 mmol). The reaction mixture is stirred and heated at 100° C. for 1 hour, followed by cooling, dilution with water (50 mL) and 2 M sulfuric acid (5 mL), and extraction with EtOAc (3×). The combined organic extracts are washed with brine (2×), dried over magnesium sulfate, filtered, and evaporated under vacuum. The product S92 is purified by silica gel column chromatography in 80% yield.




Synthesis of S94: To a solution of S86 (1 mmol) in CH2Cl2 (10 ml) is added at 0° C. acetic anhydride (1.2 mmol) and triethylamine (1.3 mmol). The reaction mixture is stirred at room temperature overnight, then washed with H2O. After drying with Na2SO4, the solvent is evaporated and the product S94 (98% yield by NMR) is used for the next reaction without further purification.
Synthesis of S95: To a stirred solution of S84 (0.5 mmol) in benzene (20 ml) is added anhydrous AlCl3 (0.6 mmol) drop-wise. The reaction mixture is refluxed for 5 hours and poured on to crushed ice (10 g). After extraction and concentration, the product S95 is purified by silica gel column chromatography in 83% yield.
Synthesis of S96: To a solution of S86 (0.1 mmol) in methanol (5 ml) is added NaI (10 mg, excess) and Chloramine-T (0.3 mmol). The reaction mixture is stirred for 30 minutes and quenched with Na2S2O3 solution. The solvent is evaporated. The product is purified by silica gel column chromatography as a mixture of mono-iodinated or di-iodinated products in a combined yield of 60%.
Synthesis of S97: S86 (3 mmol) is added to concentrated H2SO4 (2 ml). To the stirred mixture is added, slowly, concentrated HNO3 (2 ml) drop-wise. After 10 minutes, the reaction mixture is poured on to crushed ice (5 g) and neutralized with Na2CO3 to pH=7. The Boc-deprotected nitro intermediate is collected by extraction with EtOAc and converted back to S97 by reaction with Boc2O. Purification by silica gel column chromatography provides S97 in 78% yield.
Synthesis of S98: A mixture of S97 (2 mmol) and 10% Pd/C (0.1 g) in methanol (20 ml) is bubbled through with H2 gas for 2 hours. After filtration and concentration, the amine product is used for the next reactions without further purification.
Synthesis of S99 and S100: S98 (1 mmol) is dissolved in aqueous HCl (2 mmol HCl, 10 ml H2O). To this solution is added at 0° C. slowly a solution of sodium nitrite (1 mmol) in water (5 ml). The reaction mixture is stirred at 0° C. for 1 hour, then NaN3 (2 mmol) in water (2 ml) is added drop-wise at 0° C. The resulting mixture is stirred at 0° C. for 1 hour and at room temperature overnight. The product is extracted with ethyl acetate and washed with saturated sodium bicarbonate and water. The organic layer is dried over anhydrous sodium sulfate and concentrated to give crude product S98. Column purification on silica gel provide the product in 71% yield. Similarly, S99 is synthesized in 60% yield.
Example 5
High-Throughput Screening Method
Assays for screening biologically-active small molecules have been developed. These assays are based on rebinding of FKBP12 protein to RyR.
A highly-efficient assay for high-throughput screening for small molecules is developed by immobilization of FKBP12.6 (GST-fusion protein) onto a 96-well plate coated with glutathione. PKA-phosphorylated ryanodine receptor type 2 (RyR2) is loaded onto the FKBP12.6-coated plate, and incubated with JTV-519 analogues at various concentrations (10-100 nM) for 30 min. Thereafter, the plate is washed to remove the unbound RyR2, and then incubated with anti-RyR2 antibody for 30 min. The plate is again washed to remove unbound anti-RyR2 antibody, and then treated with florescent-labeled secondary antibody. The plate is read by an automatic fluorescent plate reader for binding activity.
In an alternative assay, RyR2 is PKA-phosphorylated in the presence of 32P-ATP. Radioactive PKA-phosphorylated RyR2 is loaded onto an FKBP12.6-coated, 96-well plate, in the presence of JTV-519 analogues at various concentrations (10-100 nM) for 30 min. The plate is washed to remove the unbound radiolabeled RyR2, and then read by an automatic plate reader.
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.
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.

## Claims

1. A method for the synthesis of compounds of the formula




wherein R is NH-2-Py, comprising: reacting a compound of formula S26




with a compound of formula RSO2Cl, wherein R is defined as above, to form a reaction mixture; and purifying the reaction mixture to yield a compound of the formula




wherein R is defined as above.

2. The method of claim 1, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

3. The method of claim 1, wherein the reaction mixture further comprises a base catalyst.

4. The method of claim 2, further comprising washing the reaction mixture with basic solution.

5. A method for the synthesis of compounds of the formula




wherein R is selected from the group consisting of Ph- and 4-N3-2-OH—C6H5, comprising: reacting a compound of formula S26,




with a compound of formula RCOX, wherein R is defined as above and X is selected from the group consisting of Cl and NHS, to yield a reaction mixture; and purifying the reaction mixture to yield a compound of formula




wherein R is as defined above.

6. The method of claim 5, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

7. The method of claim 5, further comprising adding a base catalyst to the reaction mixture.

8. The method of claim 6, further comprising washing the reaction mixture using basic solution.

9. The method of claim 6, further comprising washing the reaction mixture using acidic solution.

10. A method for the synthesis of compounds of the formula




wherein X is S, comprising: reacting a compound of formula S26,




with a compound of the formula C6H4—NCX, wherein X is as defined above, to yield a reaction mixture; and purifying the reaction mixture to yield a compound of formula




wherein X is as defined above.

11. The method of claim 10, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

12. The method of claim 11, wherein the solvent is a base catalyst.

13. The method of claim 12, further comprising adding additional organic solvent to the reaction mixture following the reaction.

14. The method of claim 11, further comprising washing the reaction mixture using basic solution.

15. The method of claim 11, further comprising washing the reaction mixture using acidic solution.

16. A method for the synthesis of isomers of compounds of the formula




comprising: reacting a compound of formula S26,




with phenyl methoxyphosphonyl chloride (Ph(MeO)P(O)Cl) to form a reaction mixture; and purifying and separating the product into the isomers.

17. The method of claim 16, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

18. The method of claim 17, further comprising washing the reaction mixture using basic solution.

19. A method for the synthesis of compounds of the formula




wherein X is selected from the group consisting of CH2—CH2 and




comprising: reacting a compound of formula S26,




with a compound of formula ClOC—X—COCl wherein X is defined as above, to form a reaction mixture; and purifying the reaction mixture to yield a compound of formula




wherein X is defined as above.

20. The method of claim 19, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

21. The method of claim 19, further comprising adding a base catalyst to the reaction mixture.

22. The method of claim 20, further comprising washing the reaction mixture using basic solution.

23. The method of claim 20, further comprising washing the reaction mixture using acidic solution.

24. A method for the synthesis of S38,




comprising: reacting a compound of formula




with sodium hydroxide; and purifying S38.

25. The method of claim 24, wherein the reacting takes place in a first solvent and the purifying comprises: removing the first solvent to form a residue; dissolving the residue in water to form an aqueous solution; washing the aqueous solution with an organic solvent; acidifying the aqueous solution; extracting S38 from the aqueous solution using an organic solvent to form an extract; and removing the organic solvent from the extract.

26. The method of claim 25, wherein the first solvent is an organic solvent.

27. A method for the synthesis of S44,




comprising: treating a compound of formula S36,




with thionyl chloride to form S36-Cl; reacting S36-Cl with cystamine to form a reaction mixture; and purifying the reaction mixture to yield S44.

28. The method of claim 27, wherein cystamine is mono-protected and further comprising removing the protecting group before purifying the reaction mixture.

29. The method of claim 27, wherein the treating takes place in a solvent, wherein the solvent is an organic solvent.

30. The method of claim 27, further comprising adding a base catalyst to the reaction mixture.

31. A method for the synthesis of compounds of the formula




wherein X is NHEt, comprising: treating a compound of formula S36,




with thionyl chloride to obtain S36-Cl; reacting S36-Cl with a compound of the formula HX, wherein X is defined as above; and purifying the product to yield a compound of the formula




wherein X is defined as above.

32. The method of claim 31, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

33. A method for the synthesis of compounds of the formula




wherein R is selected from the group consisting of NO2— and HO—, comprising: reacting a compound of formula S36,




with thionyl chloride to obtain S36-Cl; reacting S36-Cl with cystamine to form S36-cystamine; and reacting the S36-cystamine with an NHS activated ester of an azido compound in a solvent to yield a compound of the formula




wherein R is defined as above.

34. A method for the synthesis of compounds of the formula




wherein X is selected from the group consisting of NHEt, NHPh, NH2, and NHCH2-pyridine, comprising: reacting a compound of formula S77,




with thionyl chloride to obtain S77-Cl; reacting S77-Cl with a compound of the formula HX, wherein X is defined as above; and purifying the residue to yield a compound of the formula




wherein X is defined as above.

35. The method of claim 34, wherein reacting takes place in a solvent, wherein the solvent is an organic solvent.

36. A method for the synthesis of S84,




comprising: reacting a compound of formula S68,




with benzyl bromide to form a reaction mixture; and purifying the reaction mixture to yield S84.

37. The method of claim 36, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

38. The method of claim 36, further comprising adding a base catalyst to the reaction mixture.

39. A method for the synthesis of S86,




comprising: reacting a compound of formula S85,




with BBr3 to form a reaction mixture; quenching the reaction mixture to form a product; and purifying the product to yield S86.

40. The method of claim 39, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

41. The method of claim 39, wherein quenching the reaction mixture comprises adding methanol.

42. A method for the synthesis of S87,




comprising: reacting a compound of formula S86,




with trifluoromethylsulfonyl anhydride to form a reaction mixture; and purifying the reaction mixture to yield S87.

43. The method of claim 42, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

44. The method of claim 42, further comprising adding a base catalyst to the reaction mixture.

45. The method of claim 42, wherein purifying the reaction mixture comprises: quenching the reaction mixture with water to form an aqueous layer; extracting the aqueous layer using an organic solvent to form an organic layer; drying and concentrating the organic layer to form a residue; and purifying the residue to yield S87.

46. A method for the synthesis of compounds of the formula S88,




comprising: reacting a compound of formula S87,




with tris(dibenzylideneacetone)dipalladium(O), 2-(di-tert-butylphosphino)-biphenyl, morpholine, and K2PO3 to form a reaction mixture; and purifying the reaction mixture to yield S88.

47. The method of claim 46, wherein purifying the reaction mixture comprises: washing the reaction mixture using water to form an aqueous layer; extracting the aqueous layer using an organic solvent to form an organic layer; drying and concentrating the organic layer to form a residue; and purifying the residue to yield S88.

48. A method for the synthesis of S89,




comprising: reacting a compound of formula S87,




with benzenethiol and a catalyst to form a reaction mixture; and purifying the reaction mixture to yield S89.

49. The method of claim 48, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

50. The method of claim 48, wherein the catalyst is i-Pr2NEt.

51. The method of claim 48, wherein purifying the reaction mixture comprises: washing the reaction mixture; and drying and concentrating the reaction mixture to yield S89.

52. The method of claim 51, wherein washing the reaction mixture comprises using a basic solution and water to wash the reaction mixture.

53. A method for the synthesis of S89,




comprising: refluxing a compound of formula S87,




with benzenethiol in an organic solvent and i-Pr2Net/Pd2(dba)3/xantphos catalyst to form a product; and purifying the product to yield S89.

54. A method for the synthesis of S90,




comprising: reacting a compound of formula S87,




with a base, phenylboronic acid, and a catalyst to form a reaction mixture; and purifying the reaction mixture to yield S90.

55. The method of claim 54, wherein the base is K2PO3.

56. The method of claim 54, wherein the catalyst is Pd(Ph3P)4.

57. The method of claim 54, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

58. The method of claim 57, further comprising washing the reaction mixture using water.

59. A method for the synthesis of S92,




comprising: reacting a compound of formula S87,




with zinc cyanide and a catalyst to form a reaction mixture; and purifying the reaction mixture to yield S92.

60. The method of claim 59, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

61. The method of claim 59, wherein the catalyst is Pd(Ph3P)4.

62. The method of claim 59, wherein purifying the reaction mixture comprises: washing the reaction mixture using water to form an aqueous layer; extracting the aqueous layer using an organic solvent to form an organic extract; washing the organic extract; drying the organic extract; and concentrating the organic extract to yield S92.

63. A method for the synthesis of S94,




comprising: reacting a compound of formula S86,




with acetic anhydride to form a reaction mixture; and purifying the reaction mixture to yield S94.

64. The method of claim 63, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

65. The method of claim 63, further comprising adding a base catalyst to the reaction mixture.

66. The method of claim 64, wherein purifying the reaction mixture comprises: washing the reaction mixture using water; drying the reaction mixture; removing the solvent from the reaction mixture to yield a residue; and purifying the residue to yield S94.

67. A method for the synthesis of S95,




comprising: reacting a compound of formula S94,




with anhydrous AlCl3 to form a reaction mixture; and purifying the reaction mixture to yield S95.

68. The method of claim 67, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

69. The method of claim 68, wherein purifying the reaction mixture comprises: extracting the reaction mixture using water to form an organic layer; concentrating the organic layer to form a residue; and purifying the residue to yield S95.

70. A method for the synthesis of S96,




comprising: reacting a compound of formula S86,




with Nal and Chloramine-T to form a reaction mixture; quenching the reaction mixture; and purifying the reaction mixture to yield S96.

71. The method of claim 70, wherein the reaction takes place in a solvent, wherein the solvent is an organic solvent.

72. The method of claim 70, wherein quenching the reaction mixture comprises adding Na2S2O3 solution to the reaction mixture.

73. A method for the synthesis of S98,




comprising: hydrogenating a compound of formula S97,




to yield S98.

74. The method of claim 73, wherein hydrogenating comprises: reacting S97 with H2 and Pd/C in an organic solvent to form a reaction mixture; filtering the reaction mixture; concentrating the reaction mixture to yield a residue; and purifying the residue to yield S98.

75. A method for the synthesis of compounds of the formula




wherein R is selected from the group consisting of CH2═CH—, Me—, and p-Me—C6H4—, comprising: reacting a compound of formula S26




with a compound of formula RSO2Cl, wherein R is defined as above, to form a reaction mixture; and purifying the reaction mixture to yield a compound of the formula




wherein R is defined as above.

76. The method of claim 75, wherein the reaction takes place in a solvent, wherein the solvent is an organic solvent.

77. The method of claim 75, wherein the reaction mixture further comprises a base catalyst.

78. The method of claim 76, further comprising washing the reaction mixture with basic solution.

79. A method for the synthesis of compounds of the formula




wherein NR1R2 is selected from the group consisting of




and NBu2, comprising: reacting a compound of formula S3,




with a compound of formula HNR1R2, wherein HNR1R2 is defined as above, to form a reaction mixture; and purifying the reaction mixture to yield a compound of the formula




wherein NR1R2 is defined as above.

80. The method of claim 79, wherein the reaction takes place in a solvent.

81. The method of claim 80, wherein the solvent is an organic solvent.

82. A method for the synthesis of compounds of the formula




wherein X is O, comprising: reacting a compound of formula S26,




with a compound of the formula C6H4—NCX, wherein X is as defined above, to yield a reaction mixture; and purifying the reaction mixture to yield a compound of formula




wherein X is as defined above.

83. The method of claim 82, wherein the reacting takes place in a solvent wherein the solvent is an organic solvent.

84. The method of claim 83, wherein the solvent is a base catalyst.

85. The method of claim 84, further comprising adding organic solvent to the reaction mixture following the reaction.

86. The method of claim 83, further comprising washing the reaction mixture using basic solution.

87. The method of claim 83, further comprising washing the reaction mixture using acidic solution.

88. A method for the synthesis of compounds of the formula




wherein n is 1 and R is CH2═CH or wherein n is 2 and R is




comprising: treating an intermediate compound of formula




wherein R is defined as above, with hydrogen peroxide to form a reaction mixture; drying the reaction mixture to form a residue; and purifying the residue to yield a compound of formula




wherein R is defined as above.

89. The method of claim 88, wherein the reaction takes place in a solvent, wherein the solvent is an organic solvent.

90. The method of claim 89, wherein purifying the residue comprises comprising dissolving the residue in an organic solvent to form a mixture, washing the mixture using basic solution, drying the mixture, and removing the organic solvent from the mixture.

91. A method for the synthesis of S57,




comprising: reacting a compound of formula S26,




with methyl chlorooxoacetate to form a reaction mixture; and purifying the product to yield S57.

92. The method of claim 91, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

93. The method of claim 91, further comprising adding a base catalyst to the reaction mixture.

94. The method of claim 91, further comprising washing the reaction mixture using basic solution prior to purifying the product.

95. The method of claim 92, further comprising washing the reaction mixture using acidic solution prior to purifying the product.

96. A method for the synthesis of S36,




comprising: reacting a compound of formula S57,




in a first solvent with sodium hydroxide to form a reaction mixture; removing the first solvent to form a residue; and purifying the residue to yield S36.

97. The method of claim 96, wherein the first solvent is an organic solvent.

98. A method for the synthesis of compounds of the formula




wherein X is —OCH3, comprising: treating a compound of formula S36,




with thionyl chloride to obtain S36-Cl; reacting S36-Cl with a compound of the formula HX, wherein X is defined as above; and purifying the product to yield a compound of the formula




wherein X is defined as above.

99. The method of claim 98, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

100. A method for the synthesis of S37,




comprising: reacting a compound of formula S26,




in a solvent with 4-nitrophenyl chloroformate to form a reaction mixture; washing the reaction mixture using water; removing the solvents from the reaction mixture to form a residue; and purifying the residue to yield S37.

101. The method of claim 100, wherein the solvent is an organic solvent.

102. The method of claim 100, further comprising adding a base catalyst to the reaction mixture.

103. A method for the synthesis of compounds of the formula




wherein NR1R2 selected from the group consisting of




comprising: reacting a compound of formula




with HNR1R2, wherein NR1R2 is defined as above, in a solvent to form a reaction mixture; removing the solvent from the reaction mixture to form a residue; and purifying the residue to yield a compound of formula




wherein NR1R2 is defined as above.

104. The method of claim 103, wherein the solvent is an organic solvent.

105. A method for the synthesis of S76,




comprising: reacting a compound of formula S68,




with methyl chlorooxoacetate to form a reaction mixture; and purifying the reaction mixture to yield S76.

106. The method of claim 105, wherein the reaction takes place in a solvent, wherein the solvent is an organic solvent.

107. The method of claim 105, further comprising adding a base catalyst to the reaction mixture.

108. The method of claim 106, further comprising washing the reaction mixture using basic solution and then removing the solvent.

109. The method of claim 106, further comprising washing the reaction mixture using acidic solution and then removing the solvent.

110. A method for the synthesis of S77,




comprising: reacting a compound of formula S76,




in a first solvent with sodium hydroxide; removing the first solvent to form a residue; and purifying the residue to yield S77.

111. The method of claim 110, wherein purifying the residue comprises: dissolving the residue in water to form an aqueous solution; washing the aqueous solution with an organic solvent; acidifying the aqueous solution; extracting S77 from the aqueous solution using an organic solvent to form an extract; and removing the organic solvent from the extract.

112. The method of claim 110, wherein the first solvent is an organic solvent.

113. A method for the synthesis of S82,




comprising: reacting a compound of formula S68,




with CH2CHSO2Cl to form an intermediate product of the formula




reacting the intermediate compound with




to form a final reaction mixture; and purifying the reaction mixture to yield S82.

114. The method of claim 113, wherein the either or both reacting steps take place in a solvent, wherein the solvent is an organic solvent.

115. A method for the synthesis of S83,




comprising: reacting a compound of formula S68,




with RCOCl, wherein R is




to yield a reaction mixture; and purifying the reaction mixture to yield S83.

116. The method of claim 115, wherein the reacting takes place in a solvent, wherein the solvent is an organic solvent.

117. The method of claim 115, further comprising adding a base catalyst to the reaction mixture.

118. The method of claim 116, further comprising washing the reaction mixture using basic solution.

119. A method for the synthesis of S85,




comprising: reacting a compound of formula S26,




with Boc2O in an organic solvent to form a protected product in a reaction mixture; washing the reaction mixture using a saturated sodium bicarbonate solution to form an aqueous layer; extracting the aqueous layer using an organic solvent to form organic layers; and drying and concentrating the organic layers to yield S85.

120. The method of claim 119, further comprising adding a base catalyst to the reaction mixture.