U.S. patent application number 13/865359 was filed with the patent office on 2013-10-24 for agents for treating disorders involving modulation of ryanodine receptors.
This patent application is currently assigned to ARMGO Pharma, Inc.. The applicant listed for this patent is ARMGO PHARMA, INC., LES LABORATOIRES SERVIER. Invention is credited to Sandro BELVEDERE, Mark BERTRAND, Nicole VILLENEUVE, Yael WEBB, Jiaming YAN.
Application Number | 20130281512 13/865359 |
Document ID | / |
Family ID | 49165460 |
Filed Date | 2013-10-24 |
United States Patent
Application |
20130281512 |
Kind Code |
A1 |
YAN; Jiaming ; et
al. |
October 24, 2013 |
AGENTS FOR TREATING DISORDERS INVOLVING MODULATION OF RYANODINE
RECEPTORS
Abstract
The present invention relates to 1,4-benzothiazepine derivatives
and their use to treat conditions, disorders and diseases
associated with ryanodine receptors (RyRs) that regulate calcium
channel functioning in cells. The invention also discloses
pharmaceutical compositions comprising the compounds and uses
thereof to treat diseases and conditions associated with RyRs, in
particular cardiac, musculoskeletal and central nervous system
(CNS) disorders.
Inventors: |
YAN; Jiaming; (New York,
NY) ; BELVEDERE; Sandro; (New York, NY) ;
WEBB; Yael; (Yorktown Heights, NY) ; BERTRAND;
Mark; (Saint Jean Le Blanc, FR) ; VILLENEUVE;
Nicole; (Rueil Malmaison, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LES LABORATOIRES SERVIER
ARMGO PHARMA, INC. |
Suresnes Cedex
Tarrytown |
NY |
FR
US |
|
|
Assignee: |
ARMGO Pharma, Inc.
Tarrytown
NY
LES LABORATOIRES SERVIER
Suresnes Cedex
|
Family ID: |
49165460 |
Appl. No.: |
13/865359 |
Filed: |
April 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61625890 |
Apr 18, 2012 |
|
|
|
Current U.S.
Class: |
514/44A ;
514/211.09; 540/552 |
Current CPC
Class: |
A61P 25/14 20180101;
A61P 35/04 20180101; A61P 25/28 20180101; A61P 37/02 20180101; A61P
43/00 20180101; C07C 57/15 20130101; A61P 13/12 20180101; A61P 9/00
20180101; A61K 31/7088 20130101; A61P 9/06 20180101; A61P 29/00
20180101; C07D 281/10 20130101; A61P 9/12 20180101; A61P 21/04
20180101; A61P 37/06 20180101; A61P 19/02 20180101; C07D 285/36
20130101; A61P 3/10 20180101; A61P 19/00 20180101; A61P 9/04
20180101; A61P 11/00 20180101; A61P 25/22 20180101; A61K 31/554
20130101; A61P 21/00 20180101; A61P 35/00 20180101; A61P 19/08
20180101; A61P 25/16 20180101; A61P 13/00 20180101; A61P 25/24
20180101; A61P 9/10 20180101; A61P 25/00 20180101; A61P 13/02
20180101 |
Class at
Publication: |
514/44.A ;
540/552; 514/211.09 |
International
Class: |
C07D 285/36 20060101
C07D285/36; A61K 31/554 20060101 A61K031/554; A61K 31/7088 20060101
A61K031/7088 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2012 |
EP |
EP12167732.2 |
Claims
1. A compound represented by the structure of Formula (I):
##STR00025## wherein R is COOH; and pharmaceutically acceptable
salts thereof.
2. The compound according to claim 1, in the form of a salt with a
pharmaceutically acceptable acid or base.
3. The compound according to claim 2, wherein the salt is selected
from the group consisting of sodium, potassium, magnesium,
hemifumarate, hydrochloride and hydrobromide.
4. The compound according to claim 3, wherein the salt is the
sodium or the hemifumarate salt.
5. The compound according to claim 1, which is selected from the
group consisting of: ##STR00026##
6. The compound according to claim 1, which is represented by the
structure of Formula (1): ##STR00027## or pharmaceutically
acceptable salts thereof.
7. The compound according to claim 6, in the form of a salt with a
pharmaceutically acceptable acid or base.
8. The compound according to claim 7, wherein the salt is selected
from the group consisting of sodium, potassium, magnesium,
hemifumarate, hydrochloride and hydrobromide.
9. The compound according to claim 8, wherein the salt is the
sodium salt.
10. The compound according to claim 8, wherein the salt is the
hemifumarate salt.
11. A pharmaceutical composition comprising a compound according to
claim 1, in combination with one or more pharmaceutically
acceptable excipients or carriers.
12. A method of treating or preventing a condition selected from
the group consisting of cardiac disorders and diseases, muscle
fatigue, musculoskeletal disorders and diseases; CNS disorders and
diseases, cognitive dysfunction, neuromuscular disorders and
diseases, bone disorders and diseases, cancer cachexia, malignant
hyperthermia, diabetes, sudden cardiac death, and sudden infant
death syndrome, or for improving cognitive function, the method
comprising the step of administering to a subject in need thereof a
therapeutically effective amount of a compound according to claim 1
or a pharmaceutical composition comprising such compound, to
effectuate such treatment.
13. The method according to claim 12, wherein the condition is
associated with an abnormal function of a ryanodine receptor 1
(RyR1), a ryanodine receptor type (RyR2), a ryanodine receptor type
3 (RyR3), or a combination thereof.
14. The method according to claim 12, wherein the cardiac disorders
and diseases are selected from the group consisting of irregular
heartbeat disorders and diseases, exercise-induced irregular
heartbeat disorders and diseases, heart failure, congestive heart
failure, chronic heart failure, acute heart failure, systolic heart
failure, diastolic heart failure, acute decompensated heart
failure, cardiac ischemia/reperfusion (I/R) injury, chronic
obstructive pulmonary disease, I/R injury following coronary
angioplasty or following thrombolysis for the treatment of
myocardial infarction (MI); and high blood pressure.
15. The method according to claim 14, wherein the irregular
heartbeat disorders and diseases selected from the group consisting
of 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.
16. The method according to claim 12, wherein the muscle fatigue is
due to a skeletal muscle disease, disorder or condition.
17. The method according to claim 12, wherein the musculoskeletal
disorder, disease or condition is selected from the group
consisting of exercise-induced skeletal muscle fatigue,
exercise-induced muscle fatigue which is due to prolonged exercise
or high-intensity exercise, a congenital myopathy, muscular
dystrophy, spinal muscular atrophy (SMA), Spinal and bulbar
muscular atrophy (SBMA), age-related muscle fatigue, sarcopenia,
central core disease, cancer cachexia, bladder disorders, and
incontinence.
18. The method according to claim 17, wherein the muscular
dystrophy is selected from the group consisting of Duchenne
Muscular Dystrophy (DMD), Becker's Muscular Dystrophy (BMD),
Limb-Girdle Muscular Dystrophy (LGMD), facioscapulohumeral
dystrophy, myotonic muscular dystrophy, congenital muscular
dystrophy (CMD), distal muscular dystrophy, Emery-Dreifuss muscular
dystrophy, and oculopharyngeal muscular dystrophy.
19. The method according to claim 12, wherein the CNS disorders and
diseases are selected from the group consisting of Alzheimer's
Disease (AD), neuropathy, seizures, Parkinson's Disease (PD), and
Huntington's Disease (HD); and the neuromuscular disorders and
diseases are selected from the group consisting of Spinocerebellar
ataxia (SCA), and Amyotrophic lateral sclerosis (ALS, Lou Gehrig's
disease).
20. The method according to claim 12, wherein the cognitive
dysfunction is stress-related or age-related, or wherein the
cognitive function to be improved is short term memory, long term
memory, attention or learning, or wherein the cognitive dysfunction
is associated with a disease or disorder selected from the group
consisting of Alzheimer's disease (AD), attention deficit
hyperactivity disorder (ADHD), autism spectrum disorder (ASD),
generalized anxiety disorder (GAD), obsessive compulsive disorder
(OCD), Parkinson's Disease (PD), post-traumatic stress disorder
(PTSD), Schizophrenia, Bipolar disorder, and major depression.
21. The method according to claim 12, wherein the condition is
cancer cachexia, preferably due to a cancer having bone
metastases.
22. The method according to claim 12, wherein the compound is used
at a dose sufficient to restore or enhance binding of calstabin2 to
RyR2.
23. The method according to claim 12, wherein the compound is used
at a dose sufficient to restore or enhance binding of calstabin1 to
RyR1.
24. The method according to claim 12, wherein the compound is used
at a dose sufficient to decrease Ca.sup.2+ leak through a RyR
channel.
25. The method according to claim 12, further comprising the use of
an antisense oligonucleotide (AO) which is specific for a splicing
sequence in an mRNA of interest, for enhancing exon skipping in
said mRNA of interest.
26. A method for treating a subject that has Duchenne Muscular
Dystrophy (DMD), comprising the step of administering to said
subject a compound according to claim 1, or a pharmaceutical
composition comprising such compound, in combination with an
antisense oligonucleotide (AO) which is specific for a splicing
sequence of at least one exon of the DMD gene, preferably a
splicing sequence of exon 23, 45, 44, 50, 51, 52 and/or 53 of the
DMD gene.
27. A process for the preparation of a compound according to claim
1, comprising the step of reacting a compounds of the formula
##STR00028## with a compound of the formula ##STR00029## wherein
R.sup.a is COOR' or CN; R.sup.1 is a C.sub.1-C.sub.4 alkyl, and L
is a leaving group to afford a compound of the formula:
##STR00030## and converting the group R.sup.a to the group R so as
to afford a compound of formula (I).
Description
FIELD OF THE INVENTION
[0001] The present invention relates to 1,4-benzothiazepine
derivatives and their use to treat disorders and diseases
associated with ryanodine receptors (RyRs) that regulate calcium
channel functioning in cells. The invention also discloses
pharmaceutical compositions comprising these compounds and uses
thereof to treat diseases and conditions associated with RyRs, in
particular cardiac, skeletal muscular and central nervous system
(CNS) disorders.
BACKGROUND OF THE INVENTION
[0002] The sarcoplasmic reticulum (SR) is a structure in cells that
functions, among other things, as a specialized intracellular
calcium (Ca.sup.2+) store. RyRs are channels in the SR, which open
and close to regulate the release of Ca.sup.2+ from the SR into the
intracellular cytoplasm of the cell. Release of Ca.sup.2+ into the
cytoplasm from the SR increases cytoplasmic Ca.sup.2+
concentration. Open probability of RyRs refers to the likelihood
that a RyR is open at any given moment, and therefore capable of
releasing Ca.sup.2+ into the cytoplasm from the SR.
[0003] There are three types of RyR, all of which are highly
homologous: 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 is a tetramer.
Part of the RyR complex is formed by four RyR polypeptides in
association with four FK506 binding proteins (FKBPs) (calstabins),
specifically FKBP12 (calstabin1) and FKBP12.6 (calstabin2).
Calstabin1 binds to RyR1 and RyR3 while calstabin2 binds to RyR2.
The calstabins bind to the RyR (one molecule per RyR subunit),
stabilize the RyR function, facilitate coupled gating between
neighboring RyRs and prevent abnormal activation (Ca.sup.2+ leak)
of the channel by stabilizing the channel's closed state.
Ryanodine Receptor 2 and Cardiac Diseases
[0004] In cardiac striated muscle, RyR2 is the major Ca.sup.2+
release channel required for excitation-contraction (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 Ca.sup.2+ channels. Ca.sup.2+
influx through the open voltage-gated channels in turn initiates
Ca.sup.2+ release from the SR via RyR2. This process is known as
Ca.sup.2+-induced Ca.sup.2+ release. The RyR2-mediated
Ca.sup.2+-induced Ca.sup.2+ release then activates the contractile
proteins in the cardiac cell, resulting in cardiac muscle
contraction.
[0005] Phosphorylation of RyR2 by protein kinase A (PKA) is an
important part of the "fight or flight" response that increases
cardiac EC coupling gain by augmenting the amount of Ca.sup.2+
released for a given trigger. This signaling pathway provides a
mechanism by which activation of the sympathetic nervous system
(SNS), in response to stress, results in increased cardiac output.
Phosphorylation of RyR2 by PKA results in partial dissociation of
calstabin2 from the channel, which in turn, leads to increased open
probability, and increased Ca.sup.2+ release from the SR into the
intracellular cytoplasm.
[0006] Heart failure (HF) is characterized by a sustained
hyperadrenergic state in which serum catecholamine levels are
chronically elevated. One consequence of this chronic
hyperadrenergic state is persistent PKA hyperphosphorylation of
RyR2, such that 3-4 out of the four Ser2808 in each homotetrameric
RyR2 channel are chronically phosphorylated (Marx S O, et al. Cell,
2000; 101(4):365-376). In particular, chronic PKA
hyperphosphorylation of RyR2 is associated with depletion of the
channel-stabilization subunit calstabin2 from the RyR2 channel
macromolecular complex. Depletion of calstabin results in a
diastolic SR Ca.sup.2+"leak" from the RyR complex, which
contributes to impaired contractility (Marx et al., 2000). Due to
the activation of inward depolarizing currents, this diastolic SR
Ca.sup.2+ "leak" also is associated with fatal cardiac arrhythmias
(Lehnart et al, J Clin Invest. 2008; 118(6):2230-2245). Indeed,
mice engineered with RyR2 lacking the PKA phosphorylation site are
protected from HF progression after myocardial infarction (MI)
(Wehrens X H et al. Proc Natl Acad Sci USA. 2006; 103(3):511-518).
In addition, chronic PKA hyperphosphorylation of RyR2 in HF is
associated with remodeling of the RyR2 macromolecular complex that
includes depletion of phosphatases (Marx et al. 2000) PP1 and PP2a
(impairing dephosphorylation of Ser2808) and the cAMP-specific type
4 phosphodiesterase (PDE4D3) from the RyR2 complex. Depletion of
PDE4D3 from the RyR2 complex causes sustained elevation of local
cAMP levels (Lehnart S E, et al., Cell 2005; 123(1):25-35). Thus,
diastolic SR Ca.sup.2+ leak contributes to HF progression and
arrhythmias. Moreover, a recent report has demonstrated that
RyR2-S2808D+/+ (aspartic acid replacing serine 2808) knock-in mice,
that mimic constitutive PKA hyperphosphorylation of RyR2, show
depletion of calstabin2 and leaky RyR2. RyR2-S2808D+/+ mice develop
age-dependent cardiomyopathy, demonstrate elevated RyR2 oxidation
and nitrosylation, a reduced SR Ca.sup.2+ store content, and
increased diastolic SR Ca.sup.2+ leak. After myocardial infarction,
RyR2-S2808D+/+ mice exhibit increased mortality compared with WT
littermates. Treatment with 5107, a 1,4-benzothiazepine derivative
that stabilizes RyR2-calstabin2 interactions (WO 2007/024717),
inhibited the RyR2-mediated diastolic SR Ca.sup.2+ leak and reduced
HF progression in both WT and RyR2-S2808D+/+ mice (Shan et al., J
Clin Invest. 2010 Dec. 1; 120(12):4375-87).
[0007] Moreover, RyR2 contains about 33 free thiol residues
rendering it highly sensitive to the cellular redox state. Cysteine
oxidation facilitates RyR opening and SR Ca.sup.2+ leak. Shan et
al, 2010, demonstrated that oxidation and nitrosylation of RyR2 and
dissociation of the stabilizing subunit calstabin2 from RyR2
induces SR Ca.sup.2+ leak.
[0008] Catecholaminergic polymorphic ventricular tachycardia (CPVT)
is an inherited disorder in individuals with structurally normal
hearts. More than 50 distinct RyR2 mutations have been linked to
CPVT. CPVT patients experience syncope and sudden cardiac death
(SCD) from the toddler to adult ages, and by 35 years of age the
mortality is up to 50%. Individuals with CPVT have ventricular
arrhythmias when subjected to exercise, but do not develop
arrhythmias at rest. CPVT-associated RyR2 mutations result in
"leaky" RyR2 channels due to the decreased binding of the
calstabin2 subunit (Lehnart et al., 2008). Mice heterozygous for
the R2474S mutation in RyR2 (RyR2-R2474S mice) exhibit spontaneous
generalized tonic-clonic seizures (which occurred in the absence of
cardiac arrhythmias), exercise-induced ventricular arrhythmias, and
SCD. Treatment with 5107 enhanced the binding of calstabin2 to the
mutant RyR2-R2474S channel, inhibited the channel leak, prevented
cardiac arrhythmias and raised the seizure threshold (Lehnart et
al., 2008).
Ryanodine Receptor 1 and Skeletal Muscle Diseases
[0009] Skeletal muscle contraction is activated by SR Ca.sup.2+
release via RyR1. Depolarization of the transverse (T)-tubule
membrane activates the dihydropyridine receptor voltage sensor
(Cav1.1) that in turn activates RyR1 channels via a direct
protein-protein interaction causing the release of SR Ca.sup.2+
stores. Ca.sup.2+ binds to troponin C allowing actin-myosin
cross-bridging to occur and sarcomere shortening.
[0010] In conditions of prolonged muscular stress (e.g., during
marathon running) or in a disease such as heart failure, both of
which are characterized by chronic activation of SNS, skeletal
muscle function is impaired, possibly due to altered EC coupling.
In particular, the amount of Ca.sup.2+ released from the SR during
each contraction of the muscle is reduced, aberrant Ca.sup.2+
release events can occur, and Ca.sup.2+ reuptake is slowed (Reiken,
S, et al. 2003. J. Cell Biol. 160:919-928). These observations
suggest that the deleterious effects of chronic activation of the
SNS on skeletal muscle might be due, at least in part, to defects
in Ca.sup.2+ signaling.
[0011] The RyR1 macromolecular complex consists of a tetramer of
the 560-kDa RyR1 subunit that forms a scaffold for proteins that
regulate channel function including PKA and the phosphodiesterase
4D3 (PDE4D3), protein phosphatase 1 (PP1) and calstabin1. A-kinase
anchor protein (mAKAP) targets PKA and PDE4D3 to RyR1, whereas
spinophilin targets PP1 to the channel (Marx et al. 2000;
Brillantes et al., Cell, 1994, 77, 513-523; Bellinger et al. J.
Clin. Invest. 2008, 118, 445-53). The catalytic and regulatory
subunits of PKA, PP1, and PDE4D3 regulate PKA-mediated
phosphorylation of RyR1 at Ser2843 (Ser2844 in the mouse). It has
been shown that PKA-mediated phosphorylation of RyR1 at Ser2844
increases the sensitivity of the channel to cytoplasmic Ca.sup.2+,
reduces the binding affinity of calstabin1 for RyR1, and
destabilizes the closed state of the channel (Reiken et al., 2003;
Marx, S. O. et al., Science, 1998, 281:818-821). Calstabin1
concentrations in skeletal muscle are reported to be approximately
200 nM and that PKA phosphorylation of RyR1 reduces the binding
affinity of calstabin1 for RyR1 from approximately 100-200 nM to
more than 600 nM. Thus, under physiologic conditions, reduction in
the binding affinity of calstabin1 for RyR1, resulting from PKA
phosphorylation of RyR1 at Ser2843, is sufficient to substantially
reduce the amount of calstabin1 present in the RyR1 complex.
Chronic PKA hyperphosphorylation of RyR1 at Ser2843 (defined as PKA
phosphorylation of 3 or 4 of the 4 PKA Ser2843 sites present in
each RyR1 homotetramer) results in "leaky" channels (i.e., channels
prone to opening at rest), which contribute to the skeletal muscle
dysfunction that is associated with persistent hyperadrenergic
states such as occurs in individuals with heart failure (Reiken et
al., 2003).
[0012] Moreover, regulation of RyR1 by posttranslational
modifications other than phosphorylation, such as by nitrosylation
of free sulfhydryl groups on cysteine residues (S-nitrosylation),
as well as channel oxidation, have been reported to increase RyR1
channel activity. S-nitrosylation and oxidation of RyR1 have each
been shown to reduce calstabin1 binding to RyR1.
[0013] It was previously reported by Bellinger et al. (Proc. Natl.
Acad. Sci. 2008, 105(6):2198-2002) that during extreme exercise in
mice and humans, RyR1 is progressively PKA-hyperphosphorylated,
S-nitrosylated and depleted of PDE4D3 and calstabin1, resulting in
"leaky" channels that cause decreased exercise capacity in mice.
Treatment with 5107 prevented depletion of calstabin1 from the RyR1
complex, improved force generation and exercise capacity, and
reduced Ca.sup.2+ dependent neutral protease calpain activity and
plasma creatinine kinase levels.
[0014] Duchenne muscular dystrophy (DMD) is one of the leading
lethal childhood genetic diseases. DMD is X-linked, affecting 1 in
3,500 male births and typically results in death by .about.30 y of
age from respiratory or cardiac failure. Mutations in dystrophin
associated with DMD lead to a complete loss of the dystrophin
protein, thereby disrupting the link between the subsarcolemma
cytoskeleton and the extracellular matrix. This link is essential
for protecting and stabilizing the muscle against contraction
induced injury. Currently, there is no cure for DMD and most
treatments in the clinic are palliative. Emerging interventions in
Phase I/II clinical trials are exon skipping, myostatin inhibition,
and up-regulation of utrophin. However, problems with systemic
delivery, sustaining exon skipping, and up-regulation of utrophin
exist. In addition, in Phase I/II clinical trials, inactivation of
myostatin to increase muscle size did not show improved muscle
strength or function. Sarcolemmal instability due to mutations in
dystrophin has a cascade effect. One major effect is increased
cytosolic Ca.sup.2+ concentration, which leads to activation of
Ca.sup.2+ dependent proteases (calpains). Another effect is
inflammation and elevated iNOS activity, which can cause
oxidation/nitrosylation of proteins, lipids, and DNA. DMD muscle
pathology is progressive and far exceeds the instability of the
sarcolemma. Thus the pathology is consistent with the instability
of the sarcolemma increasing the susceptibility to further injury.
It was recently demonstrated that excessive oxidation or
nitrosylation of RyR1 can disrupt the interaction of calstabin1
with the RyR1 complex, leading to RyR1 leakiness and muscle
weakness in a mouse model of muscular dystrophy (mdx) and that
treatment with 5107 improves indices of muscle function in this
mouse model (Bellinger, A. et al. 2009, Nature Medicine,
15:325-330).
[0015] Age-related loss of muscle mass and force (sarcopenia)
contributes to disability and increased mortality. Andersson, D. et
al. (Cell Metab. 2011 Aug. 3; 14(2):196-207) reported that RyR1
from aged (24 months) mice is oxidized, cysteine-nitrosylated, and
depleted of calstabin1, compared to RyR1 from younger (3-6 months)
adults. This RyR1 channel complex remodeling resulted in "leaky"
channels with increased open probability, leading to intracellular
calcium leak in skeletal muscle. Treating aged mice with 5107
stabilized binding of calstabin1 to RyR1, reduced intracellular
calcium leak, decreased reactive oxygen species (ROS), and enhanced
tetanic Ca.sup.2+ release, muscle-specific force, and exercise
capacity.
[0016] PCT International patent publications WO 2005/094457, WO
2006/101496 and WO 2007/024717 disclose 1,4-benzothiazepine
derivatives and their use in treating cardiac, skeletal muscular
and cognitive disorders, among others.
[0017] PCT International patent publication WO 2008/060332 relates
to the use of 1,4-benzothiazepine derivatives for treating muscle
fatigue in subjects suffering from pathologies such as muscular
dystrophy, or in subjects suffering from muscle fatigue as a result
of sustained, prolonged and/or strenuous exercise, or chronic
stress.
[0018] PCT International patent publication WO 2008/021432 relates
to the use of 1,4-benzothiazepine derivatives for the treatment
and/or prevention of diseases, disorders and conditions affecting
the nervous system.
[0019] PCT International patent publication WO 2012/019076 relates
to the use of 1,4-benzothiazepine derivatives for the treatment
and/or prevention of cardiac ischemia/reperfusion injury.
Fauconnier et al., Proc Natl Acad Sci USA, 2011, 108(32): 13258-63
reported that RyR leak mediated by caspase-8 activation leads to
left ventricular injury after myocardial ischemia-reperfusion, and
that treatment with 5107 inhibited the SR Ca.sup.2+ leak, reduced
ventricular arrhythmias, infarct size, and left ventricular
remodeling at 15 days after reperfusion.
[0020] PCT International patent publication WO 2012/019071 relates
to the use of 1,4-benzothiazepine derivatives for the treatment
and/or prevention of sarcopenia.
[0021] PCT International patent publication WO 2012/037105 relates
to the use of 1,4-benzothiazepine derivatives for the treatment
and/or prevention of stress-induced neuronal disorders and
diseases.
[0022] There is a need to identify new compounds effective for
treating disorders and diseases associated with RyRs, including
skeletal muscular and cardiac disorders and diseases. More
particularly, a need remains to identify new agents that can be
used to treat RyR-associated disorders by, for example, repairing
the leak in RyR channels, and enhancing binding of calstabins to
PKA-phosphorylated/oxidized/nitrosylated RyRs, and to mutant RyRs
that otherwise have reduced affinity for, or do not bind to,
calstabins.
SUMMARY OF THE INVENTION
[0023] The present invention provides novel 1,4-benzothiazepine
derivatives, and their pharmaceutically acceptable salts. In some
embodiments, the compounds of the present invention are ryanodine
receptor (RyR) calcium channel stabilizers, sometimes referred to
as "Ryca1s.TM." The present invention further provides methods of
using these compounds for treating disorders and diseases
associated with RyRs.
[0024] The compounds of the present invention are a selection from
the 1,4-benzothiazepine derivatives described in WO 2007/024717. WO
2007/024717 describes structurally similar compounds, however, as
further described herein, these compounds have been found to be
highly unstable and thus their therapeutic utility as drugs is
limited. The problem underlying the present application is thus to
provide alternative 1,4-benzothiazepine derivatives that are not
only pharmacologically active--but also have favorable properties
such as high metabolic stability, and thus are suitable as drugs in
treating diseases and conditions associated with the RyR, for
example cardiac, skeletal muscular and central nervous system (CNS)
disorders. It has unexpectedly been discovered that compounds of
formula (I) are stable as well as pharmacologically active thus
providing a technical solution to the problem underlying the
present invention.
[0025] The compounds of the present invention are represented by
the structure of Formula (I):
##STR00001##
[0026] wherein
[0027] R is COOH;
[0028] and pharmaceutically acceptable salts thereof.
[0029] The compounds of Formula (I) may be present in the form of a
salt with a pharmaceutically acceptable acid or base. Such salts
are preferably selected from the group consisting of sodium,
potassium, magnesium, hemifumarate, hydrochloride and hydrobromide
salts, with each possibility representing a separate embodiment of
the present invention. One currently preferred salt is the sodium
salt. Another currently preferred salt is the hemifumarate
salt.
[0030] In some specific embodiments, the compound is selected from
the group consisting of compound 1, compound 4 and compound 6, and
pharmaceutically acceptable salts thereof. The structures of these
compounds are described hereinbelow.
[0031] In a preferred embodiment, the compound is represented by
the structure of compound (1):
##STR00002##
[0032] or pharmaceutically acceptable salts thereof.
[0033] In some embodiments, compound 1 is provided as the parent
compound. In other embodiments, however, compound 1 is provided in
the form of a salt with a pharmaceutically acceptable acid or base.
Preferably, such salt is selected from the group consisting of
sodium, potassium, magnesium, hemifumarate, hydrochloride and
hydrobromide salts, with each possibility representing a separate
embodiment of the present invention. One currently preferred salt
is the sodium salt. Another currently preferred salt is the
hemifumarate salt.
[0034] The present invention also provides methods for the
synthesis of compounds of the invention, and salts thereof.
[0035] The present invention also provides pharmaceutical
compositions comprising one or more of the compounds of the
invention, and at least one additive or excipient, e.g., fillers,
diluents, binders, disintegrants, buffers, colorants, emulsifiers,
flavor-improving agents, gellants, glidants, preservatives,
solubilizers, stabilizers, suspending agents, sweeteners, tonicity
agents, wetting agents, emulsifiers, dispersing agents, swelling
agents, retardants, lubricants, absorbents, and
viscosity-increasing agents. The compositions may be presented in
capsules, granules, powders, solutions, sachets, suspensions, or
tablet dosage form.
[0036] The present invention further provides methods of treating
or preventing various disorders, diseases and conditions associated
with RyRs, such as cardiac, musculoskeletal, cognitive, CNS and
neuromuscular disorders and diseases, comprising administering to a
subject in need of such treatment an amount of a compound of
Formula (I) or a salt thereof, effective to prevent or treat a
disorder or disease associated with an RyR. The present invention
also provides a method of preventing or treating a leak in RyR
(including RyR1, RyR2 and RyR3) in a subject, including
administering to the subject an amount of a compound of Formula (I)
or a salt thereof, effective to prevent or treat a leak in RyR.
[0037] In addition, the present invention provides a method of
modulating the binding of RyRs and calstabins in a subject,
including administering to the subject an amount of a compound of
Formula (I) or a salt thereof, effective to modulate the amount of
RyR-bound calstabin.
[0038] The present invention further relates to the use of a
compound of Formula (I) for the manufacture of a medicament for the
treatment and/or prevention of disorders, diseases and conditions
associated with RyRs, such as cardiac, musculoskeletal and
cognitive/CNS disorders and diseases. In another embodiment, the
present invention relates to the use of a compound of Formula (I)
for the manufacture of a medicament for preventing or treating a
leak in RyR. In another embodiment, the present invention relates
to the use of a compound of Formula (I) for the manufacture of a
medicament for modulating the amount of RyR-bound calstabins.
[0039] The methods of the invention can be practiced on an in vitro
system (e.g., cultured cells or tissues) or in vivo (e.g., in a
non-human animal or a human).
[0040] In some embodiments, the compounds of the invention are
provided in combination with exon skipping therapy, e.g., antisense
oligonucleotides (AOs) so as to enhance exon skipping in an mRNA of
interest, e.g., the DMD gene, as further described herein. Other
features and advantages of the present invention will become
apparent from the following detailed description and figures.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1A Immunoblot with calstabin2 antibody showing binding
of calstabin2 to PKA-phosphorylated RyR2 in the absence (-) or
presence of 100 nM compound 1. (+): calstabin binding to non-PKA
phosphorylated RyR2. S36 (U.S. Pat. No. 7,544,678), is used as a
positive control.
[0042] FIG. 1B Immunoblot with calstabin2 antibody showing binding
of calstabin2 to PKA-phosphorylated RyR2 in the absence (-) or
presence of 100 nM compound 2, compound 3 or compound 4. (+):
calstabin binding to non-PKA phosphorylated RyR2. S36 is used as a
positive control.
[0043] FIG. 1C Immunoblot with calstabin1 antibody showing binding
of calstabin1 to PKA-phosphorylated RyR1 in the absence (Neg) or
presence of the indicated concentrations of compound 1 or compound
4. (Pos): calstabin binding to non-PKA phosphorylated RyR1. S36 is
used as a positive control.
[0044] FIG. 2 FIG. 2A: Immunoblot with calstabin1 antibody showing
the levels of calstabin1 in immunoprecipitated RyR1 complexes from
tibialis lysates in mice administered vehicle (50:50 DMSO/PEG),
isoproterenol alone (ISO) or isoproterenol together with the
indicated concentrations of compound 1 in osmotic pumps. S36 is
used as control at 3.6 mM. FIG. 2B: quantification of % calstabin1
rebinding to RyR1.
[0045] FIG. 3 Rat chronic heart failure model induced by
ischemia-reperfusion (I/R) injury. For I/R protocol, the left
anterior descending (LAD) coronary artery was occluded for 1 h.
[0046] FIG. 4 Left ventricular (LV) volumes and ejection fraction
(EF) in rats treated with compound 1 at 5 mg/kg/d (5 MK) or 10
mg/kg/d (10 MK) in drinking water vs. vehicle (H.sub.2O)-treated
and sham-operated animals. Chronic heart failure was induced by
ischemia-reperfusion (I/R) injury. LAD artery was occluded for 1 h;
treatment started 1 week after reperfusion and continued for 3
months. Echocardiographic parameters were obtained after 1, 2 or 3
months of treatment. FIG. 4A: LV End Diastolic Volume; FIG. 4B: LV
End Systolic Volume; FIG. 4C: EF. FIGS. 4A and 4B: .sctn.
P<0.001 vs. sham; * P<0.05 vs. vehicle; .dagger. P<0.001
vs. vehicle. FIG. 4C: .sctn. P<0.001 vs. sham, .dagger.
P<0.001 vs. vehicle.
[0047] FIG. 5 FIGS. 5A-C depict body weight (BW) (5A), Infarct size
(5B), and LV weight (5C), and FIG. 5D depicts collagen content in
rats treated with compound 1 at 5 mg/kg/d (5 MK) and 10 mg/kg/day
(10 MK) in drinking water vs. vehicle (H.sub.2O)-treated and
sham-operated animals. Chronic heart failure was induced by
ischemia-reperfusion (I/R) injury. LAD artery was occluded for 1 h;
treatment started 1 week after reperfusion and continued for 3
months. Parameters were measured after 3 months of treatment. FIGS.
5A-C: not significant. FIG. 5D: .dagger..dagger..dagger.<0.001
vs. sham; * P<0.05 vs. vehicle.
[0048] FIG. 6 Invasive hemodynamics: Left ventricular systolic
pressure (LV SP) (6A), dP/dtmax (6B); and dP/dtmin (6C) in rats
treated with compound 1 at 5 mg/kg/d (5 MK) or 10 mg/kg/day (10 MK)
in drinking water vs. vehicle (H.sub.2O)-treated and sham-operated
animals. Chronic heart failure was induced by ischemia-reperfusion
(I/R) injury. LAD artery was occluded for 1 h; treatment started 1
week after reperfusion and continued for 3 months. Hemodynamic
parameters were measured after 3 months of treatment. FIG. 6A: not
significant. FIG. 6B: .sctn. P<0.05 vs. sham; * P<0.05 vs.
vehicle. FIG. 6C: .dagger. P<0.01 vs. sham; *P<0.05 vs.
vehicle.
[0049] FIG. 7 Compound 1 plasma concentrations (.mu.M) vs. time of
day.
[0050] FIG. 8 EF in rats treated with compound 1 or compound A at 5
mg/kg/d (5 MK) in drinking water vs. vehicle (H.sub.2O)-treated and
sham-operated animals. LAD artery was occluded for 1 h; treatment
started 1 week after reperfusion and continued for 3 months.
Echocardiographic parameters were obtained after 1, 2 or 3 months
of treatment. .sctn. P<0.001 vs. sham; * P<0.05 vs. vehicle;
.dagger. P<0.001 vs. vehicle.
[0051] FIG. 9 Effect of compound 1 on spontaneous physical activity
of mdx and WT mice as compared with vehicle (H.sub.2O)-treated
controls. P<0.001 for days 1-19 activity in mdx mice dosed with
10 and 50 mg/kg/day (target dose) administered in drinking water,
compared to vehicle control.
[0052] FIG. 10 Specific force-frequency relationship of EDL muscle.
(A) mdx mice treated with compound 1 (5, 10 and 50 mg/kg/d (target
dose)) administered in drinking water, as compared with vehicle
(H.sub.2O)-treated controls (n=5). p<0.05, for the 50 mg/kg/d
dose, at frequencies of 150 Hz and above. (B) WT, C57BL/6, mice
treated with compound 1 (50 mg/kg/d (target dose) administered in
drinking water, as compared with vehicle (H.sub.2O)-treated
controls (n=4).
[0053] FIG. 11 Average body weight (11A) and average water
consumption (11B) of mdx and WT mice treated with vehicle
(H.sub.2O) or compound 1 administered in drinking water.
DETAILED DESCRIPTION OF THE INVENTION
[0054] It should be understood 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.
[0055] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural references unless the
content clearly dictates otherwise. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety.
[0056] The term "Ryca1s.TM." refers to ryanodine receptor calcium
channel stabilizers, represented by compounds of the general
Formula (I) or (IA) as provided by the invention, as well as the
specific compounds designated by numerical numbers as provided by
the invention, and herein collectively referred to as "compound(s)
of the invention".
Compounds
[0057] In some embodiments, the compounds of the present invention
are represented by the structure of Formula (IA):
##STR00003##
[0058] wherein
[0059] R is COOH or a bioisostere thereof, COOR.sup.1 or CN;
and
[0060] R.sup.1 is a C.sub.1-C.sub.4 alkyl;
[0061] and pharmaceutically acceptable salts thereof.
[0062] In some preferred embodiments, R in Formula (IA) is a
carboxylic acid (COOH). In other preferred embodiments, R in
Formula (IA) is a carboxylic acid bioisostere, for example
tetrazole. Alternatively, the carboxylic acid bioisostere may be an
acidic heterocycle such as 1,2,4-oxadiazol-5(4H)-one,
1,2,4-thiadiazol-5(4H)-one, 1,2,4-oxadiazole-5(4H)-thione,
1,3,4-oxadiazole-2(3H)-thione,
4-methyl-1H-1,2,4-triazole-5(4H)-thione, 5-fluoroorotic acid, and
the like. Additional carboxylic acid bioisosteres are described in,
e.g., Hamada, Y. et al., Bioorg. Med. Chem. Lett. 2006;
16:4354-4359; Herr, R. J. et al., Bioorg. Med. Chem. 2002; 10:
3379-3393; Olesen, P. H., Curr. Opin. Drug Discov. Devel. 2001; 4:
471; Patani. G. A. et al., J. Chem. Rev. 1996; 96:3147; Kimura, T.
et al. Bioorg. Med. Chem. Lett. 2006; 16: 2380-2386; and Kohara, Y.
et al. Bioorg. Med. Chem. Lett. 1995; 5(17): 1903-1908. The
contents of each of the aforementioned references are incorporated
by reference herein.
[0063] In one preferred embodiment, the compounds of the present
invention are represented by the structure of Formula (IA) wherein
R is COOH and pharmaceutically acceptable salts thereof (i.e., a
compound of formula (I)).
[0064] In other preferred embodiments, R in Formula (IA) is at
position 4 of the phenyl ring (i.e., position 7 of the
benzothiazepine ring). Each possibility represents a separate
embodiment of the present invention. The compounds of Formula (IA)
or (I) may be present in the form of a salt with a pharmaceutically
acceptable acid or base. Such salts are preferably selected from
the group consisting of sodium, potassium, magnesium, hemifumarate,
hydrochloride and hydrobromide salts, with each possibility
representing a separate embodiment of the present invention. One
currently preferred salt is the sodium salt. Another currently
preferred salt is the hemifumarate salt.
[0065] In some specific embodiments, the compound is selected from
the group consisting of compound 1, compound 2, compound 3,
compound 4, compound 5, compound 6, compound 7, compound 8,
compound 9, compound 10, compound 11, and compound 12, and
pharmaceutically acceptable salts thereof. These compounds are
represented by the following structures:
##STR00004## ##STR00005##
CHEMICAL DEFINITIONS
[0066] The term "alkyl" as used herein refers to a linear or
branched, saturated hydrocarbon having from 1 to 4 carbon atoms
("C.sub.1-C.sub.4 alkyl"). Representative alkyl groups include, but
are not limited to, methyl, ethyl, propyl, isopropyl, butyl,
sec-butyl, and tert-butyl. The alkyl group may be unsubstituted or
substituted by one or more groups selected from halogen, haloalkyl,
hydroxy, alkoxy, haloalkoxy, cycloalkyl, aryl, heterocyclyl,
heteroaryl, amido, alkylamido, dialkylamido, nitro, amino, cyano,
N.sub.3, oxo, alkylamino, dialkylamino, carboxyl, thio, thioalkyl
and thioaryl.
[0067] Compounds of the present invention may exist in their
tautomeric form. All such tautomeric forms are contemplated herein
as part of the present invention.
[0068] All stereoisomers of the compounds of the present invention
(for example, those which may exist due to asymmetric carbons on
various substituents), including enantiomeric forms and
diastereomeric forms, are contemplated within the scope of this
invention. Individual stereoisomers of the compounds of the
invention may, for example, be substantially free of other isomers
(e.g., as a pure or substantially pure optical isomer having a
specified activity), or may be admixed, for example, as racemates,
or as mixtures enriched by one stereoisomer. The chiral centers of
the present invention may have the S or R configuration as defined
by the IUPAC 1974 Recommendations. The racemic forms can be
resolved by physical methods, such as, for example, fractional
crystallization, separation or crystallization of diastereomeric
derivatives or separation by chiral column chromatography. The
individual optical isomers can be obtained from the racemates by
any suitable method, including without limitation, conventional
methods, such as, for example, salt formation with an optically
active acid or base, followed by crystallization.
[0069] Compounds of the present invention are, subsequent to their
preparation, preferably isolated and purified to obtain a
composition containing an amount by weight equal to or greater than
about 90% of the compound, about 95% of the compound, and even more
preferably greater than about 99% of the compound ("substantially
pure" compound), which is then used or formulated as described
herein. Such "substantially pure" compounds of the present
invention are also contemplated herein as part of the present
invention.
[0070] Therapeutic Use
[0071] The present invention provides compounds that are capable of
treating conditions, disorders and diseases associated with RyRs.
More particularly, the present invention provides compounds that
are capable of fixing a leak in RyR channels, which may be RyR1,
RyR2 and/or RyR3 channels. In one embodiment, the compounds of the
invention enhance association and/or inhibit dissociation of RyR
and calstabin (e.g., RyR1 and calstabin1; RyR2 and calstabin2; and
RyR3 and calstabin1). "Conditions, disorders and diseases
associated with RyRs" means disorders and diseases that can be
treated and/or prevented by modulating RyRs and include, without
limitation, cardiac disorders and diseases, muscle fatigue,
musculoskeletal disorders and diseases, CNS disorders and diseases,
cognitive dysfunction, neuromuscular diseases and disorders,
cognitive function improvement, bone disorders and diseases, cancer
cachexia, malignant hyperthermia, diabetes, sudden cardiac death,
and sudden infant death syndrome.
[0072] Thus, in one embodiment, the present invention relates to a
method of treating or preventing a condition selected from the
group consisting of cardiac disorders and diseases, muscle fatigue,
musculoskeletal disorders and diseases, CNS disorders and diseases,
cognitive dysfunction, neuromuscular diseases and disorders, bone
disorders and diseases, cancer cachexia, malignant hyperthermia,
diabetes, sudden cardiac death, and sudden infant death syndrome,
or for improving cognitive function, the method comprising the step
of administering to a subject in need thereof a therapeutically
effective amount of a compound of Formula (I) or (IA) as described
herein, or a salt thereof, to effectuate such treatment. A
currently preferred compound is a compound of Formula (1).
[0073] In another embodiment, the present invention relates to the
use of an effective amount of compound of Formula (I) or (IA), as
described herein, or a salt thereof, for the manufacture of a
medicament for treating or preventing a condition selected from the
group consisting of cardiac disorders and diseases, muscle fatigue,
skeletal muscular disorders and diseases, CNS disorders and
diseases, neuromuscular disorder and diseases, cognitive
dysfunction, bone disorders and diseases, cancer cachexia,
malignant hyperthermia, diabetes, sudden cardiac death, and sudden
infant death syndrome, or for improving cognitive function. A
currently preferred compound is a compound of Formula (1).
[0074] In another embodiment, the present invention relates to a
compound of Formula (I) or (IA) as described herein, or a salt
thereof, for use in the manufacture of a medicament for treating or
preventing a condition selected from the group consisting of
cardiac disorders and diseases, muscle fatigue, skeletal muscular
disorders and diseases, CNS disorders and diseases, cognitive
dysfunction, neuromuscular diseases and disorders, bone disorders
and diseases, cancer cachexia, malignant hyperthermia, diabetes,
sudden cardiac death, and sudden infant death syndrome, or for
improving cognitive function. A currently preferred compound is a
compound of Formula (1).
[0075] In one embodiment, the condition, disorder or disease is
associated with an abnormal function of RyR1. In another
embodiment, the condition, disorder or disease is associated with
an abnormal function of RyR2. In another embodiment, the condition,
disorder or disease is associated with an abnormal function of
RyR3. Each possibility represents a separate embodiment of the
present invention.
[0076] Cardiac disorders and diseases include, but are not limited
to, irregular heartbeat disorders and diseases, exercise-induced
irregular heartbeat disorders and diseases, heart failure,
congestive heart failure, chronic heart failure, acute heart
failure, systolic heart failure, diastolic heart failure, acute
decompensated heart failure, cardiac ischemia/reperfusion (I/R)
injury (including I/R injury following coronary angioplasty or
following thrombolysis during myocardial infarction (MI)), chronic
obstructive pulmonary disease, and high blood pressure. 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.
[0077] The compounds of the invention are also useful in treating
muscle fatigue, which may be due to prolonged exercise or
high-intensity exercise, or may be caused by musculoskeletal
diseases. Examples of muscular disorders and diseases include, but
are not limited to, skeletal muscle fatigue, central core diseases,
exercise-induced skeletal muscle fatigue, bladder disorders,
incontinence, age-associated muscle fatigue, sarcopenia, congenital
myopathies, skeletal muscle myopathies and/or atrophies, cancer
cachexia, myopathy with cores and rods, mitochondrial myopathies
[e.g., Kearns-Sayre syndrome, MELAS (mitochondrial myopathy,
encephalopathy, lactic acidosis, and stroke) syndrome, and MERRF
(myoclonus epilepsy with ragged-red fibers) syndrome], endocrine
myopathies, muscular glycogen storage diseases [e.g., Pompe's
disease, Andersen's disease, and Cori's diseases], myoglobinurias
[e.g., McArdle's disease, Tarui disease, and DiMauro disease],
dermatomyositis, myositis ossificans, familial periodic paralysis,
polymyositis, inclusion body myositis, neuromyotonia, stiff-man
syndrome, malignant hyperthermia, common muscle cramps, tetany,
myasthenia gravis, spinal muscular atrophy (SMA), Spinal and bulbar
muscular atrophy (SBMA, also known as spinobulbar muscular atrophy,
bulbo-spinal atrophy, X-linked bulbospinal neuropathy (XBSN),
X-linked spinal muscular atrophy type 1 (SMAX1), and Kennedy's
disease (KD)), and muscular dystrophy. Preferred skeletal muscular
disorders include, but are not limited to exercise-induced skeletal
muscle fatigue, a congenital myopathy, muscular dystrophy,
age-related muscle fatigue, sarcopenia, central core disease,
cancer cachexia, bladder disorders, and incontinence.
[0078] Examples of muscular dystrophy include, but are not limited
to, Duchenne Muscular Dystrophy (DMD), Becker's Muscular Dystrophy
(BMD), Limb Girdle Muscular Dystrophy (LGMD), Congenital Muscular
Dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral
dystrophy, myotonic muscular dystrophy, Emery-Dreifuss muscular
dystrophy, and oculopharyngeal muscular dystrophy, with DMD being
currently preferred.
[0079] Congenital muscular dystrophy as used herein refers to
muscular dystrophy that is present at birth. CMD is classified
based on genetic mutations: 1) genes encoding for structural
proteins of the basal membrane or extracellular matrix of the
skeletal muscle fibres; 2) genes encoding for putative or
demonstrated glycosyltransferases, that in turn affect the
glycosylation of dystroglycan, an external membrane protein of the
basal membrane; and 3) other. Examples of CMD include, but are not
limited to Laminin-.alpha.2-deficient CMD (MDC1A), Ullrich CMG
(UCMDs 1, 2 and 3), Walker-Warburg syndrome (WWS), Muscle-eye-brain
disease (MEB), Fukuyama CMD (FCMD), CMD plus secondary laminin
deficiency 1 (MDC1B), CMD plus secondary laminin deficiency 2
(MDC1C), CMD with mental retardation and pachygyria (MDC1D), and
Rigid spine with muscular dystrophy Type 1 (RSMD1).
[0080] Cognitive dysfunction may be associated with or includes,
but is not limited to memory loss, age-dependent memory loss,
post-traumatic stress disorder (PTSD), attention deficit
hyperactivity disorder (ADHD), autism spectrum disorder (ASD),
generalized anxiety disorder (GAD), obsessive compulsive disorder
(OCD), Schizophrenia, Bipolar disorder, or major depression
[0081] CNS disorders and diseases include, but are not limited to
Alzheimer's Disease (AD), neuropathy, seizures, Parkinson's Disease
(PD), and Huntington's Disease (HD).
[0082] Neuromuscular disorders and diseases include, but are not
limited to Spinocerebellar ataxia (SCA), and Amyotrophic lateral
sclerosis (ALS, Lou Gehrig's disease).
[0083] In some embodiments, the compounds of the present invention
improve cognitive function, which may be selected from short term
memory, long term memory, attention, learning, and any combination
thereof.
[0084] In some embodiments, the compounds of the present invention
are useful in the treatment of cancer cachexia, i.e., muscle
weakness which is associated with cancer in general, and preferably
muscle weakness in metastatic cancer, such as bone metastases.
Muscle weakness and muscle atrophy (cachexia) are common
paraneoplastic symptoms in cancer patients. These conditions cause
significant fatigue and dramatically reduce patients' quality of
life. The present invention provides a method for treating and
preventing muscle weakness in a cancer patient, based, in part, on
the discovery that, in certain types of cancers, e.g., prostate and
breast cancer with bone metastases, RyR1 is oxidized which induces
it to become "leaky". It has further been found that prevention of
the leak by administration of Ryca1 compounds improves muscle
function. Exemplary cancers include, but are not limited to, breast
cancer, prostate cancer, bone cancer, pancreatic cancer, lung
cancer, colon cancer, and gastrointestinal cancer.
Exon Skipping Therapy:
[0085] In some embodiments, the compounds of the present invention
modulate (e.g., enhance) mRNA splicing by enhancing
antisense-mediated exon skipping. This modulation of splicing is
accomplished in the presence of antisense oligonucleotides (AOs)
that are specific for splicing sequences of interest. In some
embodiments of the invention, the compound of formula (I) or (IA)
and the AO can act synergistically wherein the compound of formula
(I) or (IA) enhances AO mediated exon skipping. Thus, in some
embodiments, the present invention relates to a pharmaceutical
composition for use in the treatment or prevention of any of the
conditions described herein that are associated with Leaky RyR,
further comprising the use of an antisense AO which is specific for
a splicing sequence in an mRNA sequence, for enhancing exon
skipping in the mRNA of interest.
[0086] One particular embodiment for exon skipping enhancement by
the compounds of the present invention pertains to Duchenne
Muscular Dystrophy (DMD). DMD is a lethal X-linked recessive
disease characterized by progressive muscle weakness over a
patient's lifetime. DMD is primarily caused by out of frame
multi-exon deletions in the DMD gene that ablate dystrophin protein
production. Loss of dystrophin expression alone does not explain
DMD pathophysiology. Disruption of the dystrophin-glycoprotein
complex (DGC) also results in oxidative stress, mitochondrial
Ca.sup.2+ overload and apoptosis, increased influx of Ca.sup.2+
into the muscle, and pathologic Ca.sup.2+ signaling. There are no
curative therapies for DMD, and the only demonstrated
pharmacological treatment is corticosteroids, which may prolong
ambulation, but have substantial side effects. Antisense
oligonucleotide-mediated exon skipping is a promising therapeutic
approach aimed at restoring the DMD reading frame and allowing
expression of an intact dystrophin glycoprotein complex. To date,
low levels of dystrophin protein have been produced in humans by
this method. Kendall et al. (Sci Transl Med, 2012, 4(164), p.
164ra160) reported that certain small molecules such as Dantrolene
and other RyR modulators, potentiate antisense oligomer-guided exon
skipping to increase exon skipping to restore the mRNA reading
frame, the sarcolemmal dystrophin protein, and the dystrophin
glycoprotein complex in skeletal muscle of mdx mice, a mouse model
of DMD.
[0087] Thus, in one embodiment, the present invention relates to a
method for treating DMD, by administering to a subject in need
thereof a compound of formula (I) or (IA) according to the present
invention, in combination with an antisense oligonucleotide (AO)
which is specific for a splicing sequence of one or more exons of
the DMD gene, for example exon 23, 45, 44, 50, 51, 52 and/or 53 of
the DMD gene. Preferred AOs include, but are not limited to, AOs
targeting DMD exon 23, 50 and/or 51 of the DMD gene, such as
2'-O-methyl (2'OMe) phosphorothioate or phosphorodiamidate
morpholino (PMO) AOs. Examples of such AOs include, but not limited
to, Pro051/GSK2402968, AVI4658/Eteplirsen, and PMO E23 morpholino
(5'-GGCCAAACCTCGGCTTACCTGAAAT-3').
[0088] The term an "effective amount," "sufficient amount" or
"therapeutically effective amount" of an agent as used herein
interchangeably, is that amount sufficient to effectuate beneficial
or desired results, including clinical results and, as such, an
"effective amount" or its variants depends upon the context in
which it is being applied. The response is in some embodiments
preventative, in others therapeutic, and in others a combination
thereof. The term "effective amount" also includes the amount of a
compound of the invention, which is "therapeutically effective" and
which avoids or substantially attenuates undesirable side
effects.
[0089] 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.
Pharmaceutical Compositions
[0090] 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 the invention in
admixture with a pharmaceutically acceptable diluent and/or
carrier. The pharmaceutically-acceptable carrier is preferably
"acceptable" in the sense of being compatible with the other
ingredients of the composition and not deleterious to the recipient
thereof.
[0091] The compound may be administered alone, but is preferably
administered with one or more pharmaceutically acceptable carriers.
The pharmaceutically-acceptable carrier employed herein may be
selected from various organic or inorganic materials that are used
as materials for pharmaceutical formulations and which are
incorporated as any one or more of fillers, diluents, binders,
disintegrants, buffers, colorants, emulsifiers, flavor-improving
agents, gellants, glidants, preservatives, solubilizers,
stabilizers, suspending agents, sweeteners, tonicity agents,
wetting agents, emulsifiers, dispersing agents, swelling agents,
retardants, lubricants, absorbents, and viscosity-increasing
agents.
[0092] The compounds of the present invention are administered to a
human or animal subject by known procedures including, without
limitation, oral, sublingual, buccal, parenteral (intravenous,
intramuscular or subcutaneous), transdermal, per- or
trans-cutaneous, intranasal, intra-vaginal, rectal, ocular, and
respiratory (via inhalation administration). The compounds of the
invention may also be administered to the subject by way of
delivery to the subject's muscles including, but not limited to,
the subject's cardiac or skeletal muscles. In one embodiment, the
compound is administered to the subject by way of targeted delivery
to cardiac muscle cells via a catheter inserted into the subject's
heart. In other embodiments, the compounds may be administered
directly into the CNS, for example by intralumbar injection or
intreventricular infusion of the compounds directly into the
cerebrospinal-fluid (CSF), or by intraventricular, intrathecal or
interstitial administration. Oral administration is currently
preferred.
[0093] The pharmaceutical compositions according to the invention
for solid oral administration include especially tablets or
dragees, sublingual tablets, sachets, capsules including gelatin
capsules, powders, and granules, and those for liquid oral, nasal,
buccal or ocular administration include especially emulsions,
solutions, suspensions, drops, syrups and aerosols. The compounds
may also be administered as a suspension or solution via drinking
water or with food. Examples of acceptable pharmaceutical carriers
include, but are not limited to, cellulose derivatives including
carboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose,
hydroxypropylmethylcellulose, ethyl cellulose and microcrystalline
cellulose; sugars such as mannitol, sucrose, or lactose; glycerin,
gum arabic, magnesium stearate, sodium stearyl fumarate, saline,
sodium alginate, starch, talc and water, among others.
[0094] The pharmaceutical compositions according to the invention
for parenteral injections include especially sterile solutions,
which may be aqueous or non-aqueous, dispersions, suspensions or
emulsions and also sterile powders for the reconstitution of
injectable solutions or dispersions. The compounds of the invention
may be 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.
[0095] The pharmaceutical compositions for rectal or vaginal
administration are preferably suppositories, and those for per- or
trans-cutaneous administration include especially powders,
aerosols, creams, ointments, gels and patches.
[0096] For transdermal administration, the compounds of the
invention 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 the invention and
permit the compounds to penetrate through the skin and into the
bloodstream. The compound/enhancer compositions also may be 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 is dissolved in a solvent, evaporated to the desired
viscosity and then applied to backing material to provide a
patch.
[0097] The pharmaceutical formulations of the present invention are
prepared by methods well-known in the pharmaceutical arts,
including but not limited to wet and dry granulation methods, or by
direct compression. The choice of carrier is determined by the
solubility and chemical nature of the compounds, chosen route of
administration and standard pharmaceutical practice.
[0098] The pharmaceutical compositions mentioned above illustrate
the invention but do not limit it in any way.
[0099] In accordance with the methods of the present invention, any
of these compounds may be 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 calstabin 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 the invention effective to limit or prevent a
decrease in the level of RyR-bound calstabin in the subject ranges
from about 0.01 mg/kg/day to about 100 mg/kg/day (e.g., 1, 2, 5,
10, 20, 25, 50 or 100 mg/kg/day), and/or is an amount sufficient to
achieve plasma levels ranging from about 300 ng/ml to about 5,000
ng/ml. Alternatively, the amount of compounds from the invention
ranges from about 1 mg/kg/day to about 50 mg/kg/day. Alternatively,
the amount of compounds from the invention ranges from about 10
mg/kg/day to about 20 mg/kg/day. Also included are amounts of from
about 0.01 mg/kg/day or 0.05 mg/kg/day to about 5 mg/kg/day or
about 10 mg/kg/day which can be administered.
Methods of Synthesis
[0100] The present invention provides, in a further aspect,
processes for the preparation of a compound of the invention, and
salts thereof. More particularly, the present invention provides
processes for the preparation of compounds of Formula (I) or (IA),
e.g., compound 1, compound 2, compound 3, compound 4, compound 5,
compound 6, compound 7, compound 8, compound 9, compound 10,
compound 11, and compound 12, or salts thereof. The various
synthetic routes to the compounds are described in the examples.
The general route of synthesis (ROS) is set forth in Scheme 1
below:
##STR00006##
[0101] In Scheme 1, R.sup.aCOOR.sup.1 or CN; R.sup.1 is a
C.sub.1-C.sub.4 alkyl, and L is a leaving group, which is, by way
of example, a halogen, a sulfonate (OSO.sub.2R' wherein R' is alkyl
or aryl, e.g., OMs (mesylate), OTs (tosylate)), and the like. The
amine starting material is reacted with the alkylating agent
(benzyl derivative shown above), preferably in the presence of a
base, to yield the desired product or a precursor thereof
(R.dbd.R.sup.a). If desired, such precursor may further be reacted
to convert the group R.sup.a to the group R as exemplified in the
experimental section hereinbelow, or by any other method known to a
person of skill in the art. For example, an ester precursor
(R.sup.a=COOR.sup.1 wherein R.sup.1 is a C.sub.1-C.sub.4 alkyl),
can be converted into the corresponding carboxylic acid
(R.dbd.COOH) by hydrolysis under acidic or basic conditions in
accordance with known methods. Alternatively, a nitrile precursor
(R.sup.a=CN) can be converted into a tetrazole (a carboxylic acid
isostere) by reaction with sodium azide under suitable conditions,
or to a carboxylic acid (R.dbd.COOH) by hydrolysis.
[0102] The amine starting material may be prepared in accordance
with the methods described in WO 2009/111463 or WO 2007/024717, or
by any other method known to a person of skill in the art. The
contents of all of the aforementioned references are incorporated
by reference herein. The nature of the base is not particularly
limiting. Preferred bases include, but are not limited to, hydrides
(e.g., sodium or potassium hydride) and N,N-diisopropylethylamine.
Other suitable bases include, but are not limited to an organic
base such as a tertiary amine, selected from the group consisting
of acyclic amines (e.g., trimethylamine, triethylamine,
dimethylphenylamine diisopropylethylamine and tributylamine),
cyclic amines (e.g., N-methylmorpholine) and aromatic amines
(dimethylaniline, dimethylaminopyridine and pyridine).
[0103] The reaction may be conducted in the presence or absence of
a solvent. The nature of the solvent, when used, is not
particularly limiting, with examples including solvents such an
ester (e.g., ethyl acetate), an ether (e.g., THF), a chlorinated
solvent (e.g., dichloromethane or chloroform), dimethylformamide
(DMF), and other solvents such as acetonitrile or toluene or
mixtures of these solvents with each other or with water.
[0104] Salts of compounds of formula (I) wherein R.dbd.COOH may be
prepared by reacting the parent molecule with a suitable base,
e.g., NaOH or KOH to yield the corresponding alkali metal salts,
e.g., the sodium or potassium salts. Alternatively, esters
(R.dbd.COOR.sup.1) may be directly converted to salts by reactions
with suitable bases.
[0105] Salts of compounds of formula (I) may also be prepared by
reacting the parent molecule with a suitable acid, e.g., HCl,
fumaric acid, or para-toluenesulfonic acid to yield the
corresponding salts, e.g., hydrochloride, tosylate or
hemi-fumarate.
EXAMPLES
[0106] The following examples are provided as illustrations of the
some preferred embodiments according to the invention.
Example 1
Synthesis
Instruments:
[0107] NMR: Bruker AVANCE III 400 or Varian Mercury 300
[0108] LC/MS: Waters Delta 600 equipped with Autosampler 717Plus,
Photo Diode Array Detector 2996, and Mass Detector 3100, or
Shimadzu 210
General procedure for the alkylation of
7-methoxy-2,3,4,5-tetrahydrobenzo[f][1,4]thiazepine ("Amine").
##STR00007##
[0109] Amine (structure shown above) (1 mmol) was dissolved in 3 ml
dichloromethane. To the solution was added alkylation reagent (1
mmol), followed by N,N-diisopropylethylamine (0.34 ml, 2 mmol). The
mixture was stirred at room temperature overnight. The solution was
loaded onto column directly and eluted with hexane/EtOAc (2:1,
v/v).
##STR00008##
Compound 2
[0110] Methyl
3-((7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)benzoate.
.sup.1HNMR (300 MHz, CDCl.sub.3): 7.96 (m, 2H), 7.46 (m, 3H), 6.70
(dd, J=8.4 Hz, 3.0 Hz, 1H), 6.50 (d, J=2.7 Hz, 1H), 4.09 (s, 2H),
3.90 (s, 3H), 3.72 (s, 3H), 3.57 (s, 2H), 3.35 (m, 2H), 2.72 (m,
2H). MS: 344(M+1)
##STR00009##
Compound 3
[0111] Methyl
4-((7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)benzoate:
.sup.1HNMR (300 MHz, CDCl.sub.3): 7.99 (d, J=8.4 Hz, 2H), 7.46 (d,
J=8.4 Hz, 1H), 7.37 (d, J=8.7 Hz, 2H), 6.70 (dd, J=8.4 Hz, 3.0 Hz,
1H), 6.50 (d, J=2.7 Hz, 1H), 4.09 (s, 2H), 3.90 (s, 3H), 3.72 (s,
3H), 3.57 (s, 2H), 3.35 (m, 2H), 2.72 (m, 2H). MS: 344(M+1)
##STR00010##
Compound 5
[0112] Methyl
2-((7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)benzoate:
The compound was converted to hydrochloride salt with 2M HCl in
ether. .sup.1HNMR (300 MHz, DMSO-d.sub.6): 10.33 (br, 1H), 8.08 (d,
J=7.5 Hz, 1H), 7.80-7.65 (m, 3H), 7.51 (d, J=8.1 Hz, 1H), 7.14 (s,
1H), 6.99 (dd, J=8.4, 2.1 Hz, 1H), 4.90-4.40 (m, br, 4H), 3.88 (s,
3H), 3.78 (s, 3H), 3.40 (m, 2H), 3.26 (m, 1H), 3.11 (m, 1H). MS:
344 (M+1)
##STR00011##
Compound 7
[0113]
2-((7-Methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)ben-
zonitrile: .sup.1HNMR (300 MHz, CDCl.sub.3): 7.67-7.26 (m, 5H),
6.73 (d, J=2.7 Hz, 1H), 6.74 (dd, J=2.7, 8.4 Hz, 1H), 4.14 (s, 2H),
3.78 (s, 3H), 3.70 (s, 2H), 3.36 (m, 2H), 2.76 (m, 2H). MS: 311
(M+1)
##STR00012##
Compound 8
[0114]
3-((7-Methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)ben-
zonitrile: .sup.1HNMR (300 MHz, CDCl.sub.3): 7.64-7.42 (m, 5H),
6.74 (dd, J=2.7, 8.4 Hz, 1H), 6.48 (d, J=2.7 Hz, 1H), 4.08 (s, 2H),
3.75 (s, 3H), 3.57 (s, 2H), 3.36 (m, 2H), 2.76 (m, 2H). MS: 311
(M+1)
##STR00013##
Compound 9
[0115]
4-((7-Methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)ben-
zonitrile: .sup.1HNMR (300 MHz, CDCl.sub.3): 7.64 (d, J=7.2 Hz,
2H), 7.42 (m, 3H), 6.74 (dd, J=2.7, 8.4 Hz, 1H), 6.48 (d, J=2.7 Hz,
1H), 4.08 (s, 2H), 3.75 (s, 3H), 3.58 (s, 2H), 3.36 (m, 2H), 2.76
(m, 2H). MS: 311 (M+1)
Hydrolysis of Ester (General Procedure)
[0116] Methyl ester (3 mmol) was dissolved in 30 ml of
THF/methanol/1 M NaOH (1:1:1, v/v). The mixture was stirred for 8
hours and TLC showed complete disappearance of the ester. 1 ml
Conc. HCl was added to adjust to acidic pH. The organic solvent was
removed and the formed solid was collected by filtration. The solid
was dried in the air.
##STR00014##
Compound 4
[0117]
3-((7-Methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)ben-
zoic acid: This was obtained by extraction with EtOAc as solvent.
.sup.1HNMR (300 MHz, CDCl.sub.3): 8.10 (s, 1H), 8.04 (d, J=8.4 Hz,
1H), 7.80 (br, 1H), 7.46 (m, 2H), 6.80 (m, 2H), 4.40 (s, 2H), 3.90
(s, 2H), 3.76 (s, 3H), 3.42 (s, 2H), 2.86 (s, 2H). MS: 330 (M+1),
328 (M-1).
##STR00015##
Compound 1
[0118]
4-((7-Methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)ben-
zoic acid: This was obtained by extraction with EtOAc as solvent.
.sup.1HNMR (300 MHz, CDCl.sub.3): 8.02 (d, J=8.4 Hz, 2H), 7.46 (d,
J=8.4 Hz, 1H), 7.42 (d, J=8.7 Hz, 2H), 6.70 (dd, J=8.4 Hz, 3.0 Hz,
1H), 6.50 (d, J=3.0 Hz, 1H), 4.11 (s, 2H), 3.72 (s, 3H), 3.62 (s,
2H), 3.35 (m, 2H), 2.76 (m, 2H). MS: 330 (M+1), 328 (M-1).
Compound 1, Sodium Salt
[0119] The sodium salt of compound 1 was prepared from the parent
molecule using 1 equivalent of NaOH in EtOH (m.p. of the salt:
>290.degree. C.).
[0120] .sup.1HNMR (DMSO-D6, 600 MHz), .delta. (ppm): 7.77 (2H, m),
7.41 (1H, d), 7.13 (2H, m), 6.75 (1H, dd), 6.63 (1H, d), 4.00 (2H,
s), 3.70 (3H, s), 3.49 (2H, s), 3.18 (2H, m), 2.70 (2H, m).
Compound 1, Hemifumarate Salt
[0121] 1.6 g of compound 1 (neutral form) and 265 mg of fumaric
acid were introduced in a round bottom flask. After addition of 18
mL of acetone and 2 mL of water, the reaction mixture was refluxed.
A partial solubilisation was observed (but no complete
clarification) followed by precipitation. The reaction mixture was
then refluxed overnight. After cooling the residual solid was
isolated by filtration, washed with 3 mL of acetone and dried under
vacuum (40.degree. C./10 mbars) for 4 hours.
[0122] .sup.1HNMR (DMSO-D6, 600 MHz), .delta. (ppm): 12.97 (2H,
bs), 7.90 (2H, m), 7.43 (1H, d), 7.40 (2H, m), 6.77 (1H, dd), 6.64
(1H, d), 6.62 (1H, s), 4.03 (2H, s), 3.70 (3H, s), 3.58 (2H, s),
3.20 (2H, m), 2.72 (2H, m).
##STR00016##
Compound 6
[0123]
2-((7-Methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)methyl)ben-
zoic acid: The compound was converted to hydrochloride salt with 2M
HCl in ether. .sup.1HNMR (300 MHz, DMSO-d.sub.6): 10.10 (br, 1H),
8.08 (d, J=7.5 Hz, 1H), 7.66-7.51 (m, 4H), 7.17 (d, J=2.1 Hz, 1H),
6.99 (dd, J=8.4, 2.1 Hz, 1H), 4.80-4.40 (m, br, 4H), 3.78 (s, 3H),
3.46 (m, 2H), 3.13 (m, 2H). MS: 330(M+1), 328 (M-1).
Synthesis of Tetrazole (General Procedure)
[0124] Nitrile precursor (3.22 mmol), sodium azide (830 mg, 12.9
mmol) and triethylamine hydrochloride (1.72 g, 12.9 mmol) were
stirred in 40 ml anhydrous DMF at 100.degree. C. for 5 days. The
DMF was removed under high vacuum and the residue was mixed with
water. The water solution was extracted with dichloromethane
(3.times.100 ml), The pure compound was purified by column
chromatography (EtOAc/methanol).
##STR00017##
Compound 10
[0125]
4-(2-(1H-Tetrazol-5-yl)benzyl)-7-methoxy-2,3,4,5-tetrahydrobenzo[f]-
[1,4]thiazepine
[0126] .sup.1HNMR (300 MHz, CDCl.sub.3 and a drop of CD3OD): 8.30
(d, J=8.7 Hz, 1H), 7.53 (m, 2H). 7.14 (t, J=7.8 Hz, 1H), 7.20 (d,
J=7.5 Hz, 1H), 6.84 (dd, J=2.7, 8.4 Hz, 1H), 6.69 (d, J=2.7 Hz,
1H), 4.46 (s, 2H), 3.80 (s, 2H), 3.75 (s, 2H), 3.43 (m, 2H), 2.96
(m, 2H). MS: 354(M+1), 352(M-1)
##STR00018##
Compound 11
[0127]
4-(3-(1H-Tetrazol-5-yl)benzyl)-7-methoxy-2,3,4,5-tetrahydrobenzo[f]-
[1,4]thiazepine
[0128] .sup.1HNMR (300 MHz, CDCl.sub.3): 8.16 (s, 1H), 7.90 (d,
J=7.5 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 7.20 (m, 2H), 6.74 (dd,
J=2.7, 8.4 Hz, 1H), 6.58 (d, J=2.7 Hz, 1H), 4.18 (s, 2H), 3.75 (s,
5H), 3.36 (m, 2H), 2.76 (m, 2H).). MS: 354(M+1), 352(M-1)
##STR00019##
Compound 12
[0129]
4-(4-(1H-Tetrazol-5-yl)benzyl)-7-methoxy-2,3,4,5-tetrahydrobenzo[f]-
[1,4]thiazepine .sup.1HNMR (300 MHz, CDCl3 and a drop of CD3OD):
7.99 (d, J=7.2 Hz, 2H), 7.42 (m, 3H), 6.74 (dd, J=2.7, 8.4 Hz, 1H),
6.53 (d, J=2.7 Hz, 1H), 4.10 (s, 2H), 3.71 (s, 3H), 3.58 (s, 2H),
3.36 (m, 2H), 2.76 (m, 2H).). MS: 354(M+1), 352(M-1)
Synthesis of 7-methoxy-2,3,4,5-tetrahydrobenzo[f][1,4]thiazepine
("Amine")
##STR00020##
[0130] 2-(4-Methoxyphenylthio)ethanamine (1)
[0131] 4-Methoxythiophenol (50 g, 0.357 mol), 2-chloroethylamine
monohydrochloride (39.8 g, 0.343 mol.), K.sub.2CO.sub.3(78.8 g,
0.57 mol) and diisopropyl ethylamine (32 mL, 0.178 mol) were mixed
in 200 mL of THF. The mixture was degassed for 5 min. under reduced
pressure and refluxed under argon overnight. The solvent was
removed and water (300 mL) was added to the flask. The mixture was
extracted with dichloromethane (3.times.200 mL). The organics were
collected, dichloromethane was removed and 50 mL conc. HCl was
added, followed by 200 mL of water. The solution was extracted with
1:1 EtOAc/hexane (3.times.200 mL). The aqueous layer was adjusted
to pH 10 with 2 M NaOH, and was extracted with dichloromethane
(3.times.200 mL). The combined organic solution was dried over
anhydrous sodium sulfate. Removal of solvent provided 61 g of the
target compound as a colorless liquid, with a yield of 97%.
[0132] .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.35 (d, J=8.7 Hz, 2H),
6.81 (d, J=8.7 Hz, 2H), 3.77 (s, 3H), 2.88-2.80 (m, 4H), 1.44 (s,
2H).
Benzyl 2-(4-methoxyphenylthio)ethylcarbamate (2)
First Method
[0133] To a the flask containing compound 1 (8.0 g, 43.7 mmol),
sodium bicarbonate (12.1 g, 144 mmol), water (100 mL) and
dichloromethane (200 mL) was added benzyl chloroformate (8.2 g,
48.1 mmol, diluted in 100 mL of dichloromethane) dropwise at
0.degree. C. After the addition, the mixture was stirred at r.t.
for 5 hr. The organic layer was collected and aqueous solution was
extracted with 100 mL of dichloromethane. The combined organic
solution was dried over sodium sulfate. The solvent was removed and
the resulting solid was triturated with 200 mL of THF/hexane
(1:10). The solid was collected and dried leaving the target
product (12.9 g) in the yield of 93%.
Alternative Method
[0134] To the solution of compound 1 (10 g, 54.6 mmol) and
triethylamine (15 mL, 106 mmol) in 200 mL of dichloromethane was
added benzyl chloroformate (7.24 mL, 51.5 mmol, diluted in 100 mL
of dichloromethane) dropwise at 0.degree. C. After the addition,
the solution was stirred at r.t. for one hour. The solid was
removed by filtration. The solution was extracted with 100 mL of
0.1 M HCl and 100 mL of sat. sodium carbonate, and dried over
anhydrous sodium sulfate. Removal of solvent provided a white solid
that was stirred in 200 mL of THF/hexane (1:20) for three hours.
The solid was collected by filtration to give 14.2 g of the target
compound in 87% yield.
[0135] .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.35 (m, 7H), 6.83 (d,
J=8.7 Hz, 2H), 5.07 (m, 3H), 3.77 (s, 3H), 3.10 (q, J=6.3 Hz, 2H),
2.92 (t, J=6.3 Hz, 2H).
Benzyl
7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepine-4(5H)-carboxylate
(3)
[0136] A mixture of compound 2 (7.3 g, 23 mmol), paraformaldehyde
(6.9 g 0.23 mol) and p-toluenesulfonic acid (1.45 g, 7.6 mmol) in
250 mL of toluene was stirred at 70.degree. C. overnight. After
cooling to r.t., the solid was filtered off. The solution was
extracted with sat. sodium carbonate (100 mL), and the organic
layer was dried over anhydrous sodium sulfate. The target product
(7.4 g) was obtained as a liquid after removal of the solvent in
97% yield. .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.44 (d, J=8.1 Hz,
0.77H), 7.32 (m, 5.60H), 7.07 (d, J=2.7 Hz, 0.33H), 6.68 (m,
1.30H), 5.04 (s, 2H), 4.59 (ss, 2H), 3.96 (br, 1.80), 3.80 (ss,
1.23 H), 3.55 (s, 1.97H), 2.76 (m, 2H).
7-Methoxy-2,3,4,5-tetrahydrobenzo[f][1,4]thiazepine hydrobromide
(Amine) (4 HBr salt)
First Method
[0137] A solution of HBr (33% in acetic acid, 10 mL) was added to
the compound 3 (4.2 g, 12.8 mmol). After the addition, carbon
dioxide began to develop and a white solid formed. The mixture was
let stand at r.t. for another 2 hours. Diethyl ether (150 mL) was
added to the mixture, and it was stirred for 30 min. The solid was
collected by filtration and washed with diethyl ether. The solid
was dried under vacuum to give the 3.40 g of the target compound
with the yield of 91.8%.
[0138] .sup.1H-NMR (300 MHz, DMSO-d.sub.6): 9.02 (br, 2H), 7.52 (d,
J=8.1 Hz, 1H), 7.27 (d, J=3.3 Hz, 1H), 6.92 (dd, J=8.4, 2.7 Hz,
1H), 4.41 (s, 2H), 3.77 (s, 3H), 3.53 (m, 2H), 2.96 (m, 2H).
Alternative Method (Free Base 4a)
[0139] Compound 3 (10 g, 30 mmol) was mixed with 50 mL of conc.
HCl, 50 mL of water and 30 mL of dioxane. The mixture was stirred
at 100.degree. C. overnight. After cooling to r.t., most of the
solvent and HCl was removed under reduced pressure. Water (100 mL)
was added to the solution and the solid was filtered off. The
aqueous solution was extracted with EtOAc/hexane (1:1, 3.times.100
mL) and basified by adding 15 g of NaOH. The mixture was extracted
with dichloromethane (3.times.150 mL). The combined solution was
dried over anhydrous sodium sulfate. Removal of solvent provided a
liquid that solidified after standing at rt. leaving 6.2 g of
target compound.
[0140] .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.42 (d, J=8.1 Hz, 1H),
6.78 (d, J=2.7 Hz, H), 6.68 (dd, J=2.7, 8.1 Hz, 1H), 4.08 (s, 2H),
3.96 (br, 1.80), 3.76 (s, 3H), 3.38 (m, 2H), 2.68 (m, 2H).
Example 2
Binding of Calstabin2 to PKA-Phosphorylated RyR2
[0141] Cardiac SR membranes were prepared as previously described
(Marx et al., 2000; Kaftan et al., Circ. Res., 1996, 78:990-97).
Immunoblotting of microsomes (50 .mu.g) was performed as described,
with anti-calstabin antibody (1:1,000) (Jayaraman et al., J. Biol.
Chem., 1992, 267:9474-77) for 1 hr at room temperature (Reiken et
al., Circulation, 107:2459-66, 2003). After incubation with
HRP-labeled anti-rabbit IgG (1:5,000 dilution; Transduction
Laboratories, Lexington, Ky.), the blots were developed using ECL
(Amersham Pharmacia, Piscataway, N.J.) and detected on x-ray film,
or exposed to secondary antibodies labeled with infrared Dye and
visualized on equipment from Li-Cor Biosciences (model Odyssey).
Unless otherwise stated, compounds were tested at a concentration
of 100 nM. A representative calstabin2 binding assay is presented
below.
[0142] A. PKA phosphorylation of cardiac sarcoplasmic reticulum
(CSR)
[0143] B. Reaction mixture was set up in 1.5 ml microfuge tube. 200
.mu.g of cardiac SR were added to a reaction mix of kinase buffer,
PKA and ATP to a final volume of 100 (Reaction mix below). ATP was
added last to initiate the reaction.
[0144] Reaction Mix:
20 .mu.l Sample (cardiac SR, 2 or 10 .mu.g/.mu.l) 10
.mu.l=10.times. Kinase buffer (80 mM MgCl.sub.2, 100 mM EGTA, 500
mM Tris/PIPES), pH=7.0
[0145] 20 .mu.l PKA (2units/ul) (Sigma # P2645)
10 .mu.l=10.times. ATP (1.0 mM) (Sigma A 9187)
[0146] 40 .mu.l=distilled H.sub.2O [0147] 1. The tubes were
incubated at 30.degree. C. for 30 minutes. [0148] 2. The reaction
mix was then transferred to 0.5 ml thick walled glass tubes. [0149]
3. The glass tubes containing the reaction mix were centrifuged for
10 min at 50,000.times.g in Sorvall Centrifuge RCM120EX using
S120AT3 rotor. Centrifugation at 50,000.times.g for 10 min is
sufficient to isolate the microsomes. [0150] 4. The resulting
pellet was washed 4 times with binding buffer (10 mM Imidazol 300
mM Sucrose, pH=7.4). Each time 100111 of 1.times. binding buffer
was added to the tube to wash the pellet. The pellet was
resuspended by flushing up and down using the pipette tip. After
the last spin 501.11 of binding buffer was added and the pellets
from all tubes were pooled. The reaction was stored at -20.degree.
C. [0151] 5. Phosphorylation was confirmed by separating
approximately 10 .mu.g of CSR by 6% Polyacylamide gel
electrophoresis (PAGE) and analyzing the immunoblots for both total
RyR (5029 Ab, 1:3000 dilution or Monoclonal Ab from Affinity
Bioreagents, Cat # MA3-916, 1:2000 dilution) and PKA phosphorylated
RyR2 (P2809 Ab, 1:10000 dilution). [0152] 6. Aliquots can be stored
at -80 C.
C. Calstabin Rebinding Assay
[0152] [0153] 1. PKA-phosphorylated CSR (approximately 20 .mu.g)
was incubated with 250 nM Calstabin 2 in 100111 binding buffer (as
described above) with or without compounds. [0154] 2. The reaction
was set up in 0.5 ml thick walled glass tube (Hitachi Centrifuge
ware, Catalog #B4105). [0155] 3. Calstabin2 was added as the last
reagent in the reaction mix. Reaction was carried out at room
temperature for 30 mins. [0156] 4. After the reaction, the tubes
were centrifuged for 10 min at 100,000 g. (Sorvall RCM120EX
centrifuge with S120AT3 rotor). [0157] 5. The resulting pellet was
washed 4 times in 1.times. binding buffer at 4.degree. C. After
each wash the tubes were centrifuged at 50,000 g for 10 mins at
4.degree. C. [0158] 6. After the final wash, supernatant was
discarded. [0159] 7. 20 .mu.l of sample buffer (2.times.) [6.times.
sample buffer described below] were added and the pellet was
resuspended with the tip and/or by brief vortexing. The suspension
was transferred to 1.5 ml microcentrifuge tube. [0160] 8. The tubes
were heated at 90.degree. C. for 4 min. [0161] 9. Proteins were
separated using 15% SDS/PAGE. [0162] 10. Calstabin2 binding was
detected with anti-FKBP (Jayaraman et al., J. Biol. Chem. 1992;
267:9474-77, 1:2000) primary antibody and appropriate secondary
antibody.
[0163] 6.times. Sample Buffer
[0164] 7.0 ml 4.times.Tris-HCl/SDS, pH6.8
[0165] 3.0 ml glycerol (30% final concentration)
[0166] 1.0 g SDS (10% final concentration)
[0167] 0.93 g DTT (0.6 M final)
[0168] 1 mg Bromophenol blue (0.001% final concentration)
[0169] Distilled water to 10 ml final volume.
[0170] Store in 1 ml aliquots at -70.degree. C.
Results:
[0171] FIG. 1A depicts an immunoblot with calstabin2 antibody
showing binding of calstabin2 to PKA-phosphorylated RyR2 in the
absence (-) or presence of 100 nM compound 1. (+): calstabin
binding to non-PKA phosphorylated RyR2. S36, a benzothiazepine
described in U.S. Pat. No. 7,544,678, is used as a control. As
shown, compound 1, at a concentration of 100 nM, prevented the
dissociation of calstabin2 from PKA-phosphorylated RyR2 and/or
enhanced the (re)binding of calstabin2 to PKA-phosphorylated
RyR.
[0172] As shown in FIG. 1B, the following representative compounds
were also found to prevent dissociation of calstabin2 from
PKA-phosphorylated RyR2, and/or enhance the (re)binding of
calstabin2 to PKA-phosphorylated RyR2 when tested in the
aforementioned calstabin2 rebinding assay at 100 nM: compound 2,
compound 3 and compound 4.
Example 3
Binding of Calstabin1 to PKA-Phosphorylated RyR1
[0173] SR membranes from skeletal muscle were prepared in a manner
similar to Example 2, and as further described in US patent
application publication No. 2004/0224368, the contents of which are
incorporated by reference herein. Immunoblotting of microsomes (50
.mu.g) was performed as described, with anti-calstabin1 antibody
(Zymed) (1:1,000). The blots were developed and quantified as
described in Example 2.
[0174] FIG. 1C depicts an immunoblot with calstabin1 antibody
showing binding of calstabin1 to PKA phosphorylated RyR1 in the
absence (Neg) or presence of the indicated concentrations of
compound 1 or compound 4. (Pos): calstabin binding to non-PKA
phosphorylated RyR1. S36 is used as a control. As shown, compound 1
and compound 4 prevented the dissociation of calstabin1 from PKA
phosphorylated RyR1 and/or enhanced the (re)binding of calstabin1
to PKA-phosphorylated RyR1 in a dose-dependent manner, with an
estimated EC50 of about 100 nM and 150 nM, respectively.
Example 4
Calstabin1 Rebinding to RyR1 in Isoproterenol Treated Mice
[0175] Isoproterenol, a beta adrenergic receptor agonist, induces
heart failure in mice via overstimulation of the beta adrenergic
receptor. Concurrent with this is the activation of PKA,
phosphorylation of the RyR2 on the SR, and decreased interaction of
calstabin2 (FKBP12.6) to RyR2. A similar cascade of events occurs
in skeletal muscle, wherein RyR1 is phosphorylated, leading to
decreased binding of calstabin1 (FKBP12) to RyR1.
[0176] As described in detail in International publication no.
WO2008/064264, the contents of which are incorporated by reference
herein, chronic isoproterenol treatment to a wild-type mouse offers
a fast and reliable method for inducing changes in RyR biochemistry
that could be readily quantified. These changes include increased
RyR phosphorylation and concomitant decreased calstabin
binding.
Animals and Reagents
[0177] C57Bl/6 mice were maintained and studied according to
approved protocols. The synthetic beta-adrenergic agonist,
isoproterenol (ISO) was obtained from Sigma (165627) and prepared
as a 100 mg/ml stock in water. Lysis buffer was made by adding
sucrose (1 mM), dithiothreitol (320 mM), and 1 protease inhibitor
tablet (10.times.) to 10 ml stock solution (10 mM HEPES, 1 mM EDTA,
20 mM NaF, 2 mM Na.sub.3VO.sub.4).
Osmotic Pump Preparation and Surgical Implantation
[0178] Mice were continually infused for five days with 10 mg/ml
isoproterenol (1 .mu.l/hr) by means of a subcutaneously implanted
osmotic infusion pump (Alzet MiniOsmotic pump, Model 2001, Durect
Corporation, Cupertino, Calif.).
[0179] For drug loading, the osmotic pump was held vertically and
200 .mu.l drug solution was injected into the pump via a 1 ml
syringe (attached to a cannula) that contained an excess of drug
solution (.about.250-300 .mu.l). The drug solution was injected
slowly downward, while the syringe was slowly lifted, until the
pump was overfilled. Overflow of displaced fluid upon capping the
pump confirmed that the pump was properly filled.
[0180] The loaded osmotic pumps were implanted subcutaneously by
the following steps. The recipient mouse was anesthetized with
1.5-2% isoflurane in O.sub.2 administered at 0.6 L/min, and its
weight was then measured and recorded. The mouse was then placed
chest-down on styrofoam, its face in the nose cone. The fur was
clipped on the back of the neck, extending behind the ears to the
top of the head. The area was wiped gently with 70% alcohol, and a
small incision was made at the midline on the nape of head/neck. A
suture holder was swabbed with alcohol, inserted into the cut, and
opened to release the skin from the underlying tissue. To
accommodate the pump, this opening was extended back to the
hindquarters. The loaded pump was inserted into the opening, with
its release site positioned away from the incision, and was allowed
to settle underneath the skin with minimal tension. The incision
was closed with 5.0 nylon suture, requiring about 5-6 sutures, and
the area was wiped gently with 70% alcohol. Following surgery, mice
were placed in individual cages to minimize injury and possible
activation of the sympathetic nervous system.
Skeletal Muscle Isolation
[0181] Mouse skeletal muscle tissue was isolated as follows. The
leg muscles were exposed by cutting the skin at the ankle and
pulling upward. The tissue was kept moistened with Tyrode's buffer
(10 mM HEPES, 140 mM NaCl, 2.68 mM KCl, 0.42 mM Na.sub.2HPO.sub.4,
1.7 mM MgCl.sub.2, 11.9 mM NaHCO.sub.3, 5 mM glucose, 1.8 mM
CaCl.sub.2, prepared by adding 20 mg CaCl.sub.2 to 100 ml 1.times.
buffer made from a 10.times. solution without CaCl.sub.2). The
following muscles were isolated and frozen in liquid nitrogen. The
extensor digitorum longus (EDL) was isolated by inserting scissors
between lateral tendon and the X formed by the EDL and Tibialis
tendons, cutting upward toward the knee; cutting the fibularis
muscle to expose the fan-shaped tendon of gastrocnemius; inserting
forceps under X and under the muscle to loosen the EDL tendon;
cutting the EDL tendon and pulling up the muscle; and finally
cutting loose the EDL. The soleus was isolated by removing the
fibularis muscle from top of gastrocnemius; exposing the soleus on
the underside of the gastrocnemius by cutting and lifting up the
Achilles tendon; cutting the soleus at the top of the muscle behind
the knee; and finally pulling the soleus and cutting it away from
the gastrocnemius muscle. The tibialis was isolated by cutting the
tibialis tendon from the front of ankle, pulling the tendon
upwards, and cutting it away from the tibia. The vastus (thigh
muscle) was isolated from both legs, by cutting the muscle just
above the knee and removing the muscle bundle. The samples were
frozen in liquid nitrogen.
RyR1 Immunoprecipitation from Tissue Lysates
[0182] RyR1 was immunoprecipitated from samples by incubating
200-500 .mu.g of homogenate with 2 .mu.l anti-RyR1 antibody (Zymed)
in 0.5 ml of a modified RIPA buffer (50 mM Tris-HCl (pH 7.4), 0.9%
NaCl, 5.0 mM NaF, 1.0 mM Na.sub.3VO.sub.4, 0.5% Triton-X100, and
protease inhibitors) at 4.degree. C. for 1.5 hr. The samples were
then incubated with Protein A sepharose beads (Amersham Pharmacia
Biotech, Piscatawy, N.J.) at 4.degree. C. for 1 hour, after which
the beads were washed three times with ice cold RIPA. Samples were
heated to 95.degree. C. and size fractionated by SDS-PAGE (15%
SDS-PAGE for calstabin). Immunoblots were developed using an
anti-FKBP antibody (FKBP12/12.6, Jayaraman et al., J. Biol. Chem.
1992; 267:9474-77) at a 1:2,000 dilution. The antibodies were
diluted in 5% milk or TBS-T (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl,
0.05% Tween.RTM. 20, 0.5% Triton X-100).
Results
[0183] Osmotic pumps containing isoproterenol with or without test
compound were implanted in mice as described above. The mice were
osmotically perfused for five days with either vehicle alone
(DMSO/PEG), isoproterenol alone (ISO) (0.5 mg/kg/hr), or a
combination of isoproterenol (0.5 mg/kg/hr) and compound 1 at the
indicated concentrations. At day 6, each mouse was sacrificed, and
skeletal muscle tissue was isolated and used to analyze calstabin1
binding in RyR1 immunoprecipates.
[0184] The effect of compound 1 on enhancing calstabin1 binding to
RyR1 in skeletal muscle isolated from isoproterenol treated mice is
depicted in FIGS. 2A (immunoblot) and 2B (graphical
quantification). As shown, compound 1 enhanced levels of calstabin1
bound to RyR1 in skeletal muscle membranes to a level similar to
that observed by administration of 3.6 mM S36, another
benzothiazepine derivative used as a positive control
(WO2008/064264). Similar results were obtained for compound 4 (data
not shown).
Example 5
Effect of Compound 1 in a Model of Chronic Post-Ischemic Heart
Failure in Rat
Objective
[0185] The objective of this study was to test the ability of
compound 1 to reduce cardiac dysfunction and attenuate ventricular
remodelling in a model of ischemia-reperfusion induced heart
failure.
Methodology
[0186] Chronic heart failure was induced in male wistar rats
(224-240 g, 10-11 weeks of age) by ischemia-reperfusion (I/R)
injury. For I/R protocol, the left anterior descending (LAD)
coronary artery was occluded for 1 h. Drug treatment (5 mg/kg/d or
10 mg/kg/d in drinking water) was initiated 1 week after
reperfusion and was maintained for a 3 month study period. The
efficacy of compound 1 was assessed by echocardiography at one, two
and three months after treatment began, and by invasive
hemodynamics at 3 months in comparison with vehicle-treated and
sham-operated animals. Cardiac specimens were also analyzed to
assess hypertrophy and collagen content. Blood was collected from
each rat on the final study day to assess drug plasma
concentrations as shown in FIG. 3. The study design is depicted in
FIG. 3. Experiments were performed in a blinded manner.
Statistical Methods
[0187] On parameters measured over time, comparison of Sham versus
Vehicle and comparison of drug treatments are analyzed by 2 way
ANOVAs with repeated measures. On parameters measured at sacrifice
and morphometry, comparisons of Sham versus Vehicle are analyzed by
t-test and comparisons of drug treatments by 1-way ANOVA followed
by Dunnett test.
Results
[0188] Vehicle-treated I/R animals, compared to sham-operated
animals, showed increased left ventricular (LV) end systolic (LV
ESV) and end diastolic (LV EDV) volumes (FIGS. 4 A and B),
depressed cardiac function as measured by decreased Ejection
Fraction (EF) (FIG. 4C) and increased interstitial collagen content
(FIG. 5D). compound 1, administered at 5 and 10 mg/kg/d,
significantly increased EF, as well as decreased both LVESV and
LVEDV compared to vehicle, from one to three months (FIGS. 4A-C),
as well as reduced interstitial collagen content (FIG. 5D).
[0189] Invasive hemodynamic study (at 3 months) showed a
preservation of LV dP/dt max and LV dP/dt min in the animals
treated with compound 1 at 5 and 10 mg/kg/d compared to vehicle
(FIGS. 6B and C), with no statistically significant change in LV
systolic pressure upon treatment (FIG. 6A).
[0190] No effects on body weight (BW), infarct size or hypertrophy
(LV weight) were observed upon treatment (FIGS. 5A-C). Drug plasma
concentrations are depicted in FIG. 7.
[0191] The results show that compound 1, at concentrations as low
as 5 mg/kg/d, exerts a beneficial effect on both systolic and
diastolic cardiac function in a model of chronic post-ischemic
heart failure in rat.
[0192] Compound 1 was significantly and surprisingly more active in
comparison with compound A, a structurally related benzothiazepine
derivative described in WO 2007/024717. As shown in FIG. 8,
compound A, administered at a concentration of 5 mg/kg/d for 3
months, failed to improve systolic and diastolic cardiac function
when compared with compound 1 in the chronic post-ischemic heart
failure rat model at the end of the study. Thus, beneficial effects
of compound 1, but not compound A, were observed at a dose of 5
mg/kg/d after 3 months of treatment in the rat CHF model.
##STR00021##
Example 6
Effect of Compound 1 on Muscle Function in a Mouse Muscular
Dystrophy Model (Mdx)
Objective
[0193] The objective of this study was to test whether treatment
with compound 1 improves muscle function in a dystrophin-deficient
mouse model (mdx).
[0194] Methodology
[0195] C57BL/10ScSn-DMD.sup.mdx/J (abbreviated as mdx, n=5 per
group) mice, 6 weeks and approximately 20 grams at study
initiation, were acclimated to wheel cages for six days, prior to
randomization into groups receiving treatment with either vehicle
(H.sub.2O) or target doses of 5 mg/kg/d, 10 mg/kg/d, or 50 mg/kg/d
(actual doses: 7.9 mg/kg/d; 12.8 mg/kg/d; and 61.5 mg/kg/d,
respectively, determined from weekly measured drug solution
consumption divided by body weight) of the sodium salt of compound
1 (based on the weight of the parent drug; the sodium salt is
referred to hereinafter in this Example as "compound 1")
administered in the drinking water ad libitum for 4 weeks.
Age-matched C57BL/6 (abbreviated as WT, n=4 per group) mice, were
randomized into groups receiving treatment with either vehicle
(H.sub.2O) or a target dose of 50 mg/kg/d (actual dose: 67.7
mg/kg/d) of the sodium salt of compound 1.
[0196] Voluntary activity on wheel, body weight, and average water
consumption were measured in the first 3 weeks. Specific muscle
force was measured after 4 weeks of treatment, at the end of the
study.
[0197] Distance traveled (Km/day) over a 24 hr period was analyzed
as an index of improved functional activity (see, DMD_M.2.1.002 SOP
at http://www.treat-nmd.eu/). At the conclusion of the study,
Extensor digitorum longus (EDL) muscle was isolated for muscle
force analysis as further described hereinbelow. Blood was
collected from each mouse by retro-orbital bleeds at the end of the
study (after end of dark cycle--about 7 AM) to assess drug plasma
concentrations. Experiments were blinded.
Force Measurements
[0198] At the end of the study, EDL muscle was dissected from hind
limbs for isometric force analysis using the 407A Muscle Test
System from Aurora Scientific (Aurora, Ontario, Canada). A 6-0
suture were tied to each tendon and the entire EDL muscle, tendon
to tendon, was transferred to a Ragnoti bath of O.sub.2/CO.sub.2
(95%/5%) bubbled Tyrode solution (in mM: NaCl 121, KCl 5.0,
CaCl.sub.2 1.8, MgCl.sub.2, NaH.sub.2PO.sub.4, NaHCO.sub.3 24, and
glucose 5.5). Using the sutures, one tendon was tied vertically to
a stainless steel hook connected to a force transducer The other
sutured tendon was clamped down into a moving arm on the Aurora
system. The EDL muscle was stimulated to contract using an
electrical field between two platinum electrodes. At the start of
each experiment, muscle length was adjusted to yield the maximum
force. Force-frequency relationships were determined by triggering
contraction using incremental stimulation frequencies (5-250 Hz for
200 ms at suprathreshold voltage). Between stimulations the muscle
was allowed to rest .about.3 min. At the end of the force
measurement, the length (L.sub.o) of the EDL muscle while sutured
in the Aurora system was measured excluding the tendons. The EDL
muscle was then removed from the system and weighed after clipping
the end tendons and sutures off. The EDL muscle was then frozen in
liquid nitrogen. The cross-sectional area (mm.sup.2) of the EDL
muscle was calculated by dividing the EDL muscle weight by the EDL
muscle length and the mammalian muscle density constant of 1.056
mg/m.sup.3 (Yamada, T., et al. Arthritis and rheumatism
60:3280-3289). To determine EDL specific force (kN/m.sup.2), the
absolute tetanic force was divided by the EDL muscle
cross-sectional area.
Statistical Methods
[0199] For statistical analysis of distance traveled, change from
baseline was calculated for each day by subtracting the baseline
value (defined as the mean of the two measurements obtained on Day
-1 and Day -2) from each post dose assessment. Change from baseline
was then statistically analyzed with a repeated measures analysis
of variance model with fixed effects for treatment, day and
treatment by day interaction. Baseline was included as a covariate
and mouse was included as a random effect. The most appropriate
covariance structure was determined through investigation of the
Akaike's Information Criterion (AIC) and Bayesian Information
Criterion (BIC). The covariance structures investigated were
autoregressive, compound symmetry, unstructured, and toeplitz. The
optimal covariance structure selected was compound symmetry. From
the model, point estimates and associated 95% confidence intervals
(CI) for the difference in change from baseline between each ARM210
treated mice group and vehicle treated group (for both mdx and
C57BL/6 mice) were obtained for each day, each week, and the entire
assessment period from Day 1 to Day 19. As this study is
exploratory in nature, no adjustments in multiple comparisons were
made.
[0200] Specific force was analyzed using a repeated measures
analysis of variance model with fixed effects for treatment,
frequency and treatment by frequency interaction and mouse as a
random effect. Similar to the analysis described above, the optimal
covariance structure was chosen from among autoregressive, compound
symmetry, unstructured, and toeplitz. The optimal covariance
structure selected for specific force was toeplitz. From the model,
point estimates and associated 95% confidence intervals (CI) for
the difference in specific force between each ARM210 treated mice
group and vehicle treated group (for both mdx and C57BL/6 mice)
were obtained for each frequency and across all frequencies. As
this study is exploratory in nature, no adjustments in multiple
comparisons were made.
Results
[0201] The ability of compound 1 to improve voluntary exercise in
mdx mice was tested. After acclimating the mice to the voluntary
wheel cage, mouse activity on the voluntary wheels was monitored by
a computer 24/7. Data collected was transcribed to distance
traveled per day over 3 weeks. Mdx mice treated with 10 and 50
mg/kg/d (target dose) of compound 1 traveled significantly longer
distances on the wheel compared to mdx mice treated with vehicle
(H.sub.2O) alone (P<0.001 from day 1 to day 19). Treatment
effect observed as early as 2-3 days after treatment initiation,
and continued throughout the activity monitoring period. No effect
of compound 1 on travel distance was observed with WT mice treated
with 50 mg/kg/d compound 1 (FIG. 9). In addition, as determined by
in vitro force measurements in EDL muscle (FIG. 10), compound 1
treatment increased specific force in mdx muscle dose-dependently.
At stimulation frequencies of 150 Hz and above the 50
mg/kg/d-treated mdx mice showed statistically significant increase
in specific muscle force (P<0.05). No effect of compound 1
treatment on specific muscle force was observed in WT mice.
[0202] As shown in FIG. 11, compound 1 treatment did not affect
body weight. No dose-dependent effects on water consumption were
observed. Morning blood exposure of compound 1 was (average.+-.SEM)
3.3.+-.0.4 .mu.M for the 5 mg/kg/d-dosed mdx mice, 10.7.+-.0.9
.mu.M for the 10 mg/kg/d-dosed mdx mice, 52.8.+-.1.7 .mu.M for the
50 mg/kg/d-dosed mdx mice and 72.8.+-.7.0 .mu.M for the 50
mg/kg/d-dosed WT mice.
[0203] Taken together, the results show that, as compared with
vehicle-treated controls, treatment with compound 1 at 10 mg/kg/d
and 50 mg/kg/d (target dose) improved voluntary wheel exercise
after 3 weeks and specific muscle force after 4 weeks in mdx mice,
a murine model of Duchenne muscular dystrophy (DMD), thereby
demonstrating the utility of compound 1 and its analogs as claimed
herein, in the treatment of muscular dystrophy.
Example 7
Metabolic Stability
[0204] The metabolic stability of compound 1, a representative
Ryca1.TM. according to the present invention, was compared to
compound B and compound C, structurally related benzothiazepine
derivative described in WO 2007/024717.
A. Metabolic Stability in Human Hepatic Microsomes
Methods:
[0205] Compound solubilization: Stock solutions were made in DMSO,
and working solutions in water containing 1 mg/ml BSA.
[0206] Prediction of metabolic bioavailability: Metabolic
bioavailability predictions (MF %) were based on in vitro metabolic
stability measurements with hepatic microsomes assuming total
absorption. Briefly, unchanged drugs were quantified by LC-MS-MS
following incubation (10.sup.-7M) with rat and human hepatic
microsomes (0.33 mg protein/ml) after 0, 5, 15, 30 and 60 min of
incubation in presence of NADPH (1 mM). Enzymatic reaction was
stopped with methanol (v/v) and proteins were precipitated by
centrifugation. The in vitro intrinsic clearances (Clint_mic)
expressed as ml/min/g protein were the slope (after LN
linearization) of the unchanged drug remaining concentration versus
incubation time. In vitro Clint were then scaled up to in vivo
whole body (vivoClint) using 0.045 mg prot/kg of liver and liver
weight of 11 g for the rat and 1.2 kg for Man. In vivo Clint were
then transformed into hepatic clearances (HepCl) using the
well-stirred model (HepCl=vivoClint*HBF/(vivoClint+HBF) where HBF
(hepatic blood flow) were taken as 22 ml/min for the rat and 1500
ml/min for Man. The MF % were then deducted from the extraction
ratio with the following equation (MF %=1-HepCl/HBF). The results
are presented in Table 1:
TABLE-US-00001 TABLE 1 Stability in human microsomes Rat microsomes
Human microsomes Clint_mic MF Clint_mic MF rat mic man mic Compound
Structure ml/min/gprot % Class ml/min/gprot % Class B ##STR00022##
823 5 very low 285 6 very low C ##STR00023## 1926 2 very low 1326 2
very low 1 ##STR00024## 101 30 Inter- mediate 9.1 75 high a.
Clint_mic: in vitro intrinsic clearance in ml/min/g protein b. MF
%: metabolic bioavailability in %
B. Metabolic Stability in Rat and Human Hepatic Hepatocytes
[0207] Compound solubilization: Stock solutions were made in DMSO,
and working solutions in William medium containing 1/10 rat plasma
or 1/4 human plasma.
[0208] Metabolic stability determination: Compounds were incubated
at 10.sup.-7 M with isolated hepatocytes (6E+5 cells/ml for rat
hepatocytes and 4E+5 cells/ml for human hepatocytes) at 37.degree.
C. in plasma from the same species diluted in Wiliams medium (1/10
dilution for rat and 1/4 dilution for human). Sampling times were
performed at 0, 10, 20, 30, 60 and 120 min and enzymatic reaction
stopped with methanol (v/v). Proteins were precipitated by
centrifugation and the supernatant was analyzed by LC/MS/MS. Clint
expressed as ml/min/g protein were calculated as for hepatic
microsomes using a ratio of 0.134 mg protein/ml for 4E+5 cells/ml
for human and 0.201 mg protein/ml for 6E+5 cells/ml for rat. The
presence of the reference drug and the potential metabolite was
checked by LC/MS/MS during the assay in each sample. The results
are presented in Table 2:
TABLE-US-00002 TABLE 2 Stability in rat and human hepatocytes Rat
hepatocytes Human hepatocytes Clint Clint Compound (ml/min/gprot)
MF rat % Q cellules/ml (ml/min/gprot) MF human % Q cellules/ml B
1334 3 6.00E+05 693 3 4.00E+05 1 5 90 6.00E+05 0 100 4.00E+05 C
2610 2 6.00E+05 100 16 4.00E+05 a. Clint_mic: in vitro intrinsic
clearance in ml/min/gprotein b. MF %: metabolic bioavailability in
% c. Q: cells quantity per ml
C. Metabolic Stability in Mouse and Rat Microsomes
Materials and Methods
[0209] Dilution Buffer:
[0210] 0.1M Tris HCl buffer at pH 7.4 containing 5 mM EDTA.
[0211] NADPH Cofactor Solution:
[0212] To a 50 mL falcon tube containing 2.79 mL of dilution buffer
were added 0.429 mL of NADPH-regenerating soln. A and 0.079 mL of
NADPH-regenerating soln. B
[0213] Microsome Preparation:
[0214] (1.5 mg/mL solution) A 50 mL falcon tube containing 3.32 mL
of dilution buffer was prewarmed at 37.degree. C. for 15 min. (at
least 10 min.) 0.178 mL of microsome (24.6 mg/mL) were added to the
prewarmed dilution buffer. The protein concentration of this
microsome preparation was 1.25 mg/mL.
[0215] Sample (Test Compound)--Original and Intermediate Stock
Solutions:
[0216] A 1 mg/mL (0.5 mg/mL was used for compound 1) solution of
the test compound in methanol was prepared. 100 .mu.M intermediate
solution of the test compound from the original stock solution were
prepared using the dilution buffer. A 5 .mu.M solution was prepared
by diluting the 100 .mu.M intermediate solution using dilution
buffer.
[0217] Experiment:
[0218] (The experiments were conducted in 1.5 mL eppendorf micro
centrifuge tubes)
[0219] 0 minutes incubation. Procedure:
[0220] a. Add 100 .mu.L of prewarmed microsomes
[0221] b. Add 50 .mu.L of 5 .mu.M solution of the test
compound.
[0222] c. Add 500 .mu.L of cold stop solution (ice cold
Methanol)
[0223] d. Add 100 .mu.L of NADPH cofactor solution to the
eppendorf.
[0224] a. Vortex mix the eppendorf.
[0225] "t" minutes incubation
[0226] b. Add 100 .mu.L of NADPH cofactor solution to the
eppendorf.
[0227] c. Add 50 .mu.L of 5 .mu.M solution of the test
compound.
[0228] d. Add 100 .mu.L of prewarmed microsomes
[0229] e. Incubate the eppendorf at 37.degree. C. 300 rpm for `t`
min. on a thermomixer.
[0230] f. Remove the eppendorf from thermomixer
[0231] g. Add 500 .mu.L of cold stop solution (ice cold
Methanol)
[0232] h. Vortex mix the eppendorf.
[0233] Both the `0` and `t` minutes incubated samples were
centrifuged at 15, 000 rcf at 4.degree. C. for 15 min. 500 .mu.L of
the supernatant solution was removed and subject it to LC/MS
analysis (SIM--Selected Ion Monitoring)
[0234] Results are expressed as % test compound remaining=(MS Area
response of T min sample/MS Area response of `0` min sample)*100.
The MS area used is an average of duplicate injections.
[0235] Time points=0, 15, 30 and 60 min. for each test compound
[0236] Positive Control:
[0237] 2 .mu.M Imipramine--5 min. and 2 .mu.M Imipramine--15 min.
incubation was used as a positive control for the rat and mouse
liver microsome stability experiments.
[0238] The results are presented in Table 3:
TABLE-US-00003 TABLE 3 Stability in mouse and rat microsomes In
Vitro Metabolic Compound Compound Compound Stability (1) (B) (C)
Rat microsomes (% remaining) 15 min 54% 1% 0% 30 min 17% 0% 0% 1 h
2% 0% 0% Mouse microsomes (% remaining) 15 min 99% 0% 0% 30 min 98%
0% 0% 1 h 82% 0% 0%
[0239] Surprisingly, as seen in Tables 1-3, compound 1 was
significantly more stable in mouse, rat and human microsomes, and
in rat and human hepatocytes, as compared with the structural
analogs compounds B and C disclosed in WO 2007/024717, both of
which have been found to possess poor in-vitro metabolic stability
in the tested systems, making these compounds unsuitable for
development as drug candidates. Surprisingly and unexpectedly, the
replacement of the H or OH moieties in the prior art compounds with
a COOH moiety resulted in compound 1, which displayed high
metabolic stability in all tested systems. The increased metabolic
stability of Compound 1 compared with its structural analogs was
indeed surprising and substantiates the unexpected benefits of this
compound over compounds known in the art.
[0240] 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.
[0241] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying current knowledge, readily modify and/or adapt for
various applications such specific embodiments without undue
experimentation and without departing from the generic concept,
and, therefore, such adaptations and modifications should and are
intended to be comprehended within the meaning and range of
equivalents of the disclosed embodiments. It is to be understood
that the phraseology or terminology employed herein is for the
purpose of illustration and not of limitation. The means,
materials, and steps for carrying out various disclosed functions
may take a variety of alternative forms without departing from the
invention.
Sequence CWU 1
1
1125DNAArtificial SequenceSynthesized nucleic acid 1ggccaaacct
cggcttacct gaaat 25
* * * * *
References