4(RS)-4-F4-Neuroprostane Derivatives (4-F4T-NEUROP) and Their Use in Treating Ventilator-Induced Diaphragmatic Dysfunction and Other Disorders

Abstract

Inactivity of respiratory muscle contraction during mechanical ventilation (MV) induces diaphragm muscle weakness, a condition called ventilator-induced diaphragmatic dysfunction (VIDD). Among different hypotheses explaining VIDD, one involves remodeling of the sarcoplasmic reticulum (SR) calcium release channel / ryanodine (RyR1) in the diaphragm as the proximal mechanism of VIDD. The present inventors have developed novel derivatives that prevent VIDD with high efficiency in both mouse and porcine VIDD models. This effect is associated with normalization of RyR function. Thus, the present invention relates to novel compounds and their use in the treatment of diseases associated with ryanodine receptor (RyR) dysfunction.

Description

[Technical Field]

[0001] The present invention relates to novel compounds, processes for their preparation, and their use in the treatment of diseases associated with ryanodine receptor (RyR) dysfunction. [Background technology]

[0002] Skeletal muscle contraction is mediated by Ca release from the sarcoplasmic reticulum (SR) via the ryanodine receptor calcium release channel. 2+ RyRs are channels within the SR that open and close to release Ca from the SR into the cell's cytoplasm. 2+ Regulates Ca release from the SR into the cytoplasm 2+ Release of cytoplasmic Ca 2+ The open probability of an RyR is the probability that an RyR will open at any given moment, thereby allowing Ca ions to enter the cytoplasm from the SR. 2+The term "RyR" refers to the ability of RyRs to release ATP. There are three highly homologous RyRs: RyR1, RyR2, and RyR3. RyR1 is found primarily in skeletal muscle, among other tissues. The RyR1 macromolecular complex consists of a 560-kDa RyR1 subunit tetramer that forms a scaffold of proteins that regulate channel function, including protein kinase A (PKA), phosphodiesterase 4D3 (PDE4D3), protein phosphotase 1 (PP1), and calstabin1. Chronic PKA hyperphosphorylation of RyR1 at Ser2843 (defined as PKA phosphorylation of three or four of the four PKA Ser2843 sites present in each RyR1 homotetramer) results in "leaky" channels (i.e., channels that tend to open at rest), which contributes to skeletal muscle dysfunction associated with sustained hyperadrenergic states, such as those occurring in individuals with heart failure. Furthermore, modulation of RyR1 by post-translational modifications other than phosphorylation, such as nitrosylation of free sulfhydryl groups at cysteine ​​residues (S-nitrosylation) and channel oxidation, has been reported to increase RyR1 channel activity. S-nitrosylation and oxidation of RyR1 have each been shown to reduce the binding of calstabin1 to RyR1. Thus, RyR dysfunction is a hallmark of various diseases, and several methods and pharmaceutical compositions have been described for the treatment of pre-existing conditions, such as ventilator-induced diaphragmatic dysfunction (VIDD). 1 .

[0003] Mechanical ventilation (MV) remains the most efficient treatment for respiratory failure in the intensive care unit (ICU). However, MV-induced respiratory muscle contractile inactivity (i.e., unloading of the respiratory muscles) can lead to diaphragmatic weakness, atrophy, and injury in a time-dependent manner, a pathological condition known as ventilator-induced diaphragmatic dysfunction (VIDD). 2~4 have various adverse effects on cells, leading to 5 VIDD increases the difficulty of MV weaning. 6 , which has a significant impact on medical costs 7Therefore, identifying the cellular pathways involved in diaphragm weakness is an important issue for identifying potential therapeutic targets and pharmacological treatments.

[0004] Although the pathophysiological mechanisms underlying VIDD are not yet fully understood, oxidative stress is thought to be the primary pathophysiological mechanism of VIDD. 8~11 , mitochondria are an essential source of reactive oxygen species (ROS) 11~13 The reason for the increased production of mitochondrial ROS after the release of respiratory muscles induced by MV is unknown. An imbalance in energy homeostasis associated with rapid substrate oversupply is a potential source of mitochondrial dysfunction. 12 .

[0005] We have developed the first VIDD mouse model that exhibits a 30% reduction in force output after 6 hours of ventilation without histological signs of injury or necrosis. 14 Using this model, we demonstrate the role of Ca in the establishment of VIDD. 2+ We show that early alterations in homeostasis are crucial, occurring via structural and functional remodeling of the type 1 sarcoplasmic reticulum calcium channel, ryanodine receptor (RyR1), induced by oxidative stress. 15 This cellular pathway has been shown to be involved in different animal models of VIDD. 8、16~18 and human patients 3~4 observed in Ca 2+ This may be a direct mechanism of RyR1-dependent proteolysis and muscle atrophy. Interestingly, this remodeling of RyR1 has been consistently observed in ventilated mice and piglets, as well as in ventilated ICU patients. 15、19、20 This suggests that directly or indirectly stabilizing RyR1 function is a potential therapeutic target in VIDD. We previously reported that the omega-3 fatty acid docosahexaenoic acid (DHA, C22:6 n-3) can exert cardiac antiarrhythmic effects due to stabilization of the cardiac isoform of the ryanodine receptor, RyR2. 21 Interestingly, the active molecule is not DHA itself, but a non-enzymatic metabolite of DHA (NEO-DHA).21 , 4(RS)-4-F 4t - Neuroprostane (4-F 4t -NeuroP) 22 WO 2015197562 describes a therapeutically effective amount of 4F 4t The present invention describes a method for treating a disease associated with RyR dysfunction in a subject in need of such treatment, comprising administering -NeuroP. However, the synthesis of the compound involves several steps, which limits its therapeutic development. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2015197562 [Patent Document 2] WO2014086819 [Non-patent literature]

[0007] [Non-Patent Document 1] Kushnir et al. (Mol. Cell. Res., 2018, 1865, pp. 1687-1697) [Non-patent document 2] Matecki et al., PNAS, 2016, 113(2), pp. 9069-9074 [Non-patent document 3] Matecki et al., J. Muscle Res Cell Motil., 2017, 38, pp. 17-24 [Non-patent document 4] Oger C. et al., Chem. Eur. J., 2010, 16, 13976 Summary of the Invention [Problem to be solved by the invention]

[0008] There remains a need to identify new compounds that are effective for treating disorders and diseases associated with RyR dysfunction. More particularly, there remains a need to identify new agents that can be used to treat RyR-related disorders, such as RyR1 destabilization. [Means for solving the problem]

[0009] The present invention relates to novel compounds and methods for preparing said compounds. The present invention also relates to methods and pharmaceutical compositions for the treatment of diseases associated with ryanodine receptor (RyR) dysfunction. In particular, the present invention relates to a method for treating diseases associated with RyR dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a novel compound according to the present invention.

[0010] Specifically, the invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present inventors have developed 4(RS)-4-F 4t - Neuroprostane (4-F 4t -NeuroP) derivatives were designed, which were 4-F 4t -Compared to NeuroP, it has fewer reaction steps and is easier to synthesize.

[0012] For example, the compound 17,18,19,20,21,22-hexanol-4(RS)-4-F 4t -NeuroP(VB558) can be obtained in only 17 steps (4-F 4t -NeuroP has 24 steps).

[0013] The present inventors have surprisingly found that novel compounds prevent VIDD with high efficiency in both mouse and porcine VIDD models, an effect that is associated with normalization of RyR function.

[0014] A first object of the present invention is a compound of formula I or a pharmaceutically acceptable salt thereof for use in a method of treatment of the human or animal body.

[0015] [ka]

[0016] (In the formula, R1 represents H or OH; R2 is a linear C1-C 10 Alkyl group or C2-C 10 represents an alkyl group, However, R2 is

[0017] [ka]

[0018] When R1 represents H,

[0019] A second object of the present invention is a method for treating a disease associated with RyR dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof.

[0020] [ka]

[0021] (In the formula, R1 represents H or OH; R2 is a linear C1-C 10 Alkyl group or C2-C 10 represents an alkyl group, However, R2 is

[0022] [ka]

[0023] When R1 represents H,

[0024] In other words, a second object of the present invention relates to a compound of formula I, or a pharmaceutically acceptable salt thereof, as defined herein for use in treating a disease associated with RyR dysfunction in a subject in need thereof.

[0025] Linear C1-C 10 "Alkyl group" means methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl. Straight-chain alkyl groups are especially present in ethyl.

[0026] Linear C2-C 10 An "alkyl group" is a linear C2-C alkyl group containing one or more double bonds in its chain. 10 It means an alkyl group. 10 The expression "alkenyl group" includes C2-C8 alkenyl groups, C2-C6 alkenyl groups, C2-C4 alkenyl groups, C4-C 10 Alkenyl groups, C6-C 10 Alkenyl groups, C8-C 10 An alkenyl group or a C4-C8 alkenyl group should also be understood.

[0027] As defined herein, the term "pharmaceutically acceptable salt" is intended to mean a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. Such salts are compounds in which an acidic proton present in the parent compound is either replaced by a metal ion, such as an alkali metal ion, alkaline earth metal ion, or aluminum ion, or is coordinated with a pharmaceutically acceptable organic or inorganic base.

[0028] Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine, etc. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, and sodium hydroxide.

[0029] In certain embodiments, an alkenyl group contains one or two double bonds.

[0030] In particular, the double bonds in R2 are separated from the double bond to which R2 is attached by a -CH2- group and / or from each other when the number of double bonds in R2 is two by a -CH2- group.

[0031] According to a preferred embodiment, the double bond contained in the alkenyl group of R2 is in the Z configuration.

[0032] According to a preferred embodiment, the present invention relates to the method as described above, wherein the compound of formula I is such that R2 is a radical selected from the group of formulae a, b and c.

[0033] [ka]

[0034] The compounds of formula I contain several asymmetric carbon atoms in their structure.

[0035] [ka]

[0036] The asymmetric carbon atoms contained in the five-membered ring (carbons 7, 8, 10 (when R1 = OH), and 11) are preferably as shown in the structure above.

[0037] In certain embodiments, the compounds of formula I have a diastereomeric excess of greater than 90%, particularly greater than 95%, with respect to the carbon atoms contained in the five-membered ring (carbons 7, 8, 10 (when R1 = OH), and 11).

[0038] In another particular embodiment, the compound of formula I has an enantiomeric excess of greater than 90%, in particular greater than 95%, relative to the carbon atoms contained in the five-membered ring (carbons 7, 8, 10 (when R1 = OH) and 11).

[0039] The asymmetric carbon atom 4, in the gamma position relative to the carboxylic acid, can be in the R or S configuration, or a mixture of the R and S configurations.

[0040] According to an embodiment of the present invention, the asymmetric carbon atom at the γ position relative to the carboxylic acid is a mixture of R and S configurations and the compound of formula I is a mixture of diastereoisomers.

[0041] In an advantageous embodiment, the compound of formula I is

[0042] [ka]

[0043] wherein the compound of formula I has the structure of formula II, formula III, formula IV, formula S-IV, or formula R-IV.

[0044] The compound of formula II, 17,18,19,20,21,22-hexanol-4(RS)-4-F 4t -NeuroP(VB558) is 4-F 4t - a shorter derivative of NeuroP, containing an alkenyl chain of 5 carbon atoms and having one double bond in the Z configuration (with respect to formula I, R1 is -OH and R2 is a group of formula a (ethyl)).

[0045] In certain embodiments, when the compound is a compound of formula II, the asymmetric carbon atom at the γ position relative to the carboxylic acid is a mixture of R and S configurations, and said compound of formula II is a mixture of diastereoisomers, particularly wherein the diastereomeric excess is less than 95%, more particularly less than 90%.

[0046] The compound of formula III, 20,21,22-triol-4(RS)-4-F 4t -NeuroP(VB574) is 4-F 4t- a shorter derivative of NeuroP, containing an alkenyl chain of 8 carbon atoms and having two conjugated double bonds in the Z configuration (with respect to formula I, R1 is -OH and R2 is a group of formula b).

[0047] The compound of formula IV is 4-F 4t - is a monohydroxylated derivative of NeuroP, in which the five-membered ring is substituted with one hydroxyl substituent. The compound of formula IV further comprises an alkenyl chain of 11 carbon atoms and has three conjugated double bonds in the Z configuration (with respect to formula I, R1 is -H and R2 is a group of formula c).

[0048] The compound of formula S-IV (VB581) is a specific diastereoisomer of compound IV, in which the carbon atom on the γ-position of the carboxylic acid functional group is in the (S)-configuration.

[0049] The compound of formula R-IV (VB582) is a specific diastereoisomer of compound IV, in which the carbon atom on the γ-position of the carboxylic acid functional group is in the (R)-configuration.

[0050] According to a preferred embodiment, the compound of formula I has the structure of formula II defined above, i.e., 17,18,19,20,21,22-hexanol-4(RS)-4-F 4t -NeuroP(VB558).

[0051] In certain embodiments, the compounds of the invention are suitable for stabilizing the calstabin1 / RyR1 complex.

[0052] As used herein, the term "subject" refers to mammals such as rodents, cats, dogs, and primates. Preferably, a subject according to the present invention is a human.

[0053] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, and further includes the suppression of clinical recurrence. Treatment can be administered to a subject with a medical disorder or who may eventually acquire a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurring disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of disease treatment, e.g., the pattern of medication used during treatment. The therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. An induction regimen may utilize (in part or in whole) a "loading regimen," which may involve administering a higher dose of drug than a physician utilizes during a maintenance regimen, administering a drug more frequently than a physician administers during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during disease treatment, e.g., to keep the patient in remission over an extended period of time (months or years). Maintenance regimens may utilize continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., treatment interruptions, intermittent treatment, treatment upon relapse, or treatment when certain predetermined criteria are met (e.g., signs of disease, etc.)).

[0054] As used herein, "disease associated with RyR dysfunction" means any disorder or disease that can be treated and / or prevented by modulating RyR receptors that regulate calcium channels functioning in cells.

[0055] Diseases associated with RyR dysfunction have been well described, and those skilled in the art can easily identify such diseases. For example, Kushnir et al. (Mol. Cell. Res., 2018, 1865, 1687-1697) discloses the relationship between certain diseases and RyR dysfunction.

[0056] In addition, whether a disorder or disease is associated with RyR dysfunction can be easily determined empirically. Typically, two tests can be performed individually to establish a link between a disease and RyR dysfunction, but preferably both tests are performed (Matecki et al., PNAS, 2016, 113(2), pp. 9069-9074 and J. Muscle Res Cell Motil., 2017, 38, pp. 17-24).

[0057] The first test is based on RyR1 immunoprecipitation techniques and will identify a "biochemical signature" of leaky RyR1 channels, making it possible to establish a correlation between specific diseases and RyR dysfunction.

[0058] The second test was based on electrophysiological techniques, in which single-channel currents were recorded to determine the RyR open probability. The measurement involved an experimental setup in which RyR channels were incorporated into a lipid bilayer. RyR channels were obtained by biopsy, followed by preparation of SR vesicles. Single-channel currents were recorded using a bilayer-clamp BC-535 amplifier.

[0059] Thus, "diseases associated with RyR dysfunction" include, without limitation, cardiac disorders and diseases, skeletal muscle disorders and diseases, cognitive disorders and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome.

[0060] In some embodiments, the disorder or disease is associated with abnormal function of RyR1.

[0061] In some embodiments, the methods of the present invention are particularly suitable for treating a disease selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, central nervous system (CNS) disorders and diseases, cognitive disorders, bone disorders and diseases, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, or for improving cognitive function. Cardiac disorders and diseases include, but are not limited to, irregular heart rhythm disorders and diseases, exercise-induced irregular heart rhythm 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 revascularization or thrombolysis during myocardial infarction (MI)), chronic obstructive pulmonary disease, and hypertension. Irregular heart rhythm disorders and diseases include, but are not limited to, atrial and ventricular arrhythmias, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmias, atrial and ventricular tachyarrhythmias, catecholamine-induced polymorphic ventricular tachycardia (CPVT) and its exercise-induced variants.

[0062] In some embodiments, the methods of the present invention are directed to treating skeletal muscle fatigue, central core disease, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, sleep apnea, age-related muscle fatigue, sarcopenia, congenital myopathies, cancer cachexia, myopathies involving core and rods, mitochondrial myopathies (e.g., Kearns-Sayre syndrome, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis and stroke-like episodes) syndrome, and MERRF (myoclonic epilepsy with ragged-red fibers) syndrome), endocrine myopathies, muscle-type glycogen storage diseases (e.g., Pompe disease, Andersen disease, and Cori disease), myoglobinuria (e.g., McArdle disease, Tarui disease, and DiMauro disease), and The compounds are particularly suitable for the treatment of muscle diseases, including, but not limited to, dermatomyositis, myositis ossificans, familial periodic paralysis, polymyositis, inclusion body myositis, neuromyotonia, stiff-body syndrome, malignant hyperthermia, generalized muscle cramps, muscle tegmentation, myasthenia gravis, and muscular dystrophies. Examples of muscular dystrophies include, but are not limited to, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral muscular atrophy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy. Congenital muscular dystrophy, as used herein, refers to muscular dystrophies present at birth. CMDs are classified based on genetic mutations: 1) genes encoding structural proteins of the basement membrane or extracellular matrix of skeletal muscle fibers, 2) genes encoding putative or proven glycosyltransferases affecting the glycosylation of dystroglycan, an outer membrane protein of the basement membrane, and 3) others. Examples of CMDs include, but are not limited to, laminin α2-deficient CMD (MDC1A), Ullrich CMD (UCMD1, 2, and 3), Walker-Warburg syndrome (WWS), myo-oculoencephalopathy (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 stiff spine with type 1 muscular dystrophy (RSMD1).

[0063] In some embodiments, the methods of the present invention are particularly suitable for treating cognitive disorders. Cognitive disorders, diseases, or dysfunctions include, but are not limited to, Alzheimer's disease, memory loss, age-related memory loss, post-traumatic stress disorder (PTSD), neurological disorders, and epilepsy. The cognitive dysfunction can be stress-related, age-related, or a combination thereof. Alternatively, the cognitive dysfunction can be associated with a disease or disorder, including, but not limited to, 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), Huntington's disease, Rett syndrome, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS or motor neuron disease), schizophrenia, bipolar disorder, and major depression.

[0064] In some embodiments, the methods of the present invention are particularly suitable for treating ventilator-induced diaphragmatic dysfunction.

[0065] As used herein, the term "ventilator-induced diaphragmatic dysfunction" or "VIDD" has its ordinary meaning in the art and refers to a condition in which diaphragmatic atrophy and contractile dysfunction occurs after prolonged controlled mechanical ventilation. 24 Ventilator-induced diaphragmatic dysfunction can result from prolonged controlled mechanical ventilation (MV), e.g., greater than 12 hours. However, such prolonged MV is not limited to a particular length of time. For example, in some embodiments, prolonged MV comprises a period of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100 hours to at least about 1, 10, 20, 50, 75, 100, or more hours, days, or years. In other embodiments, prolonged MV comprises a period of at least about 5, 6, 7, 8, 9, or 10 hours to at least about 10, 20, or 50 hours. In some embodiments, prolonged MV is for a period of at least about 10-12 hours to any period greater than 10-12 hours.

[0066] In some embodiments, the subject requires ventilatory support due to respiratory and / or cardiac failure, which may be exacerbated by sepsis, metabolic disorders, neuromuscular disease, or surgery, including post-surgical recovery.

[0067] Typically, a subject will suffer from a disease whose symptoms worsen, resulting in the subject requiring artificial respiratory support (i.e., mechanical ventilation). For example, some pulmonary diseases, such as chronic obstructive pulmonary disease (COPD), pneumonia, sepsis (including severe sepsis and septic shock), acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), and cystic fibrosis (CF), typically require some form of ventilatory support to achieve clinical improvement. The majority of patients with severe coronavirus disease 2019 (COVID-19) are associated with acute respiratory failure and the need for mechanical ventilation. COVID-19 is a respiratory syndrome that presents with clinical pathology similar to mild upper respiratory tract illness (common cold-like symptoms), but occasionally develops severe lower respiratory tract disease and extrapulmonary manifestations, leading to multiple organ failure and death. Of the more than 430 million people infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many have required mechanical ventilation. It is estimated that 5 percent of patients infected with SARS-CoV-2 will require advanced respiratory support. 37 Thus, in some embodiments, the subject has COVID-19.

[0068] In some embodiments, the subject has suffered a traumatic injury. Pulmonary dysfunction in trauma patients is multifactorial and can be the result of direct contusion of lung tissue, lung injury from rib fractures, loss of function of the chest wall, fat embolism into the lung from long bone fragments, aspiration of blood or gastric contents, and systemic inflammatory response syndrome (SIRS) activated by shock, reperfusion, and transfusion therapy.

[0069] In some embodiments, the compound of formula I is administered before MV, shortly after MV initiation, during MV, and / or shortly after MV. In some embodiments, administration of the compound of formula I according to the present invention occurs at any time during MV.

[0070] The compounds of formula I according to the present invention are also suitable for preventing the risks associated with ventilator-induced diaphragmatic dysfunction. The risks associated with ventilation dependence include increased patient discomfort and risk of secondary diseases (e.g., pneumonia, pulmonary fibrosis, aspiration, acute renal failure, cardiac arrhythmias, sepsis, vocal cord dysfunction, and acute lung injury secondary to barotrauma or volutrauma), increased morbidity and mortality, increased healthcare costs, and prolonged treatment duration. Although patients with chronic ventilator dependence (CVD) account for only 5% to 10% of patients in intensive care units, these patients consume approximately 50% of total ICU resources, measured by staff time and equipment usage. Specifically, it has been estimated that weaning patients consume approximately 41% of the total ventilation time of intensive care unit patients. The economic costs of long-term MV dependence are enormous. Episodes of long-term MV dependence can be financially devastating for families and healthcare organizations, placing a financial burden on private insurers and government healthcare resources.

[0071] According to the present invention, the compound of Formula I is administered in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount of the compound of Formula I sufficient to treat the target disease at a reasonable benefit / risk ratio applicable to any medical treatment. The total daily use of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the severity of the disorder or injury being treated; the activity of the specific compound employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound employed; the duration of treatment; drugs used in combination with or concomitantly with the specific compound employed; and other factors well known in the medical field. For example, it is well within the skill of one of ordinary skill in the art to start administering the compound at a lower level than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In particular, compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the active ingredient, for symptomatic adjustment of the dosage to the subject being treated. Medicaments typically contain from about 0.01 mg to about 500 mg of the active ingredient, particularly from 1 mg to about 100 mg. An effective amount of the drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0072] The compound of Formula I is typically administered in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable excipient, and optionally a sustained-release matrix, such as a biodegradable polymer. "Pharmaceutically" or "pharmaceutically acceptable" refers to a molecular entity and composition that, when properly administered to a mammal, particularly a human, will not produce adverse, allergic, or other unexpected reactions. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or auxiliary formulation. In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active ingredient can be administered to animals or humans alone or in combination with another active ingredient in a unit dosage form, in a mixture with a conventional pharmaceutical support. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, sprays, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and nasal dosage forms, and rectal dosage forms. Typically, pharmaceutical compositions contain a pharmaceutically acceptable vehicle for injectable formulations. These may be, in particular, isotonic, sterile, saline solutions (such as monosodium phosphate or disodium phosphate, sodium chloride, calcium chloride, or magnesium chloride, or mixtures of such salts), or dried, especially lyophilized, compositions, which can optionally be made into an injectable solution upon addition of sterile water or saline. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions containing the compounds of the invention as free bases or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in oils, glycerol, liquid polyethylene glycols, and mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The antibody can be formulated into the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and those formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, and mandelic acids. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, and procaine. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption in the composition, such as aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active antibody in the required amount in an appropriate solvent, along with some of the other ingredients listed above, as needed, followed by filtration and purification. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof. After formulation, the solution is administered in a manner compatible with the dosage formulation and in an amount that is therapeutically effective.The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, although drug-release capsules and the like are also available. For oral administration in aqueous solution, for example, the solution is suitably buffered, if necessary, and the liquid diluent is first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those of skill in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy solution or injected at the indicated infusion site. Some dosage variation will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for the individual subject.

[0073] Therefore, a third object of the present invention relates to a pharmaceutical composition comprising a compound of formula I as defined above, or a pharmaceutically acceptable salt, and a pharmaceutically acceptable excipient.

[0074] A fourth object of the present invention is to provide a compound of formula IA:

[0075] [ka]

[0076] (In the formula, R1 represents H or OH; R2 is a linear C1-C 10 Alkyl group, C2-C7 alkenyl group containing one double bond, or C2-C7 alkenyl group containing two double bonds 10 represents an alkenyl group, However, R2 is

[0077] [ka]

[0078] When R1 represents H, or a pharmaceutically acceptable salt thereof.

[0079] In one embodiment, R2 has formulas a, b, and c:

[0080] [ka]

[0081] is a group selected from the group

[0082] The novel compounds are in particular

[0083] [ka]

[0084] wherein the compound of formula IA has the structure of formula IIA, formula IIIA, formula IVA, formula S-IVA, or formula R-IVA.

[0085] A fifth object of the invention relates to compounds of formula IA as defined above, for use in a method of treatment of the human or animal body.

[0086] A sixth object of the present invention relates to a pharmaceutical composition comprising a compound of formula IA as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0087] A seventh object of the present invention is a process for preparing a compound of formula I or of formula IA as defined above, comprising the steps of: Step A: oxidation of an alcohol of formula V to obtain an aldehyde of formula VI;

[0088] [ka]

[0089] wherein R3 is an alcohol protecting group, particularly a silyl protecting group, more particularly a t-butyldimethylsilyl protecting group; R4 is methyl or ethyl; R5 is H or OR3, where R3 is as defined above. Step B: olefination of the aldehyde of formula VI to obtain an olefin of formula VII;

[0090] [ka]

[0091] wherein R3, R4 and R5 are as defined in Step A; R2 is as defined above for Formula I. Step C: deprotection of the hydroxyl group in formula VII to give an ester of formula VIII;

[0092] [ka]

[0093] wherein R4 and R5 are as defined in Step A; R1 and R2 are as defined above for Formula I. Step D, which involves hydrolysis of the ester of formula VIII to obtain the compound of formula I;

[0094] [ka]

[0095] wherein R4 is as defined in Step A, R1 and R2 are as defined above. The method includes:

[0096] In the process according to the invention, the protecting group R3 is selected so that it is not removed under the reaction conditions of Steps A and B.

[0097] In addition, the protecting group R3 is chosen such that it can be selectively removed in the presence of an ester (R4 = methyl or ethyl).

[0098] In a preferred embodiment, the protecting group R3 is selected from silyl protecting groups, preferably tert-butyldimethylsilyl (tBDMS) or triisopropylsilyl (TIPS), in particular tert-butyldimethylsilyl.

[0099] Step A - Oxidation In the process of the present invention, selective oxidation of hydroxyl groups to aldehyde groups can be achieved under conditions that prevent side reactions such as overoxidation and undesired removal of R3 and R4 groups.

[0100] Examples of reagents and reactions that can be used in Step A include periodinane reagents, particularly Dess-Martin periodinane (DMP), pyridinium chlorochromate (PCC), and Swern oxidation ((COCl)2 / DMSO).

[0101] In certain embodiments, step A is carried out using DMP in dichloromethane at room temperature.

[0102] "Room temperature" should be understood as a temperature comprised between 15 and 30°C.

[0103] The aldehyde VI obtained after step A can be purified using, for example, column chromatography or can be used in step B as a crude product.

[0104] The starting material V can be obtained using literature procedures (Oger C. et al., Chem. Eur. J., 2010, 16, 13976). A typical synthesis is shown in FIG.

[0105] Step B - Olefination Conversion of the aldehyde group to an olefin can be achieved by a reaction that allows for the formation of an olefin with selectivity for the Z configuration.

[0106] Selectivity towards the Z isomer is particularly achieved by the Wittig reaction in which an aldehyde reacts with a phosphorus ylide, which is formed by deprotonation of a phosphonium salt using a base.

[0107] In particular, a suitable phosphonium salt is deprotonated with NaHMDS in dry THF at a temperature below −50° C., in particular −78° C., after which a compound of formula VI is added.

[0108] Step C - Deprotection Deprotection of the hydroxyl group, ie, removal of the R3 protecting group, can be achieved under conditions that depend on the nature of the protecting group.

[0109] In particular, when R3 is a silyl protecting group, such as a tert-butyldimethylsilyl protecting group, a fluorine-based reagent such as tetrabutylammonium fluoride (TBAF) can be used.

[0110] Deprotection of the hydroxyl may result in the formation of a by-product lactone (VII-lactone) during the reaction or purification, which is formed by an intramolecular transesterification reaction.

[0111] This lactone can also be hydrolyzed in Step D to give the compound of Formula I.

[0112] [ka]

[0113] Step D - Hydrolysis Hydrolysis of the ester functionality (and / or lactone group) to give the carboxylic acid can be achieved using a base such as, for example, NaOH, KOH or LiOH, especially LiOH.

[0114] Intermediate products and compounds of formula I can be purified using techniques such as column chromatography.

[0115] The intermediate products and compounds of formula I can be characterized using techniques known to those skilled in the art, such as NMR, elemental analysis and mass spectrometry.

[0116] Purity can be determined, for example, by using HPLC and chiral HPLC.

[0117] In certain embodiments, the starting material V can be used as a mixture of isomers, where the configuration of the carbon atom at position 4 is a mixture of R and S.

[0118] In another particular embodiment, the starting material V can be used as a single isomer, where the configuration of the carbon atom at position 4 is R or S.

[0119] Therefore, a seventh object of the present invention relates to a process for preparing novel compounds of formula IA, wherein the process is as defined above for compounds of formula I.

[0120] Despite the structural complexity of the compounds disclosed herein, the inventors, after considerable research, were able to develop simple and efficient synthetic methods to obtain these compounds.

[0121] In addition, the present inventors have surprisingly found that these compounds are biologically active against diseases associated with RyR dysfunction.

[0122] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0123] [Figure 1]Ca2+ spark frequency was recorded in saponin-treated EDL muscle fibers from control (n=5), post-myocardial infarction (PMI, n=3), and post-MI mice incubated with 1 μM 4F4t-NeuroP (PMI + 4F4t-NeuroP, n=3). *, p<0.05. [Figure 2A] Figure 1 shows that 4F4t-NeuroP prevents diaphragmatic weakness induced after 6 hours of mechanical ventilation in anesthetized mice. Force-frequency relationship in diaphragms after 6 hours of mechanical ventilation in control conditions (n=8) or with (n=6) or without (n=6) iv injection of 4F4t-NeuroP. Mean maximal force production recorded at 120 Hz in control diaphragms, VIDD after 6 hours of mechanical ventilation, and VIDD after treatment with 4F4t-NeuroP. Data are presented as mean ± SEM. *, p<0.05. [Figure 2B]

[0033] Figure 1 shows the dose-response relationship of the protective effect of 4F4t-NeuroP after 6 hours of mechanical ventilation. Mice were treated at the time of induction of anesthesia by IV injection of 4F4t-NeuroP at concentrations ranging between 0.01 and 100 μM. Each value corresponds to the maximum force produced by the diaphragm at a frequency of 120 Hz. [Figure 2C] Biochemical remodeling of RyR1 after VIDD: Effect of 4F4t-NeuroP. The mean values ​​of DNP / RyR1, P-RyR1 / RyR1, and Karastabin1 / RyR1 reflect RyR1 oxidation, phosphorylation at ser2844, and Karastabin1 interaction, respectively. VIDD negatively affected all parameters, and this post-translational remodeling was prevented by 4F4t-NeuroP treatment. [Figure 3A]Figure 3 shows the force-frequency relationship of mouse diaphragm muscle after 12 hours of mechanical ventilation with or without treatment with 10 μM 4F4t-NeuroP. Furthermore, 10 μM 4F4t-NeuroP prevents diaphragm muscle fiber atrophy after 12 hours of mechanical ventilation, independent of fiber phenotype. Representative immunostaining of diaphragm fast and slow fibers in mice. Mouse diaphragm cryosections were immunostained using antibodies against fast and slow forms of myosin ATPase. Muscle membranes were counterstained with a dystrophin antibody. Staining was performed on diaphragm muscle 12 hours after MV with or without 4F4t-NeuroP. The average cross-sectional area was quantified for each condition, including all muscle fiber types (Figure 3B) or by distinguishing between slow and fast fibers (Figure 3C). [Figure 3D] Figure 1 shows the average distribution of fast-twitch versus slow-twitch fibers in each condition. (*p<0.05 vs. control) [Figure 4A] A short derivative of 4F4t-NeuroP, VB558, prevents VIDD. Figure 1 shows a comparison of the structure and synthesis of VB558. [Figure 4B] VB558, a short derivative of 4F4t-NeuroP, prevents VIDD. Figure 1 shows the effect of VB558 on the force-frequency relationship in mice ventilated for 6 hours. (Control n=2, MV n=3, VB558 1 μM n=6, VB558 10 μM n=6, *p<0.05 vs MV) [Figure 5A] Figure 1 shows that VB558 prevents VIDD in piglets after 72 hours of ventilation. Protocol for IV injection of VB558 or methanol (placebo). Figure 2 shows the evolution of plasma VB558 concentrations after bolus injection in three piglets. [Figure 5B] VB558 prevents VIDD in piglets after 72 hours of ventilation. Protocol for IV injection of VB558 or methanol (placebo). Figure 1 shows the protocol for IV injection during 72 hours of mechanical ventilation. [Figure 5C] ~ [Figure 5D]VB558 prevents VIDD after 72 hours of ventilation in piglets. Protocol for IV injection of VB558 or methanol (placebo). Transdiaphragmatic pressure was measured at different frequencies of stimulation of the phrenic nerve at the onset of anesthesia (t0) and 72 hours and before mechanical ventilation (S) in VB558-treated animals (D, n=6) and in the placebo group (C, n=6) that received the same volume of vehicle (methanol, V). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, t72 vs t0). [Figure 6] Effect of hexanol on diastolic calcium levels in DMD and control hIPSc-derived ventricular cardiomyocytes. Cardiomyocytes were loaded with indo-1, and intracellular calcium levels were assessed ratiometrically. As previously demonstrated, diastolic calcium increases reflect the leaky behavior of RyR2. [Figure 6A] Compared to control cells, calcium levels are elevated in DMD, which is prevented by hexanol (1 μM). [Figure 6B] Repeating the experiment under isoproterenol (Iso 1 μM) stimulation of the cells showed complete normalization of resting calcium under these conditions. [Figure 7] 1 is a graph showing changes in intercellular calcium transients and changes in peak cell contraction analyzed in Example 7. 10 nM Iso (isoproterenol) administration promotes the onset of ESV (premature ventricular contractions). [Figure 8] 1 is a graph showing the amount of ryanodine-dependent arrhythmogenic cells in the presence of 10 nM, 100 nM, or 1 μM of compound 20,21,22-trinol-4(RS)-4-F4t-NeuroP (compound of Formula III, VB574), as measured in Example 7. The amount of cells is expressed as a percentage of total cells, and includes a control (no VB574). [Figure 9]1 is a graph showing the amount of ryanodine-dependent arrhythmogenic cells in the presence of 1 μM of the monohydroxylated derivative of 4-F4t-NeuroP (compound of Formula IV) as measured in Example 7. The amount of cells is expressed as a percentage of total cells and includes a control (no compound of Formula IV). [Example]

[0124] Materials and Methods All reactions requiring anhydrous conditions were carried out in dried glassware (oven- or flame-dried at 120°C) under a nitrogen atmosphere with magnetic stirring unless otherwise noted. All anhydrous solvents and reagents were dried under nitrogen or argon according to standard procedures or used as received from commercial suppliers (Aldrich, Thermo Fisher Scientific). Reactions were monitored by TLC using precoated silica gel 60 plates with a fluorescent reagent at 254 nm (Merck). Reaction components were visualized using a UV lamp at 254 nm or treatment with acidic p-anisaldehyde stain followed by gentle heating. Column chromatography was performed using 40-63 μm silica gel, 230-400 mesh, Macherey-Nagel, or spherical silica gel Si60, 30 μm, Interchim. Pentane (P), EtO, EtOAc, and MeOH were used for chromatographic separation. Optical rotations were recorded on a JASCO P2000 series instrument, and concentrations c for optical rotation data are given in g / 100 mL. Infrared spectra were recorded on a Spectrum one (Perkin Elmer) using an ATR device for neat samples, and absorbance was measured in wavenumbers (cm). -1) are shown. MS (ESI) and high-resolution MS (HRMS) spectra were measured on a Q-Tof microspectrometer (resolution 100,000, Waters) or a mass spectrometer Synapt G2-S (Waters). Data were acquired by direct injection between 100 and 1500 Da using positive or negative electrospray ionization. NMR spectra were recorded on a Bruker AMX300 or Bruker Avance 500 MHz spectrometer in CDCl3 or CD3OD. 1H NMR chemical shifts were provided using internal standards at δ = 7.26 ppm for CDCl3 and δ = 3.31 ppm for CD3OD and are reported as follows: chemical shift in ppm [multiplicity, coupling constant J in Hz, relative integral, assignment]. Multiplicities are defined as follows: br = broad, m = multiplet, s = singlet, d = doublet, t = triplet, q = quadruplet, quint = quintuplet or combinations thereof. 13 C NMR chemical shifts were referenced to residual solvent of the central peak (δ = 77.16 ppm for CDCl3 and δ = 49.0 ppm for CD3OD. Connectivity is 1 H- 1 Determined by H COSY experiments, carbons were assigned according to HSQC and HMBC NMR experiments.

[0125] Mouse models of heart failure Seven-week-old male C57Bl / 6 mice (Janvier, France) were placed in a post-myocardial infarction state (PMI mice) after left coronary artery ligation. Briefly, left thoracotomy was performed under anesthesia with cardiac monitoring (2% isoflurane / O, Aerrane®, Baxter, France). The artery was ligated using an 8-0 suture 1-2 mm past its emergence from the left atrial apex. For post-operative analgesia, buprenorphine solution (0.3 mg / ml) was administered. -1) was administered subcutaneously. To confirm successful ligation, electrocardiograms were systematically performed before the animals were included. Only animals surviving on postoperative day 5 and with appropriate electrocardiogram parameters at this time were included in the study to limit bias due to differences in size. All procedures were in accordance with European Parliament Directive 2010 / 63 / EU and the Council of September 22, 2010 on Animal Protection, and were approved by the Institutional Animal Research Committee (the Departmental Council for the Protection of Population and Animal Health (Ethics for Animal Welfare and Environmental Protection, No. A34-485) and the National Ethics Committee for Animal Experiments, Languedoc Roussillon, No. CE-LR-0714). 10–12 weeks after ligation, mice were euthanized by cervical dislocation, and the EDL muscle was excised for calcium spark recording.

[0126] Calcium spark measurement Diaphragm muscle samples were dissected and stored in HEPES-buffered physiological solution (in mM: 119 NaCl, 5 KCl, 1.25 CaCl2, 1 MgSO4, 10 glucose, 1.1 mannitol, 10 HEPES, pH 7.4). The muscle was then quickly placed in a dissection chamber, and the solution was replaced with relaxation solution (in mM: 140 K-glutamate, 10 HEPES, 10 MgCl2, 0.1 EGTA, pH 7.0). Bundles of 5–10 EDL fibers were manually disentangled and permeabilized for 30 s in relaxation solution containing 0.01% saponin. After washing with saponin release solution, the solution was changed to internal imaging medium for spark acquisition (in mM): 140 K-glutamate, 5 Na2ATP, 10 glucose, 10 HEPES, 4.4 MgCl2, 1.1 EGTA, 0.3 CaCl2, and 0.05 Fluo-4 pentapotassium salt (Invitrogen), pH 7.0. Potential sparks were empirically identified using an automated detection algorithm. The mean fluorescence (F0) value of the image was calculated by summing and averaging the interim F values ​​at each spatial location, ignoring potential spark regions. This F0 value was then used to create a smoothing routine, and potential spark locations were visualized and analyzed for spatiotemporal characteristics. Image analysis was performed using IDL (v5.5, Research Systems, Inc.). Statistical comparisons were performed using an analysis of variance test with a significance level of P<0.05 (Graphpad Prism v8.4).

[0127] VIDD mouse model Thirty-five adult male C57 / BL6 mice (10–12 weeks old, 25–30 g) were divided into five groups. Three groups were intubated with a 22-gauge angiocatheter and mechanically ventilated for 6 h using a volume-controlled small animal ventilator (Minivent®, Harvard Apparatus, Saint-Laurent, Canada). The tidal volume was set at 10 μl / mg body weight, the respiratory rate was 150 breaths / min, the positive end-expiratory pressure (PEEP) level was 2–4 cm H2O, and the fraction of inspired oxygen was 0.21. Involuntary ventilation was defined as the absence of diaphragmatic contraction and evidenced by repetitive stereotypic deflections observed in the airway pressure curve.

[0128] The mice were divided into two groups: the first group (control) received methanol and the second group (4F 4t A second group of mice received an equivalent amount of NeuroP delivered intravenously (IV) over 5 minutes, 20 minutes before the start of MV. 4t -NeuroP was administered intravenously (IV) over 5 minutes 20 minutes before the start of MV. The two groups were intubated and treated in the same manner (see Matecki et al., 2016 for details). 15 (See below for more details).

[0129] Contractile function in mouse muscle preparations At the end of the MV protocol, the entire diaphragm was surgically removed and the mice were euthanized by exsanguination. Isometric stretch properties were assessed as previously described in detail (Matecki et al., 2016). 15). Excised diaphragm strips were mounted in a coated tissue bath chamber filled with equilibrated, oxygenated Krebs solution. Muscles were stimulated supramaximally using square-wave pulses (Model S48, Grass Instruments, West Warwick, RI). Force-frequency relationships were determined by sequentially stimulating the muscle for 600 ms at 10, 20, 30, 50, 60, 80, 100, and 120 Hz, with a 1-minute interval between each stimulus. After measuring the contractile properties, the muscles were measured at Lo (the length at which the muscle produces maximal isometric tension), dried, and weighed. For comparative purposes, force production by the diaphragm was normalized for the total cross-sectional area of ​​the muscle strip and expressed in N.cm. -2 The total cross-sectional area of ​​the muscle strip was calculated by multiplying the muscle weight by its length and tissue density (1.056 g / cm 3 ) was determined by dividing by

[0130] The remainder of the diaphragm was sectioned, one portion was flash frozen in liquid nitrogen for secondary use in biochemical analysis, and the other portion was frozen for Ca 2+ It was used fresh for spark measurements.

[0131] Biochemical analysis of RyR1 Muscle biopsies were homogenized in 150 μl of a buffer containing 5% SDS, 5% beta-mercaptoethanol, 10% glycerol, 10 mM EDTA, and 50 mM Tris / HCl buffer (pH = 8.0). Each sample was immediately denatured at 90°C for 4 min. After centrifugation (5000 rpm) at 4°C, supernatant protein concentrations were measured in duplicate using a BCA protein assay, equilibrated to the same concentration by dilution with loading buffer, and aliquoted at 2 μg / μl. RyR1 was immunoprecipitated from 250 μg of homogenate using an anti-RyR antibody (4 μg of RyR1-1327) in 0.5 ml of modified RIPA buffer (50 mM Tris-HCl pH 7.4, 0.9% NaCl, 5.0 mM NaF, 1.0 mM Na3VO4, 1% Triton-X100, and protease inhibitors) for 1 h at 4°C. Immune complexes were incubated with protein A Sepharose beads (Amersham Pharmacia) for 1 hour at 4°C, and the beads were washed three times with buffer. Proteins were separated on an SDS-PAGE gel (4-20% gradient) and transferred to a nitrocellulose membrane (SemiDry transfer blot, Bio-Rad) at 200 mA for 2 hours. To prevent nonspecific antibody binding, the membrane was incubated with blocking solution (LICOR Biosciences) and washed with Tris-buffered saline with 0.1% Tween-20.

[0132] Blots were incubated with primary antibodies against RyR1 (RyR1-1327, an affinity-purified rabbit polyclonal antibody raised against a KLH-conjugated peptide with the amino acid sequence CAEPDTDYENLRRS, corresponding to residues 1327–1339 of mouse skeletal RyR1, with an additional cysteine ​​residue added at the amino terminus), affinity-purified together with the unconjugated peptide. We also used an antibody against calstabin1 (1:2500 in blocking buffer, LICOR Biosciences), a human antibody phosphorylated at Ser-2808 (1:5,000) that detects PKA-phosphorylated mouse RyR1 (on Ser-2844) and RyR2 (on Ser-2808), and a phospho-epitope-specific antibody against S-nitrosylated cysteine ​​residues (1:1000, Sigma). To determine RyR1 oxidation, immunoprecipitates were treated with 2,4-dinitrophenylhydrazine, and derivatized carbonyls were detected using the OxyBot Protein Oxidation Detection Kit (Catalog S7150, Chemicon International Inc.). After three washes, membranes were incubated with infrared-labeled secondary antibodies. Control samples were analyzed on each gel for normalization, and total RyR1 levels did not differ between groups.

[0133] Piglet model As mentioned earlier 18、25 Twelve piglets (15–20 kg) were divided into two groups of six animals each. In both groups, oxygenation was maintained with a fraction of inspired oxygen (FIO2) ranging from 25–35% and a tidal volume of 10–12 ml / kg body weight. -1During this time, the respiratory frequency was 15-30 cycles / min, and normocapnia was maintained. In the first group (MV group), the piglets were mechanically ventilated using CMV with a positive end-expiratory pressure level of 5 cm H2O. The absence of spontaneous breathing was verified with a ventilator trend graph, and the absence of diaphragmatic electrical activity was confirmed by measuring the electromyographic activity of the diaphragm. In the second group (control group), the piglets were ventilated using ASV with settings appropriate for the piglet's weight (0.3 L min). -1 The piglets were then ventilated using a 500-kJ / kg / day (inspiratory flow trigger at 100-150%, percentage of mechanical ventilation between 100-150%, positive end-expiratory pressure level at 5 cm H2O, and expiratory trigger at 25% of peak inspiratory flow). Electromyographic activity of the diaphragm was assessed to verify whether the piglets were able to breathe spontaneously. Both groups received identical treatments, except for the mode of MV.

[0134] Briefly, piglets were given sodium pentobarbital at 5–6 mg kg -1 The animals were anesthetized by intravenous injection of propofol 15–20 mg kg , intubated with a cuffed endotracheal tube, and mechanically ventilated (Galileo®, Hamilton Medical AG, Rhazuns, Switzerland). -1 , midazolam 0.1 to 0.3 mg / kg -1 and ketamine 3–4 mg / kg -1 .time -1 Sedation was maintained using continuous intravenous infusion. Sedation levels were monitored using a bispectral index system (BIS®, Aspect, Norwood, MA). A heating pad was used to maintain a normal body temperature of 38.5–39.5°C as needed. A carotid catheter (PiCCO®, Pulsion, Munich, Germany) was inserted to monitor heart rate, arterial blood pressure, and cardiac output. The absence of spontaneous breathing in the MV group was verified using ventilator trend graphs and diaphragm electromyographic activity measurements.

[0135] Arterial carbon dioxide tension was monitored using a capnograph (Deltatrac®, Datex-Ohmeda, Helsinki, Finland) and verified by arterial blood gas analysis (iSTAT®, Abbott, Abbott Park, IL). -1 .day -1 Parenteral nutrition was administered from day 1 to provide a diet containing 10% glucose, 20% amino acid solution and Hyperamine 20®, Braun, Boulogne Billancourt, France. All procedures were performed aseptically. All animals received intravenous antibiotic prophylaxis (amoxicillin-clavulanate 100 mg.kg 3 times daily). -1 .day -1 ).

[0136] The contractile function of the diaphragm is measured in vivo by transdiaphragmatic pressure (Pdi) 18 The phrenic nerve was stimulated by supramaximal stimulation using 2-second and 150-millisecond trains at frequencies ranging from 20 to 120 Hz, adjusted to induce diaphragmatic contraction.

[0137] The experimental and placebo solutions were administered at 0.250 mg / kg immediately before intubation of the animals. -1 IV injection at a concentration of 0.025 mg.kg followed by a maintenance dose of 0.025 mg.kg -1 was injected every 12 hours.

[0138] For these experiments, the surgeons treating the piglets were unaware of whether they were being injected with placebo or VB558. Analyses were performed blindly by a third party, and the affiliations of the piglets were later revealed.

[0139] statistics Three statistical analyses were performed: 1) unpaired Student's t-test to compare a given parameter (cross-sectional area, % of fibers) between two conditions (after mechanical ventilation or not); 2) one-way ANOVA followed by post hoc Tukey's test to compare a given parameter (spark, maximum diaphragm force at 120 Hz, coimmunoprecipitation ratio) between three conditions (control, VIDD, VIDD+VB588); and 3) two-way repeated measures ANOVA followed by post hoc Bonferroni's test to compare the relationship between force or Pdi frequency.

[0140] synthesis

[0141] Example 1 Compound II - [17,18,19,20,21,22]-hexanol-4F4 t Synthesis of Neuroprostane (VB558) Compound II was prepared according to Scheme 1.

[0142] [ka]

[0143] Scheme 1: a) DMP, CH2Cl2, RT, b) BrPh3P(CH2)2CH3, NaHMDS, THF, -78 °C to RT, 81% (2 steps), c) TBAF, THF, RT, 40%, d) LiOH, THF / H2O (1:1), RT, 36%.

[0144] Example 1a Synthesis of (E)-methyl 6-((1S,2R,3R,5S)-3,5-bis((tert-butyldimethylsilyl)oxy)-2-((Z)-pent-2-en-1-yl)cyclopentyl)-4-((tert-butyldimethylsilyl)oxy)hex-5-enoate 2

[0145] [ka]

[0146] To a solution of alcohol 1 (390 mg, 0.62 mmol, 1 eq) in DCM (10 mL) was added DMP (2.43 mL, 0.38 M in DCM, 0.93 mmol, 1.5 eq) at RT. After stirring for 30 min at RT, the reaction was quenched by the addition of 10% aqueous NaSO / NaHCO (1:1, v / v) solution (approximately 10 mL). The organic phase was separated, and the aqueous phase was extracted with EtO (3 × 10 mL). The combined organic phases were washed with 10% aqueous NaSO / NaHCO (1:1, v / v) solution (approximately 10 mL), then with brine, dried over MgSO, and concentrated in vacuo. The crude product was used in the next synthetic step.

[0147] To a solution of propyltriphenylphosphonium bromide (550 mg, 1.42 mmol, 2.3 eq) in dry THF was added NaHMDS (682 mL, 2 M in THF, 1.36 mmol, 2.2 eq) at RT, and the mixture was stirred at RT for 1 h. The reaction was cooled to -78 °C, and a solution of the aldehyde (390 mg, 0.62 mmol, 1 eq) in dry THF (4 mL) was added via cannula. The reaction was allowed to warm to RT overnight, quenched with 10% aqueous NH4Cl (approximately 10 mL), extracted with Et2O (3 × 10 mL), and the combined organic phases were washed with 10% aqueous NH4Cl (approximately 20 mL), brine, dried over MgSO4, and concentrated in vacuo. The crude extract was purified by SiO2 column chromatography (pentane / Et2O 95:5) to give 2 (260 mg, 81% over two steps) as a colorless oil. R f = 0.66 (pentane / Et2O 95:5). 1H NMR (300 MHz, CDCl3) δ 5.48 - 5.24 (m, 4H), 4.15 - 4.13 (m, 1H), 3.96 - 3.70 (m, 2H), 3.64 (s, 3H), 2.68 - 2.48 (m, 1H), 2.41 - 2.22 (m, 3H), 2.16 - 1.38 (m, 8H), 0.94 (t, J = 7.5, 3H), 0.91 - 0.80 (s, 27H), 0.00 (s, 18H). 13 C NMR (75 MHz, CDCl3) δ 174.40, 134.94 dia 1, 134.86 dia 2, 132.31 dia1, 132.21 dia 2, 129.37 dia 1, 128.90 dia 2, 127.92, 76.30, 76.08, 72.21 dia 1, 72.05 dia 2, 52.60 dia 1 52.46 dia 2, 51.64, 50.35 dia 1, 50.21 dia 2, 33.32, 29.73, 26.01, 20.82, 18.20, 14.41, -4.20, -4.41, -4.58.

[0148] Example 1b Synthesis of (E)-methyl 6-((1S,2R,3R,5S)-3,5-dihydroxy-2-((Z)-pent-2-en-1-yl)cyclopentyl)-4-hydroxyhex-5-enoate 3

[0149] [ka]

[0150] To compound 2 (260 mg, 0.4 mmol, 1 eq) was added TBAF (4.76 mL, 1 M in THF, 4.76 mmol, 12 eq). After stirring at RT for 1 h, the reaction was quenched by adding DOWEX-50W resin (2.5 g), CaCO (2.5 g), and MeOH (75 mL), stirred at RT for 1 h, filtered through a pad of Celite®, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give 3 as a colorless oil, as an ester / lactone mixture in a 72:28 ratio. R f = 0.30 (100% EtOAc).

[0151] ester: 1 H NMR (300 MHz, CDCl3) δ 5.65 - 5.23 (m, 4H), 4.15 - 3.85 (m, 3H), 3.64 (s, 3H), 2.75 (dt, J = 14.0, 7.4 Hz, 2H), 2.58 - 2.28 (m, 4H), 2.11 - 1.96 (m, 3H), 1.96 - 1.94 (m, 1H), 1.94 - 1.82 (m, 2H), 1.82 - 1.75 (m, 1H), 1.65 - 1.52 (m, 1H), 1.13 (s, 1H), 0.92 (t, J = 7.5Hz, 3H). 13 C NMR (75 MHz, CDCl3) δ 174.47, 135.18, 133.12, 130.42, 127.22, 76.19, 76.07, 75.97, 72.05, 53.26, 51.78, 50.87, 42.32, 42.14, 31.94, 30.15, 28.79, 28.56, 26.69, 20.73, 14.24.

[0152] Lactone:

[0153] [ka]

[0154] 1 H NMR (300MHz, CDCl3): 5.61-5.55 (m, 2H), 5.44-5.24 (m, 2H), 4.92-4.86 (m, 1H), 4.12-3.92 (m, 4H), 2.83-2.79 (m, 1H), 2.53-2.30 (m, 5H), 2.18-2.15 (m, 1H), 2.00-1.93 (m, 4H), 1.67-1.60 (m, 1H), 0.93 (s, 3H).

[0155] Example 1c Synthesis of (E)-6-((1S,2R,3R,5S)-3,5-dihydroxy-2-((Z)-pent-2-en-1-yl)cyclopentyl)-4-hydroxyhex-5-enoic acid II

[0156] [ka]

[0157] To a solution of 2 (50 mg, 0.18 mmol, 1 eq) in 4 mL of THF / HO (1:1, v / v) was added LiOH (45 mg, 1.07 mmol, 6 eq) at RT. After stirring for 1 h, the reaction was quenched with 1 M NaHSO to acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give compound II (27 mg, 36%) as a colorless oil. R f = 0.13 (100% EtOAc). 11H NMR (300 MHz, MeOD) δ 5.60 - 5.47 (m, 2H), 5.42 - 5.31 (m, 2H), 4.16 - 4.02 (m, 1H), 4.00 - 3.92 (m, 1H), 3.85 (dt, J = 7.0, 5.0 Hz, 1H), 2.67 (s, 1H), 2.46 (dt, J = 14.6, 7.4 Hz, 1H), 2.35 (dd, J = 8.2, 7.0 Hz, 2H), 2.15 - 1.93 (m, 5H), 1.76 (td, J = 7.7, 6.6 Hz, 2H), 1.51 (dt, J = 14.2, 5.0 Hz, 1H), 0.94 (t, J = 7.5 Hz, 3H). 13 13C NMR (75 MHz, MeOD) δ 176.04, 134.62, 131.93, 129.12,​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Scheme 2: a) DMP, CHCl, RT; b) (Z)-hex-3-en-1-iodide yltriphenylphosphonium, NaHMDS, THF, −78 °C to RT, 83% (2 steps); c) TBAF, THF, RT, 59%; d) LiOH, THF / HO (1:1), RT, 69%.

[0161] Example 2a Synthesis of (E)-methyl 6-((1S,2R,3R,5S)-3,5-bis((tert-butyldimethylsilyl)oxy)-2-((2Z,5Z)-octa-2,5-dien-1-yl)cyclopentyl)-4-((tert-butyldimethylsilyl)oxy)hex-5-enoate 5

[0162] [ka]

[0163] To a solution of alcohol 4 (467 mg, 0.74 mmol, 1 eq) in DCM (10 mL) was added DMP (2.31 mL, 0.38 M in DCM, 1.11 mmol, 1.5 eq) at RT. After stirring at RT for 30 min, the reaction was quenched by the addition of 10% aqueous NaSO / NaHCO (1:1, v / v) solution (approximately 10 mL). The organic phase was separated, the aqueous phase was extracted with EtO (3 × 10 mL), and the combined organic phase was washed with 10% aqueous NaSO / NaHCO (1:1, v / v) solution (approximately 10 mL), then with brine, dried over MgSO, and concentrated in vacuo. The crude product was used in the next synthetic step.

[0164] To a solution of (Z)-hex-3-en-1-iodide yltriphenylphosphonium (803 mg, 1.7 mmol, 2.3 eq) in dry THF at RT was added NaHMDS (815 μL, 2 M in THF, 1.63 mmol, 2.2 eq), and the mixture was stirred at −50° C. for 0.5 h. The reaction was cooled to −78° C., and a solution of the aldehyde (465 mg, 0.74 mmol, 1 eq) in dry THF (5 mL) was added via cannula and stirred for 3 h, quenched with 10% aqueous NH4Cl (approximately 10 mL), extracted with Et2O (3 × 10 mL), and the combined organic phases were washed with 10% aqueous NH4Cl (approximately 20 mL), brine, dried over MgSO4, and concentrated in vacuo. The crude extract was purified by SiO2 column chromatography (pentane / Et2O 95:5) to give 5 (172 mg, 83% over two steps) as a colorless oil. R f = 0.60 (pentane / Et2O 95:5). 1 H NMR (300 MHz, CDCl3) δ 5.57 - 5.16 (m, 6H), 4.14 (m, 1H), 3.98 - 3.74 (m, 2H), 3.63 (s, 3H), 2.66 (m, 3H), 2.42 - 2.19 (m, 3H), 2.17 - 1.62 (m, 7H), 1.51 (dt, J = 13.9, 5.0 Hz, 1H), 0.94 (t, J = 7.5Hz, 3H), 0.89 (s, 27H), 0.00 (s, 18H). 13 C RMN (100 MHz, CDCl3): 174.21, 134.87, 131.88, 129.08, 128.60, 128.51, 125.55, 76.15, 75.96, 72.00, 52.32, 51.50, 50.00, 44.36, 33.18, 29.58, 26.05, 25.85, 25.69, 20.58, 18.06, 14.32, -4.57. HRMS (ESI + ) C 38 H 74 O5Si3[M]+ Calculated value: 695.4922, measured value: 695.4918.

[0165] Example 2b Synthesis of 5-((E)-2-((1S,2R,3R,5S)-3,5-dihydroxy-2-((2Z,5Z)-octa-2,5-dien-1-yl)cyclopentyl)vinyl)dihydrofuran-2(3H)-one 6

[0166] [ka]

[0167] To compound 5 (170 mg, 0.247 mmol, 1 eq) was added TBAF (1.48 mL, 1 M in THF, 1.48 mmol, 6 eq). After stirring at RT for 1 h, the reaction was quenched by adding DOWEX-50W resin (1.2 g), CaCO (0.4 g), and MeOH (10 mL), stirred at RT for 1 h, filtered through a pad of Celite®, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give 6 (47 mg, 59%) as a colorless oil. R f =0.38 (100% EtOAc). 1 H NMR (300 MHz, CDCl3) δ 5.61 (dt, J = 5.0, 1.8 Hz, 2H), 5.45 - 5.18 (m, 4H), 4.89 (td, J = 7.4, 3.2 Hz, 1H), 4.00 (m, 2H), 2.77 (m, 3H), 2.59 - 2.29 (m, 4H), 2.26 - 1.87 (m, 6H), 1.65 (dd, J = 10.5, 4.2 Hz, 1H), 0.95 (t, 3H). 13C RMN (100 MHz, CDCl3): 177.09, 132.69, 132.39, 130.22, 129.81, 127.97, 126.74, 80.47, 76.29, 53.37, 50.85, 42.36, 28.82, 28.58, 26.85, 25.72, 20.63, 14.31. HRMS (ESI + ) C 19 H 29 O4 [M+H] + Calculated value: 321.2066, measured value: 321.2068.

[0168] Example 2c Synthesis of (E)-6-((1S,2R,3R,5S)-3,5-dihydroxy-2-((2Z,5Z)-octa-2,5-dien-1-yl)cyclopentyl)-4-hydroxyhex-5-enoic acid III

[0169] [ka]

[0170] To a solution of 6 (47 mg, 0.15 mmol, 1 eq) in 3.6 mL THF / HO (1:1, v / v) was added LiOH (37 mg, 0.88 mmol, 6 eq) at RT. After stirring for 1 h, the reaction was quenched with 1 M NaHSO to acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give compound III (34 mg, 69%) as a colorless oil. R f = 0.13 (100% EtOAc). 1H NMR (300 MHz, CD3OD) δ 5.58 - 5.47 (m, 2H), 5.43 - 5.20 (m, 4H), 4.11 - 4.01 (m, 1H), 3.95 (dt, J = 7.3, 4.9 Hz, 1H), 3.85 (dt, J = 7.7, 5.0 Hz, 1H), 2.82 - 2.65 (m, 3H), 2.45 (dt, J = 14.5, 7.4 Hz, 1H), 2.34 (td, J = 7.5, 2.1 Hz, 2H), 2.16 - 1.96 (m, 5H), 1.83 - 1.70 (m, 2H), 1.52 (dt, J = 14.2, 5.1 Hz, 1H), 0.94 (t, J = 7.6 Hz, 3H). 13 C RMN (100 MHz, CDCl3): 179.93, 136.15, 134.35, 132.76, 131.49, 130.45, 128.32, 82.82, 76.19, 75.93, 53.41, 51.41, 43.57, 33.45, 29.71, 27.31, 26.62, 21.49, 14.69. HRMS (ESI - ) C 19 H 30 O5 [MH] - Calculated value: 337.2015, measured value: 337.2007.

[0171] Example 3 Synthesis of .ALPHA.-chain synthons for monohydroxylated derivatives IV Silyl protected intermediate 11 was prepared according to the reaction in Scheme 3.

[0172] [ka]

[0173] Scheme 3: α-chain synthon 11 of monohydroxylated derivative IV

[0174] Example 3a Synthesis of (E)-methyl 6-((1R,2R,5S)-2-(2-acetoxyethyl)-5-((tert-butyldimethylsilyl)oxy)cyclopentyl)-4-oxohex-5-enoate 8

[0175] [ka]

[0176] To a solution of alcohol 7 (1 g, 3.16 mmol, 1 eq) in DCM (30 mL) was added DMP (12.5 mL, 0.38 M in DCM, 4.74 mmol, 1.5 eq) at RT. After stirring at RT for 30 min, the reaction was quenched by the addition of 10% aqueous NaSO / NaHCO (1:1, v / v) solution (approximately 20 mL). The organic phase was separated, the aqueous phase was extracted with EtO (3 × 200 mL), and the combined organic phases were washed with 10% aqueous NaSO / NaHCO (1:1, v / v) solution (approximately 20 mL) and brine, dried over MgSO, and concentrated in vacuo. The crude product was used in the next synthetic step.

[0177] To a solution of methyl 5-(dimethoxyphosphoryl)-4-oxopentanoate (3.1 g, 13 mmol, 3.4 eq) in dry THF was added NaHMDS (6 mL, 2 M in THF, 12.16 mmol, 3.2 eq) at 0 °C, and the mixture was stirred at 0 °C for 1 h. The reaction was cooled to -78 °C, and a solution of the aldehyde (1.2 g, 3.8 mmol, 1 eq) in dry THF (10 mL) was added via cannula. The mixture was stirred for 14 h, quenched with brine (approximately 20 mL), extracted with EtO (3 × 10 mL), and the combined organic phases were washed with brine, dried over MgSO, and concentrated in vacuo. The crude extract was purified by SiO column chromatography (pentane / EtO 9:1) to give 8 (1.3 g, 80% over two steps) as a colorless oil. R f = 0.64 (pentane / Et2O 9:1). 1H NMR (300 MHz, CDCl3) δ 6.53 (dd, J = 15.7, 10.3 Hz, 1H), 6.08 (d, J = 15.7 Hz, 1H), 4.01 - 3.85 (m, 3H), 3.59 (s, 3H), 2.78 (t, J = 6.7 Hz, 2H), 2.58 - 2.44 (m, 3H), 2.31 (q, J = 8.1 Hz, 1H), 1.93 (s, 5H), 1.66 - 1.33 (m, 3H), 1.33 - 1.16 (m, 1H), 0.77 (s, 9H), -0.07 (d, J = 1.2 Hz, 6H).

[0178] Example 3b Synthesis of (E)-methyl 6-((1R,2R,5S)-2-(2-acetoxyethyl)-5-((tert-butyldimethylsilyl)oxy)cyclopentyl)-4-hydroxyhex-5-enoate 9

[0179] [ka]

[0180] To enone 8 (1.2 g, 2.8 mmol, 1.0 eq) in 30 mL of dry MeOH was added CeCl (1.04 g, 2.8 mmol, 1.0 eq). After 15 min, the mixture was cooled to 0 °C and NaBH (53 mg, 1.4 mmol, 0.5 eq) was added. After 20 min, the reaction was complete, and 30 mL of EtOAc and 30 mL of saline were added. The aqueous phase was extracted with 3 × 20 mL of EtOAc. The organic layer was extracted with 20 mL of saline, dried over MgSO, filtered, and the solvent was removed under reduced pressure. Alcohol 9 was obtained and used directly in the reaction without further purification (1.15 g, 96%). R f = 0.35 (pentane / Et2O 1:1). 1H NMR (300 MHz, CDCl3) δ 5.56 - 5.42 (m, 1H), 5.42 - 5.22 (m, 1H), 4.18 - 3.80 (m, 4H), 3.62 (s, 3H), 2.43 - 2.30 (m, 3H), 2.22 (dt, J = 17.0, 7.5 Hz, 1H), 1.97 (dd, J = 3.5, 1.9 Hz, 5H), 1.85 - 1.69 (m, 3H), 1.64 - 1.40 (m, 3H), 1.20 (td, J = 7.2, 3.7 Hz, 1H), 0.81 (s, 9H), -0.03 (d, J = 1.0 Hz, 6H).

[0181] Example 3c Synthesis of (E)-methyl 6-((1R,2R,5S)-2-(2-acetoxyethyl)-5-((tert-butyldimethylsilyl)oxy)cyclopentyl)-4-((tert-butyldimethylsilyl)oxy)hex-5-enoate 10

[0182] [ka]

[0183] To a solution of alcohol 9 (1.1 g, 2.6 mmol, 1.0 eq) in 100 mL of CHCl was added TBSCl (1.2 g, 7.7 mmol, 3.0 eq), imidazole (0.7 g, 10.3 mmol, 4.0 eq), and 4-DMAP (cat.). After 4.5 h, the reaction was complete, and 100 mL of EtO and 100 mL of saline were added. The aqueous phase was extracted with 3 × 20 mL of EtO. The organic layer was extracted with 50 mL of saline, dried over MgSO, and the solvent was removed under reduced pressure. Compound 10 was obtained (1.3 g, quant.) and used directly in the reaction without further purification. R f = 0.89 (pentane / Et2O 1:1). 1H NMR (300 MHz, CDCl3) δ 5.61 - 5.07 (m, 2H), 3.96 (m, 4H), 3.59 (s, 3H), 2.26 (dd, J = 7.5, 3.6 Hz, 4H), 2.08 - 1.05 (m, 11H), 0.97 - 0.59 (m, 18H), 0.31 - -0.13 (m, 12H).

[0184] Example 3d Synthesis of (E)-methyl 4-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)cyclopentyl)hex-5-enoate 11

[0185] [ka]

[0186] To a solution of acetate 10 (1.3 g, 2.4 mmol, 1 eq) in 35 mL of dry MeOH was added K2CO3 (1.3 g, 9.6 mmol, 4 eq) and stirred at RT. After 3 h, the reaction was quenched by adding 20 mL of a 1:1 Et2O / H2O solution and stirred for 1 h. The aqueous phase was extracted with Et2O (3 × 20 mL), and the combined organic layers were washed with brine, dried over MgSO4, and concentrated in vacuo.

[0187] The crude extract was purified by SiO column chromatography (20–50% EtO / pentane gradient) to give 11 as follows: 4S-epimer (S)-11 (0.47 g, 37%), 4-R / S-epimer mixture (0.15 g, 12%), and 4R-epimer (R)-11 (0.28 g, 23%). R f =0.50 (pentane / Et2O 1:1) 4S-epimer; R f =0.40 (pentane / Et2O 1:1) 4R-epimer

[0188] [ka]

[0189] (E)-Methyl 4S-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)cyclopentyl)hex-5-enoate (S)-11 1 H NMR (300 MHz, CDCl3) δ 5.46 - 5.20 (m, 2H), 4.11 (d, J = 5.7 Hz, 1H), 3.88 (dt, J = 5.9, 2.7 Hz, 1H), 3.64 - 3.50 (m, 5H), 2.35-2.31 (m, 4H), 1.90-1.04 (m, 8H), 0.82 (m, 18H), 0.07 - -0.44 (m, 12H). 13 C NMR (125 MHz, CDCl3) δ 174.32, 134.51, 128.76, 79.32, 71.84, 62.13, 55.10, 51.50, 37.05, 34.75, 33.67, 33.10, 29.53, 26.02, 18.17, -4.41.

[0190] [ka]

[0191] (E)-Methyl 4R-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)cyclopentyl)hex-5-enoate (R)-11 1H NMR (300 MHz, CDCl3) δ 5.55 - 5.09 (m, 2H), 4.08 (q, J = 6.1 Hz, 1H), 3.95 (dt, J = 5.9, 2.2 Hz, 1H), 3.72 - 3.44 (m, 5H), 2.32 (m, 4H), 2.03 - 1.03 (m, 8H), 0.84 (s, 18H), -0.01 (s, 12H). 13 C NMR (125 MHz, CDCl3) δ 174.27, 134.84, 128.85, 79.06, 72.34, 62.15, 55.29, 51.60, 36.99, 34.82, 33.63, 33.29, 29.71, 26.04, 18.18, -4.24.

[0192] Example 4 Synthesis of Compound IV Compounds IV, both (S)-IV and (R)-IV), were prepared from the synthon obtained in Example 3 according to Synthetic Scheme 4.

[0193] [ka]

[0194] Scheme 4. 4(S)-4F 4t - The monohydroxylated C22 analog of neuroprostane and its C4 epimer, 4(R)-4-F 4t -Neuroprostane

[0195] Example 4a Synthesis of (S,E)-methyl 4-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-((2Z,5Z,8Z)-undec-2,5,8-trien-1-yl)cyclopentyl)hex-5-enoate (S)-12

[0196] [ka]

[0197] To a solution of (S)-11 (0.4 g, 0.79 mmol, 1 eq) in DCM (15 mL) was added DMP (0.5 mg, 1.18 mmol, 1.5 eq) and the reaction was allowed to stir at RT. After 30 min, the reaction was quenched by adding 10% aqueous NaHCO3 / Na2S2O3 (1 / 1, v / v) solution. The aqueous phase was extracted with Et2O (3 × 20 mL), washed with brine, dried over MgSO4, and concentrated in vacuo to give the crude aldehyde (0.39 g, 99%).

[0198] To the previously described solution of triphenylphosphonium iodide (0.91 g, 1.78 mmol, 2.25 eq) in THF (10 mL) at −40 °C under N2, NaHMDS (0.83 mL, 1.66 mmol, 2 M / THF, 2.1 eq) was slowly added. After stirring at −40 °C for 1 h, the bright orange ylide was cooled to −78 °C and slowly added via cannula to the previously prepared solution of crude aldehyde in THF (4 mL) and stirred for 1 h. The mixture was poured onto silica gel and eluted with a gradient of pentane / Et2O (1:0 to 9:1). Careful evaporation of the solvent at RT and 700 mbar afforded (S)-12 (126 mg, brsm (based on recovered starting material), 41%, 2 steps) as a colorless oil. R f = 0.80 (pentane / Et2O 9:1). 1 H NMR (300 MHz, CDCl3) δ 5.66 - 5.01 (m, 8H), 4.13 (q, J = 5.6 Hz, 1H), 3.92 (dd, J = 5.9, 3.1 Hz, 1H), 3.69 - 3.55 (m, 3H), 2.76 (dd, J = 5.0, 3.3 Hz, 4H), 2.50 - 1.68 (m, 11H), 1.64 - 1.42 (m, 1H), 1.24-1.20 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H), 0.85 (s, 18H), 0.01 (s, 12H). 13C NMR (125 MHz, CDCl3) δ 174.41, 134.62, 131.97, 129.48, 129.25, 128.36, 128.20, 128.03, 127.15, 79.07, 72.23, 55.16, 51.46, 41.20, 33.71, 33.23, 29.65, 29.39, 28.50, 25.90, 25.78, 25.64, 20.57, 18.10, 14.29, -4.28. [α] D 20 (MeOH) = +7.86 (c = 1.26).

[0199] Example 4b Synthesis of (R,E)-methyl 4-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-((2Z,5Z,8Z)-undec-2,5,8-trien-1-yl)cyclopentyl)hex-5-enoate (R)-12

[0200] [ka]

[0201] To a solution of (R)-11 (0.25 g, 0.5 mmol, 1 eq) in DCM (10 mL) was added DMP (0.32 g, 0.75 mmol, 1.5 eq), and the reaction was allowed to stir at RT. After 30 min, the reaction was quenched by adding 10% aqueous NaHCO3 / Na2S2O3 (1 / 1, v / v) solution. The aqueous phase was extracted with Et2O (3 × 20 mL), washed with brine, dried over MgSO4, and concentrated in vacuo to give the crude aldehyde (0.244 g, 98%).

[0202] To the previously described solution of triphenylphosphonium iodide (0.58 g, 1.12 mmol, 2.25 eq) in THF (10 mL) was slowly added NaHMDS (0.5 mL, 1 mmol, 2 M / THF, 2.1 eq) at −40 °C under N2. After stirring at −40 °C for 1 h, the bright orange ylide was cooled to −78 °C and slowly added via cannula to the previously prepared solution of crude aldehyde in THF (4 mL) and stirred for 1 h. The mixture was poured onto silica gel and eluted with a gradient of pentane / Et2O (1:0 to 9:1). Careful evaporation of the solvent at RT and 700 mbar afforded (R)-12 (66 mg, 30% BRSM, 30%, 2 steps) as a colorless oil. R f = 0.84 (pentane / Et2O 9:1). 1 H NMR (300 MHz, CDCl3) δ 5.64 - 5.16 (m, 8H), 4.12 (q, J = 6.0 Hz, 1H), 3.97 (dd, J = 5.8, 2.8 Hz, 1H), 3.63 (s, 3H), 2.77 (q, J = 5.6, 5.2 Hz, 4H), 2.54 -1.75 (m, 11H), 1.61 - 1.50 (m, 1H), 1.37 - 1.20 (m, 2H), 0.95 (t, J = 7.5 Hz, 3H), 0.86 (s, 18H), 0.01 (s, 12H). 13 C NMR (125 MHz, CDCl3) δ 174.20, 134.61, 132.05, 129.50, 129.15, 128.56, 128.37, 128.20, 127.20, 78.85, 72.05, 55.21, 51.49, 41.29, 33.75, 33.23, 29.63, 29.24, 28.45, 26.05, 25.65, 25.54, 20.57, 18.19, 14.32, -4.24. [α] D 20 (MeOH) = +18.2(c=5).

[0203] Example 4c Synthesis of (S)-5-((E)-2-((1R,2S,5R)-2-hydroxy-5-((2Z,5Z,8Z)-undeca-2,5,8-trien-1-yl)cyclopentyl)vinyl)dihydrofuran-2(3H)-one (S)-13

[0204] [ka]

[0205] To compound (S)-12 (126 mg, 0.2 mmol, 1 eq) was added TBAF (0.84 mL, 1 M in THF, 0.84 mmol, 4 eq). After stirring at RT for 2 h, the reaction was quenched by adding DOWEX-50W resin (1.2 g), CaCO (0.4 g), and MeOH (15 mL), stirred at RT for 1 h, filtered through a pad of Celite®, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give (S)-13 (51 mg, 72%) as a colorless oil as a 1:1 mixture of lactone / ester. R f =0.34(EtOH).

[0206] Example 4d Synthesis of (R)-5-((E)-2-((1R,2S,5R)-2-hydroxy-5-((2Z,5Z,8Z)-undeca-2,5,8-trien-1-yl)cyclopentyl)vinyl)dihydrofuran-2(3H)-one (R)-13

[0207] [ka]

[0208] To compound (R)-12 (60 mg, 0.1 mmol) was added TBAF (0.4 mL, 1 M in THF, 0.4 mmol). After stirring at RT for 2 h, the reaction was quenched by adding DOWEX-50W resin (0.6 g), CaCO (0.2 g), and MeOH (10 mL), stirred at RT for 1 h, filtered through a pad of Celite®, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give (R)-13 (40 mg, 92%) as a colorless oil as a 83:17 mixture of ester / lactone. R f =0.32(EtOH).

[0209] Example 4e Synthesis of (S,E)-4-hydroxy-6-((1R,2S,5R)-2-hydroxy-5-((2Z,5Z,8Z)-undeca-2,5,8-trien-1-yl)cyclopentyl)hex-5-enoic acid (S)-IV

[0210] [ka]

[0211] To a solution of (S)-13 (51 mg, 0.15 mmol, 1 eq) in 4 mL of THF / HO (1:1, v / v) was added LiOH (38 mg, 0.89 mmol, 6 eq) at RT. After stirring for 1 h, the reaction was quenched with 1 M NaHSO solution to acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give compound (S)-IV (38 mg, 72%) as a white powder. R f = 0.15 (100% EtOAc). 1H NMR (300 MHz, CDCl3) δ 5.70 - 5.14 (m, 8H), 4.13 (d, J = 5.4 Hz, 1H), 4.01 (q, J = 5.8 Hz, 1H), 2.76 (dd, J = 5.8, 5.2 Hz, 4H), 2.56 - 2.30 (m, 3H), 2.28 - 2.13 (m, 1H), 2.14 - 1.84 (m, 7H), 1.63 - 1.45 (m, 1H), 1.43 - 1.15 (m, 2H), 0.97 (t, J = 7.5 Hz, 3H). 13 C NMR (125 MHz, CDCl3) δ 177.97, 134.40, 132.11, 130.50, 129.06, 128.53, 128.04, 127.05, 80.66, 72.25, 54.54, 41.23, 32.75, 31.59, 30.27, 29.20, 28.12, 25.83, 25.57, 20.59, 18.09, 14.32. HRMS (ESI - ) C 22 H 33 O4[MH] - Calculated value: 361.2379, measured value: 361.2376. [α] D 20 (MeOH) = +22.6 (c = 3.8).

[0212] Example 4f Synthesis of (R,E)-4-hydroxy-6-((1R,2S,5R)-2-hydroxy-5-((2Z,5Z,8Z)-undeca-2,5,8-trien-1-yl)cyclopentyl)hex-5-enoic acid (R)-IV

[0213] [ka]

[0214] To a solution of (R)-13 (50 mg, 0.18 mmol) in 4 mL of THF / HO (1:1, v / v) was added LiOH (45 mg, 1.07 mmol, 6 eq) at RT. After stirring for 1 h, the reaction was quenched with 1 M NaHSO solution to acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO, and concentrated in vacuo. The crude product was purified by SiO column chromatography (100% EtOAc) to give compound (R)-IV (27 mg, 36%) as a white powder. R f = 0.13 (100% EtOAc). 1 H NMR (300 MHz, CDCl3) δ 5.80 - 5.16 (m, 8H), 4.17 (m, 1H), 4.03 (m, Hz, 1H), 2.76 (t, J = 5.9 Hz, 4H), 2.58 - 2.33 (m, 3H), 2.20 - 1.80 (m, 8H), 1.65 - 1.15 (m, 3H), 0.94 (t, J = 7.5 Hz, 3H). HRMS (ESI - ) C 22 H 33 O4[MH] - Calculated value: 361.2379, measured value: 361.2376. [α] D 20 (MeOH) = -4(c=2).

[0215] Example 5 VIDD prevention research result 4th floor 4t -NeuroP prevents RyR1-mediated Ca leak Spontaneous Ca 2+ release events (i.e., Ca 2+ The term "spark" refers to the spontaneous local Ca2+ release from RyR clusters in resting muscle fibers. 2+ refers to a single Ca release event 2+ Also called Ca release units (CRUs). 2+Spark measurements allow direct in situ measurement of the opening behavior of RyRs belonging to a single CRU. 2+ The frequency of sparks provides a good estimate of the RyR1 opening frequency and represents an index of RyR-mediated SR calcium leak. We previously reported RyR1 defects in peripheral muscles of animal models with heart failure. In previous studies, we found that 4F, a lipid mediator produced by nonenzymatic free radical peroxidation of docosahexaenoic acid (DHA), is involved in the RyR1 opening frequency. 4t Neuroprostanes, such as those by NeuroP, stimulate RyR2-mediated Ca2+ upregulation in cardiac myocytes. 2+ It has been demonstrated that leakage can be prevented.

[30] 4F 4t To test the efficacy of -NeuroP in preventing functional remodeling of RyR1, we administered 1 μM of 4F to the mouse fast-twitch extensor digitorum longus (EDL) muscle. 4t -Testing short-term application of NeuroP and Ca 2+ The functional consequences for RyR1 were assessed by measuring spark frequency. Thus, we confirmed RyR1 dysfunction in EDL muscle, as indicated by a significant increase in spark frequency, in a mouse model of heart failure obtained after permanent left coronary artery ligation (PMI). PMI muscle fibers were treated with 1 μM 4F 4t We observed that this RyR1-dependent leakage behavior was completely prevented when the cells were briefly incubated with -NeuroP (Figure 1). 4t -Confirmed the ability of NeuroP to prevent RyR1 dysfunction.

[0216] 4th floor 4t -NeuroP prevents VIDD following brief MV in mice Mice were anesthetized and mechanically ventilated for 6 hours. Mice were placed in a 4F 4t They were randomized to receive a single IV injection of 4F or not, and to define the optimal concentration, dose-response curves of force-frequency relationships were generated at concentrations ranging from 0.01 to 100 μM. 4tA complete prevention of VIDD by -NeuroP could be observed, with a maximal effect observed at approximately 0.3 μM (Figures 2A-B).

[0217] 4th floor 4t -NeuroP stabilizes the Calstabin1 / RyR1 complex Diaphragm samples from the above experiments were frozen and solubilized to further investigate the biochemical properties of the RyR1 macromolecular complex. After 6 hours of mechanical ventilation, immunoprecipitation of RyR1 revealed that VIDD was responsible for the oxidation of the channel, as well as phosphorylation at ser2844 and depletion of calstabin1. This biochemical signature of leaky RyR1 was 4F 4t - was completely prevented by NeuroP (Fig. 2C).

[0218] 4th floor 4t -NeuroP prevents VIDD and diaphragm muscle atrophy following prolonged MV in mice Six hours of mechanical ventilation is also a relatively short duration, making it of particular interest for exploring the central pathophysiological mechanisms of VIDD. As previously reported, six hours of mechanical ventilation is capable of inducing force deficits without histological changes. However, 12 hours of mechanical ventilation induces RyR1-dependent SR Ca upregulation. 2+ Therefore, we investigated the effect of 4F after 12 hours of mechanical ventilation on muscle atrophy. 4t The beneficial effects of NeuroP under these conditions were examined here. 4t The protective effect of a single injection of 4F-NeuroP (10 μM) was similar to that observed after 6 h of ventilation (Figure 3A). Surprisingly, after 12 h of mechanical ventilation, 10 μM of 4F 4t -NeuroP also prevented muscle fiber remodeling (i.e., atrophy), a hallmark of VIDD. 4t -NeuroP prevented the reduction of cross-sectional area (CSA) regardless of muscle fiber phenotype (Figures 3B-3C) and without altering the distribution of fiber types (slow vs. fast, Figure 3D).

[0219] 4th floor 4t-Neuro short analog VB558 prevents VIDD Due to its efficiency in stabilizing RyR, 4F 4t -NeuroP (WO2015197562) was a very interesting biomolecule. However, its synthesis is rather long and expensive, thus limiting its use for therapeutic purposes. For this reason, the present inventors have developed a method for the synthesis of 4F 4t To identify compounds that can demonstrate at least similar efficiency to NeuroP, but whose synthesis may be more feasible, 4F 4t Several analogs of -NeuroP were screened. Screening was performed in an in vitro model of arrhythmia characterized by RyR dysfunction (as described in WO2014086819). VB558, whose synthesis is 4t -It was selected as the most promising candidate because it involves only 17 steps compared to 24 steps for NeuroP (Fig. 4A).

[0220] 4th floor 4t For 4F4t-NeuroP, mice were intravenously injected with VB558 at concentrations of 1 μM and 10 μM before being ventilated for 6 hours, with both concentrations demonstrating significant protection against mechanical ventilation in a range similar to that obtained with 4F4t-NeuroP (Figure 4B).

[0221] VB558 prevents VIDD after 72 hours of ventilation in piglets To test the potential clinical utility of neuroprostanes in preventing VIDD, we decided to test the efficacy of VB558 in a piglet VIDD model, as previously published. 17、18 .

[0222] In the first set of experiments, VB558 (0.250 mg kg -1A pharmacokinetic study was performed on three control pigs injected with a single bolus of 0.250 mg.kg. Blood samples were collected periodically over a 12-hour period to assess plasma concentrations of VB558. This demonstrated a rapid plasma peak during the first 5-10 minutes that dissipated rapidly over the course of 1 hour, suggesting a rapid biodistribution of the compound (Figure 5A). To test the preventive effect of VB558 in the piglet model, the IV injection protocol was slightly adapted to the duration of mechanical ventilation (i.e., 72 hours). We administered a bolus of 0.250 mg.kg. -1 , followed by a maintenance dose of 0.025 mg.kg -1 every 12 hours (Figure 5B). The VB558 group was compared to a piglet (placebo) group receiving the same volume of vehicle (methanol) (Figure 5B).

[0223] By measuring transdiaphragmatic pressure at different stimulation frequencies of the phrenic nerve, we were able to generate a Pdi-frequency relationship at the onset of anesthesia (DO), which is the equivalent of the in vivo force-frequency relationship. It is clear that in control piglets, 72 hours of mechanical ventilation induced a large and significant decrease in Pdi, which was completely prevented by VB558.

[0224] Interaction of VB558 with prostaglandin receptors Prostaglandin (or prostanoid) receptors are a family of cell surface membrane receptors known to primarily bind and respond to polyunsaturated fatty acids (PUFAs), metabolic products of arachidonic acid. Some of these are known to regulate cAMP production or intracellular calcium concentration. Based on the chemical structure of neuroprostanes (i.e., DHA derivatives), the inventors performed in vitro binding assays to specifically evaluate the effect of VB558 as a potential agonist or antagonist on established prostanoid receptors. Results of greater than 50% inhibition (or stimulation for assays performed under basal conditions) are considered to indicate a significant effect of the test compound.

[0225] No such effect was observed at any of the receptors studied here, suggesting that the effects of VB558 detailed above are independent of prostanoid receptors.

[0226] Consideration Although the majority of intensive care patients are easily weaned from mechanical ventilation (MV), approximately 25% of patients experience difficult weaning and require prolonged mechanical ventilation. However, the use of MV leads to significant respiratory muscle damage, characterized by progressive muscle weakness and histological remodeling of the respiratory muscles, further reducing the likelihood of weaning. This is even more so in patients with complicating factors such as malnutrition, chronic electrolyte abnormalities, hyperglycemia, excessive resistive and elastic loads, exposure to corticosteroids and muscle relaxants, sepsis, and cardiac dysfunction. 28 To date, no therapeutic strategies have proven effective in preventing VIDD in intensive care patients. Our team demonstrated that RyR1 remodeling appears to be a direct pathophysiological mechanism in mouse and piglet models of VIDD, as well as in patients. 15、19 More specifically, all VIDD models retained biochemical signatures of leaky RyR1 channels (i.e., phosphorylation, oxidation, and dissociation of calstabin1) and intracellular Ca 2+ Thus, RyR1 dysfunction is driven by the β-adrenergic signaling pathway in synergy with MV-induced oxidative stress, which has been extensively studied in VIDD. 4 Indeed, RyRs are highly sensitive to oxidative / nitrosative stress in skeletal muscle and in other tissues. Therefore, this RyR remodeling also occurs in other chronic or genetic diseases, including heart failure, diabetes, and Duchenne muscular dystrophy. 23~27 Post-translational modifications of RyR1 also progress with age and may partially contribute to age-related muscle weakness. 24 .

[0227] In this study, the inventors 4tOxidized derivatives of DHA, including -NeuroP, and its short derivative VB558, have been shown to block VDD with high efficiency in both mouse and pig VIDD models. This effect is associated with normalization of RyR function. 4t Although we cannot exclude a direct effect of NeuroP and VB558 on RyR, we also found that, in contrast to the effect of Rycal, 28 We also observed that both compounds prevented the oxidation and phosphorylation of RyR, suggesting a direct effect on RyR as previously reported by the present inventors. 21、29 Rather, an upstream mechanism has been suggested.

[0228] Although at present, only spontaneous ventilation cycles and possibly phrenic nerve stimulation appear to reduce the severity of VIDD in humans, several avenues are currently being investigated using animal models to identify pharmacological options. We provide here proof-of-concept that VB558 may be an effective molecule for preventing VIDD in patients.

[0229] The need for such therapies is even more urgent in the context of the current COVID-19 pandemic, where the number of patients receiving intensive care has exploded during the peak of the outbreak.

[0230] Finally, such pharmacological strategies may be of interest in any pathological situation in which RyR function is altered, as described above (i.e., heart disease, muscle atrophy, sarcopenia, cachexia, diabetes, neurodegenerative disorders).

[0231] Example 6 DMD research We then examined the effect of VB558 on diastolic calcium levels in ventricular cardiomyocytes derived from Duchenne muscular dystrophy (DMD). The results are shown in Figure 6. These data demonstrate that RyR2 dysfunction in DMD can be prevented by VB558, supporting its therapeutic benefit in this pathology (Figures 6A-B).

[0232] Example 7 Studies of calcium transients and cell contraction Compound 20,21,22-triol-4(RS)-4-F 4t -NeuroP (compound of formula III, VB574) and 4-F 4t Monohydroxylated derivatives of -NeuroP (Compound IV) were evaluated for their antiarrhythmic properties.

[0233] Calcium transients and cell contractions were measured by the photometric system ionOptix®.

[0234] a) General principles of measurement The IonOptix® photometric system allows for simultaneous acquisition of fluorescence photometry and sarcomere length measurements in real time. To study isolated myocytes, the system includes a pacemaker, which allows full control of the duration, frequency, and voltage of the stimulation pulses. Thus, the IonOptix® photometric system correlates calcium transients with shortening of electrically stimulated myocytes (1 Hz, 20 V, Figure 7). Calcium was studied by using a ratiometric fluorescent calcium probe (indo-1-AM, excitation wavelength 360 ± 10 nm, emission wavelengths 405 ± 10 nm and 485 ± 10 nm).

[0235] b) Protocol The first step in measuring intracellular calcium was to contact ventricular myocytes with a certain amount of indo-1-AM. To render the myocytes lipid-soluble, indo-1-AM was esterified with its carboxyl functional group to yield indo-1-AM. Once present in the cells, esterases allowed Indo-1 to be released, and the carboxyl functional group allowed it to bind to intracellular calcium. The uptake time was preset to 30 min. This time was chosen based on previous experiments using different incubation times; 30 min was found to provide a good ratio / noise ratio without causing obvious disruption to cellular calcium homeostasis. The probe incubation time is crucial because too little loading will produce a weak signal, while excessive exposure will over-expose the cytosolic calcium buffering capacity.

[0236] Once loaded, the cells were placed in the measurement system via a reservoir containing Tyrode's solution (450 μL), and the probe was excited by a xenon lamp at a wavelength of 360 nm. Through optical wavelength filters, the instrument collected the fluorescence emitted by Indo-1 at two different wavelengths: 405 ± 10 nm and 485 ± 10 nm. The first wavelength represents the fluorescence of the calcium-bound probe, while the second wavelength represents the fluorescence of the free probe. The ratio of these two wavelengths reflects the intracellular calcium concentration. In this way, it is possible to observe calcium transients during ventricular myocyte contraction, but also diastolic calcium when stimulation ceases.

[0237] To measure contractions, the striations of cardiac cells due to the presence of sarcomeres were used. These striations can be scanned and acquired by computer. The recorded signal resembles a sine function. This function is then mathematically processed by Fourier transformation, which makes it possible to obtain the sine length, i.e. the period of the sarcomere length.

[0238] The experimental protocol further addresses changes in intracellular calcium transients and peak cell contraction by analyzing sarcomere shortening during 30 seconds of electrical stimulation at 1 Hz followed by 30 seconds of interspersed pauses (Figure 7).

[0239] The goal with this protocol is to identify arrhythmic events, i.e., irregularities in the rate of contractions, and then study the effects of the 4-(RS)-4-F4T-neuroprostan derivative on these events. To reproduce the sympathetic tone that characterizes the mouse model, cells are subjected to 10 nM isoproterenol. This molecule, upon stimulation of the beta-adrenergic system, then leads to an increase in the amplitude of contractions, but also promotes the onset of premature ventricular contractions (ESVs), as shown in Figure 7.

[0240] These ESVs are consistent with abnormal function of type 2 ryanodine receptors (RyRs).

[0241] Therefore, to assess the RyR stabilizing effect of potential 4-F4T-neuroprostan derivatives (compounds III and IV), the compounds were applied and the percentage of arrhythmic cells was measured.

[0242] Compound III (FIG. 8) and Compound IV (FIG. 9) were found to exhibit antiarrhythmic properties, indicative of an interaction with RyR.

[0243] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into the present disclosure.

[0244] (References) [Table 1] [Table 2] [Table 3] Table 4

Claims

1. A method for treating a disease associated with RyR dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. 【Chemistry 1】 (In the formula, R 1 represents H or OH, R 2 is a linear C 1 ~C 10 Alkyl group or C 2 ~C 10 represents an alkyl group, However, R 2 but 【Chemistry 2】 When R represents 1 represents H)

2. The compound of formula I is R 2 is a group selected from the group of formulae a, b and c 【Transformation 3】 The method of claim 1.

3. The compound of formula I is 【Chemistry 4】 3. The method of claim 1 or 2, wherein the compound of formula I is selected from the group consisting of: and the compound of formula I has the structure of formula II, formula III, formula IV, formula S-IV, or formula R-IV.

4. 4. The method of claim 1, wherein the disease associated with RyR dysfunction is ventilator-induced diaphragmatic dysfunction.

5. 5. The method of claim 4, wherein the subject requires ventilatory support due to respiratory and / or cardiac failure, which may be exacerbated by sepsis, metabolic disorders, neuromuscular disease, or surgery, including post-surgical recovery.

6. 5. The method of claim 4, wherein the subject is suffering from a disease whose worsening symptoms have led to the subject requiring mechanical ventilation, in particular, the disease is selected from the group consisting of chronic obstructive pulmonary disease (COPD), pneumonia, sepsis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), and cystic fibrosis (CF).

7. 7. The method of claim 6, wherein the severe acute respiratory syndrome is COVID-19.

8. The method of claim 4, wherein the subject has suffered a traumatic injury.

9. 9. The method of any one of claims 4 to 8, wherein the ventilator-induced diaphragmatic dysfunction results from prolonged controlled mechanical ventilation (MV) of more than 12 hours, and in particular the compound of formula I is administered before MV, shortly after the initiation of MV, during MV, and / or shortly after MV.

10. The disease is selected from the group consisting of cardiac disorders and diseases, muscle fatigue, musculoskeletal disorders and diseases, central nervous system (CNS) disorders and diseases, cognitive dysfunction, bone disorders and diseases, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome, and in particular the cardiac disorder is selected from the group consisting of exercise-induced irregular heart rhythm 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 revascularization or thrombolysis during myocardial infarction (MI)), chronic The present invention relates to a method for treating musculoskeletal disorders and diseases, including but not limited to, obstructive pulmonary disease, hypertension, and irregular heart rhythm disorders and diseases, such as atrial and ventricular arrhythmias, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmias, atrial and ventricular tachyarrhythmias, catecholamine-induced polymorphic ventricular tachycardia (CPVT) and its exercise-induced variants, and in particular, to musculoskeletal disorders and diseases, including but not limited to skeletal muscle fatigue, central core disease, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, age-dependent muscle fatigue, sarcopenia, congenital myopathy, cancer cachexia, core and rod syndrome. Myopathies associated with mitochondrial myopathies [e.g., Kearns-Sayre syndrome, MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, and MERRF (myoclonic epilepsy with ragged-red fibers) syndrome], endocrine myopathies, muscle-type glycogen storage diseases [e.g., Pompe disease, Andersen disease, and Cori disease], myoglobinuria [e.g., McArdle disease, Tarui disease, and DiMauro disease], dermatomyositis, myositis ossificans, familial periodic paralysis, polymyositis, inclusion body myositis, neuromyostenosis is selected from the group consisting of muscular dystrophy, stiff-person syndrome, malignant hyperthermia, generalized muscle spasms, muscle rigidity, myasthenia gravis, and muscular dystrophy, in particular, the muscular dystrophy is selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD), distal muscular dystrophy, facioscapulohumeral muscular atrophy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy; or The cognitive disorder is selected from the group consisting of Alzheimer's disease (AD), memory loss, age-related memory loss, post-traumatic stress disorder (PTSD), neurological disorders, epilepsy, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), generalized anxiety disorder (GAD), obsessive-compulsive disorder (OCD), Parkinson's disease (PD), schizophrenia, bipolar disorder, and major depression.

4. The method according to any one of claims 1 to 3.

11. A compound of formula IA or a pharmaceutically acceptable salt thereof 【Transformation 5】 (In the formula, R 1 represents H or OH, R 2 is a linear C 1 ~C 10 Alkyl groups, C containing one double bond 2 ~C 7 Alkenyl group or C containing two double bonds 2 ~C 10 represents an alkenyl group, However, R 2 but 【Transformation 6】 When R represents 1 represents H, In particular, R 2 is a radical selected from the group of formulae a, b and c: 【Transformation 7】 )。

12. The compound of formula IA is 【Transformation 8】 12. The compound of claim 11 , selected from the group consisting of: wherein the compound of formula IA has the structure of formula IIA, formula IIIA, formula IVA, formula S-IVA, or formula R-IVA.

13. 13. A compound of formula IA as defined in claim 11 or 12, or a compound of formula I as defined in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, for use in a method of treatment of the human or animal body.

14. a compound of formula I, or a compound of formula IA, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; The compound of formula I is as defined in any one of claims 1 to 3, wherein the compound of formula IA is as defined in claim 11 or 12. Pharmaceutical compositions.

15. A method for preparing a compound of formula I, wherein said compound of formula I is defined in any one of claims 1 to 3, the method comprising: Step A: oxidation of an alcohol of formula V to obtain an aldehyde of formula VI; 【Chemistry 9】 (In the formula, R 3 is an alcohol protecting group, specifically a silyl protecting group, more specifically a t-butyldimethylsilyl protecting group; R 4 is methyl or ethyl, R 5 is H or OR 3 where R 3 is as defined above) Step B: olefination of the aldehyde of formula VI to obtain an olefin of formula VII; 【Chemistry 10】 (In the formula, R 3 , R 4 and R 5 is as defined in step A, R 2 is as defined in claims 1 to 3 for formula I) Step C: deprotection of the hydroxyl group in formula VII to give an ester of formula VIII; 【Chemistry 11】 (In the formula, R 4 and R 5 is as defined in step A, R 1 and R 2 is defined in claims 1 to 3 for formula I) Step D: Hydrolysis of the ester of formula VIII to obtain the compound of formula I 【Chemistry 12】 (In the formula, R 4 is as defined in step A, R 1 and R 2 is as defined in claims 1 to 3 for formula I) and

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