4 (RS)-4-F4-Neuroprostaglandin Derivatives (4-F4T-NEUROP) and their use in treatment of breathing machine induced diaphragm dysfunction and other diseases

Abstract

Respiratory muscle contraction inactivity during mechanical ventilation (MV) results in diaphragm weakness, which is called ventilator induced diaphragm dysfunction (VIDD). In different hypotheses to interpret the VIDD, one of the hypotheses involves the remodeling of the myoplasmic reticulum (SR) calcium release channel / Ryanodine receptor (RyR1) in the diaphragm as a proximal mechanism of the VIDD. The inventor has developed a novel derivative which can efficiently prevent VIDD in VIDD mouse and pig models. The effect is related to the homogenization 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 new compounds, methods for their preparation and their use in the treatment of diseases associated with Ryanodine receptor (RyR) dysfunction. Background Art

[0002] Skeletal muscle contraction is activated by the release of sarcoplasmic reticulum (SR) Ca 2+ from the ryanodine receptor calcium release channel. RyR is a channel in the SR that opens and closes to regulate the release of Ca 2+ from the SR into the intracellular cytoplasm of the cell. The Ca released from the SR into the cytoplasm 2+ increases the cytoplasmic Ca 2+ concentration. The open probability of RyR refers to the likelihood that RyR is open at any given moment and thus able to release Ca 2+ from the SR into the cytoplasm. There are three types of RyR, all of which are highly homologous: RyR1, RyR2 and RyR3. RyR1 is mainly present in skeletal muscle and other tissues. The RyR1 macromolecular complex consists of a tetramer of 560-kDa RyR1 subunits, which forms a scaffold for proteins that regulate channel function, including protein kinase A (PKA) and phosphodiesterase 4D3 (PDE4D3), protein phosphatase 1 (PP1) and calstabin 1. Chronic PKA hyperphosphorylation of RyR1 at Ser2843 (defined as phosphorylation of 3 or 4 of the 4 PKA Ser2843 sites present in each RyR1 homotetramer) results in "leaky" channels (i.e., channels that are prone to opening at rest), which leads to skeletal muscle dysfunction and is associated with a persistent adrenergic hyperactive state, such as in patients with heart failure. In addition, it has been reported that RyR1 can be regulated by non-phosphorylated post-translational modifications, such as nitrosylation (S-nitrosylation) of free sulfhydryl groups on cysteine residues and channel oxidation, to increase RyR1 channel activity. It has been demonstrated that both S-nitrosylation and oxidation of RyR1 can reduce the binding of Calstabin1 to RyR1. Thus, RyR dysfunction is a hallmark of various diseases, and several methods and pharmaceutical compositions have been reported for the treatment of such diseases (such as ventilator-induced diaphragmatic dysfunction (VIDD)) 1 。

[0003] Mechanical ventilation (MV) remains the most effective treatment for respiratory failure in the intensive care unit (ICU). However, the respiratory muscle inactivity induced by MV (i.e., unloading of the respiratory muscles) has various adverse cellular effects, leading to diaphragmatic weakness, atrophy and injury in a time-dependent manner 2-4 ,which is a pathological condition called ventilator-induced diaphragmatic dysfunction (VIDD) 5。Since VIDD increases the difficulty of MV weaning 6 and has a significant impact on healthcare costs 7 therefore, identifying the cellular pathways involved in diaphragmatic muscle weakness is an important issue for determining potential therapeutic targets and / or pharmacological treatments

[0004] The pathophysiological mechanisms leading to VIDD are still not fully understood, but oxidative stress is considered to be the main pathophysiological mechanism of VIDD 8-11 and mitochondria are the main source of reactive oxygen species (ROS) 11-13 。It is not clear why the production of mitochondrial ROS increases after MV-induced respiratory muscle discharge. Imbalance of energy homeostasis and acute substrate excess are potential sources of mitochondrial dysfunction 12 。

[0005] The inventors developed the first VIDD mouse model, in which the force decreased by 30% after 6 hours of ventilation, without any histological signs of injury or necrosis 14 。Using this model, the inventors have confirmed that early alterations in Ca 2+ homeostasis play a decisive role in the establishment of VIDD. This occurs through the type 1 sarcoplasmic reticulum calcium channel and the structural and functional remodeling of the ryanodine receptor (RyR1) induced by oxidative stress 15 。This cellular pathway can be considered as the proximal mechanism of Ca 8,16-18 -dependent proteolysis and muscle atrophy observed in different animal models 3-4 of VIDD 2+ and human patients 15,19,20 。Interestingly, this RyR1 remodeling has been continuously observed in ventilated mice and piglets as well as ventilated ICU patients 21 indicating that directly or indirectly stabilizing RyR1 function represents a potential therapeutic target in VIDD. They previously reported that due to the stabilization of the cardiac isoform of the Ryanodine receptor RyR2, ω-3 fatty acid docosahexaenoic acid (DHA, C22:6n-3) can exert a cardiac antiarrhythmic effect 21 。Interestingly, the active molecule is not DHA itself, but the non-enzymatic metabolite of DHA (NEO-DHA) 4t ,4(RS)-4-F 4t -neuroprostaglandin (4F 22 -NeuroP) 4t 。WO2015197562 describes a method for treating diseases associated with RyR dysfunction in a subject in need, including administering a therapeutically effective amount of 4F

[0006] There is a need to identify new compounds for the effective treatment of conditions and diseases associated with RyR dysfunction. More particularly, there is still a need to identify new agents that can be used to treat RyR1-related conditions by, for example, destabilizing RyR1.

[0007] Brief Description of the Invention

[0008] The present invention relates to new compounds and methods for preparing said compounds. The present invention also relates to methods and pharmaceutical compositions for treating diseases associated with Ryanodine receptor (RyR) dysfunction. In particular, the present invention relates to a method for treating a disease associated with RyR dysfunction in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a new compound according to the present invention.

[0009] In particular, the present invention is defined by the claims. Detailed Description of the Invention

[0011] The inventors designed derivatives of 4(RS)-4-F 4t -neuroprostaglandin (4-F 4t -NeuroP). Compared with 4-F 4t -NeuroP, said derivatives are more easily synthesized with fewer reaction steps.

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

[0013] The inventors unexpectedly found that said new compounds efficiently prevent VIDD in both murine and porcine models of VIDD. This effect is associated with the normalization of RyR function.

[0014] A first object of the present invention is a compound of formula I or a pharmaceutically acceptable salt thereof, which is used in a method for treating the human or animal body,

[0015]

[0016] wherein:

[0017] R1 represents H or OH,

[0018] R2 represents a straight-chain C1-C 10 alkyl or C2-C 10 alkenyl,

[0019] provided that when R2 represents then R1 represents H.

[0020] The second object of the present invention is a method for treating a disease associated with RyR dysfunction in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof:

[0021]

[0022] wherein:

[0023] R1 represents H or OH,

[0024] R2 represents a straight-chain C1-C 10 alkyl or C2-C 10 alkenyl,

[0025] provided that when R2 represents then R1 represents H.

[0026] In other words, the second object of the present invention relates to a compound of formula I or a pharmaceutically acceptable salt thereof as defined herein, which is used for treating a disease associated with RyR dysfunction in a subject in need thereof.

[0027] "Straight-chain C1-C 10 alkyl" refers to a group selected from methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl. In particular, the straight-chain alkyl is ethyl.

[0028] "Straight-chain C2-C 10 alkenyl" refers to a straight-chain C2-C 10 alkyl containing one or more double bonds in its chain. The expression "C2-C 10 alkenyl" should also be understood as C2-C8 alkenyl, C2-C6 alkenyl, C2-C4 alkenyl, C4-C 10 alkenyl, C6-C 10 alkenyl, C8-C 10 alkenyl or C4-C8 alkenyl.

[0029] As defined herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt that has the desired pharmacological activity of the parent compound. Such salts are compounds in which the acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, an alkaline earth metal ion or an aluminum ion; or coordinated with a pharmaceutically acceptable organic or inorganic base.

[0030] 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.

[0031] In a specific embodiment, the alkenyl contains 1 or 2 double bonds.

[0032] In particular, if the number of double bonds in R2 is 2, 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 by a -CH2- group.

[0033] According to a preferred embodiment, the double bonds contained in the alkenyl group of R2 are of the Z-configuration.

[0034] According to a preferred embodiment, the present invention relates to the method as described above, wherein R2 in the compound of formula I is a group selected from formulae a, b and c:

[0035]

[0036] The compound of formula I contains several asymmetric carbon atoms in its structure.

[0037]

[0038] 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 above structure.

[0039] In a specific embodiment, the compound of formula I has a diastereomeric excess of greater than 90%, particularly greater than 95%, relative to the carbon atoms contained in the five-membered ring (carbons 7, 8, 10 (when R1 = OH) and 11).

[0040] In another specific embodiment, the compound of formula I has an enantiomeric excess of greater than 90%, particularly greater than 95%, relative to the carbon atoms contained in the five-membered ring (carbons 7, 8, 10 (when R1 = OH) and 11).

[0041] The asymmetric carbon atom 4 relative to the γ-position of the carboxylic acid can be of the R or S configuration, or can be a mixture of the R and S configurations.

[0042] According to an embodiment of the present invention, when the asymmetric carbon atom relative to the γ-position of the carboxylic acid is a mixture of the R and S configurations, the compound of formula I is a mixture of diastereoisomers.

[0043] In an advantageous embodiment, the compound of formula I is selected from:

[0044]

[0045] The compound of formula I has the structure of formula II, formula III, formula IV, formula S-IV or formula R-IV.

[0046] Compound of formula II, 17,18,19,20,21,22-hexanor-4(RS)-4-F4t -NeuroP(VB558) is 4-F 4t -a shorter derivative of NeuroP, which contains an alkenyl chain of 5 carbon atoms and has 1 double bond with a Z-configuration (for formula I, R1 is -OH and R2 is a group of formula a (ethyl)).

[0047] In a specific embodiment, 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 the compound of formula II is a mixture of diastereoisomers, wherein the diastereoisomeric excess is particularly less than 95%, more particularly less than 90%.

[0048] The compound of formula III, 20,21,22-trinor-4(RS)-4-F 4t -NeuroP(VB574) is 4-F 4t -a shorter derivative of NeuroP, which contains an alkenyl chain of 8 carbon atoms and has 2 conjugated double bonds with a Z-configuration (for formula I, R1 is -OH and R2 is a group of formula b).

[0049] The compound of formula IV is 4-F 4t -a monohydroxylated derivative of NeuroP, wherein the 5-membered ring is substituted with 1 hydroxyl substituent. The compound of formula IV also contains an alkenyl chain of 11 carbon atoms, which has 3 conjugated double bonds with a Z-configuration (for formula I, R1 is -H and R2 is a group of formula c).

[0050] The compound of formula S-IV (VBS81) is a specific diastereoisomer of compound IV, wherein the carbon atom at the γ-position of the carboxylic acid functional group has an (S)-configuration.

[0051] The compound of formula R-IV (VB582) is a specific diastereoisomer of compound IV, wherein the carbon atom at the γ-position of the carboxylic acid functional group has an (R)-configuration.

[0052] According to a preferred embodiment, the compound of formula I has the structure of formula II as defined above, i.e., 17,18,19,20,21,22-hexanor-4(RS)-4-F 4t -NeuroP(VB558).

[0053] In a specific embodiment, the compounds of the present invention are suitable for stabilizing the complex calstabin1 / RyR1.

[0054] As used herein, the term "subject" refers to a mammal, such as a rodent, feline, canine, and primate. Preferably, the subject according to the present invention is a human.

[0055] As used herein, the terms "treatment / treat" refer to prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treating patients at risk of having an infectious disease or suspected of having an infectious disease, as well as patients having or diagnosed with a disease or medical condition, and including suppression of clinical recurrence. The treatment can be administered to a subject having a medical condition or ultimately likely to acquire the condition to prevent, cure, delay the onset of the condition or recurrence of the condition, reduce the severity of the condition or recurrence of the condition or improve the onset of one or more symptoms of the condition or recurrence of the condition, or to extend the survival period of the subject beyond the expected survival period without the treatment. A "treatment regimen" refers to a pattern of treating a disease, such as a dosing pattern used during treatment. A treatment regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or part of a treatment regimen) used for the initial treatment of a disease. The overall purpose of an induction regimen is to provide a high level of drug to a patient during the initial stage of the treatment regimen. An induction regimen can employ (in part or in whole) a "loading regimen", which can include administering a higher dose of a drug than that used by a doctor during a maintenance regimen, administering the drug at a higher frequency than that used by a doctor during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient during the treatment of a disease, such as keeping a patient in remission for a long period (months or years). A maintenance regimen can employ continuous treatment (e.g., administering a drug at regular intervals, such as weekly, monthly, annually, etc.) or intermittent treatment (e.g., intermittent treatment, treatment at recurrence, or treatment when a specific predetermined criterion [e.g., disease manifestation, etc.] is reached).

[0056] As used herein, the term "disease associated with RyR dysfunction" refers to any disorder and disease that can be treated and / or prevented by modulating the RyR receptor that regulates calcium channel function in cells.

[0057] Diseases associated with RyR dysfunction have been well demonstrated, and those skilled in the art can easily identify these diseases. For example, Kushnir et al (Mol. Cell. Res., 2018, 1865, p. 1687-1697) disclosed the relationship between specific diseases and RyR dysfunction.

[0058] In addition, it can be easily determined experimentally whether a disorder or disease is associated with RyR dysfunction. Typically, to establish a link between a disease and RyR dysfunction, 2 tests can be performed separately, but preferably both simultaneously (Matecki et al., PNAS, 2016, 113(2), p. 9069-9074 and J. Muscle Res Cell Motil., 2017, 38, p. 17-24).

[0059] The first test relies on RyR1 immunoprecipitation technology, which can identify the "biochemical signature" of leaky RyR1 channels and allows the establishment of a correlation between specific diseases and RyR dysfunction.

[0060] The second test relies on electrophysiological techniques to determine the open probability of RyR by recording single-channel currents. The measurement involves an experimental setup in which RyR channels are incorporated into a lipid bilayer. RyR channels are obtained by biopsy and SR vesicles are subsequently prepared. Single-channel currents are recorded using a Bilayer clamp BC-535 amplifier.

[0061] Accordingly, "diseases associated with RyR dysfunction" include, but are not limited to, cardiac disorders and diseases, skeletal muscle disorders and diseases, cognitive dysfunction and diseases, malignant hyperthermia, diabetes, and sudden infant death syndrome.

[0062] In some embodiments, the disorder or disease is associated with abnormal function of RyR1.

[0063] In some embodiments, the methods of the invention are particularly useful for treating diseases 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, skeletal 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, arrhythmia disorders and diseases, exercise-induced arrhythmia 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 after coronary angioplasty or thrombolysis during myocardial infarction (MI)), chronic obstructive pulmonary disease, and hypertension. Arrhythmia disorders and diseases include, but are not limited to, atrial and ventricular arrhythmias, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmias, atrial and ventricular tachycardia, catecholaminergic polymorphic ventricular tachycardia (CPVT) and its exercise-induced variants.

[0064] In some embodiments, the methods of the invention are particularly useful for treating muscle diseases, including but not limited to 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, myopathy with cores and rods, mitochondrial myopathies [e.g., Kearns-Sayre syndrome, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) syndrome, and MERRF (myoclonic epilepsy with ragged red fibers) syndrome], endocrine myopathies, glycogen storage myopathies [e.g., Pompe disease, Andersen disease, and Cori disease], myoglobinurias [e.g., McArdle disease, Tarui disease, and DiMauro disease], dermatomyositis, myositis ossificans, familial periodic paralysis, polymyositis, inclusion body myositis, neuromyotonia, stiffness syndromes, malignant hyperthermia, common muscle cramps, tetany, myasthenia gravis, and muscular dystrophy. Examples of muscular dystrophy 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 dystrophy, myotonic muscular dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy. As used herein, congenital muscular dystrophy refers to muscular dystrophy that is present at birth. CMD is classified based on gene mutations into: 1) genes encoding structural proteins of the basement membrane or extracellular matrix of skeletal muscle fibers; 2) genes encoding putative or confirmed glycosyltransferases, which in turn affect the glycosylation of dystroglycan (the outer membrane protein of the basement membrane); and 3) others. Examples of CMD include but are not limited to laminin-α2-deficient CMD (MDC1A), Ullrich CMD (UCMD1, 2, and 3), Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), Fukuyama-type CMD (FCMD), CMD plus secondary laminin deficiency type 1 (MDC1B), CMD plus secondary laminin deficiency type 2 (MDC1C), CMD with mental retardation and myoclonus (MDC1D), and rigid spine muscular dystrophy type 1 (RSMD1).

[0065] In some embodiments, the methods of the present invention are particularly applicable to the treatment of cognitive impairment. Cognitive impairments, disorders or dysfunctions include but are not limited to Alzheimer's disease, memory loss, age-related memory loss, post-traumatic stress disorder (PTSD), neuropathy and seizures. The cognitive dysfunction can be stress-related, age-related or a combination thereof. Alternatively, the cognitive dysfunction is 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 severe depression.

[0066] In some embodiments, the methods of the present invention are particularly applicable to the treatment of ventilator-induced diaphragmatic dysfunction.

[0067] As used herein, the expression "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 occur after prolonged controlled mechanical ventilation. 24 . Ventilator-induced diaphragmatic dysfunction can be caused by prolonged controlled mechanical ventilation (MV), for example greater than 12 hours. However, such prolonged MV is not limited to any specific length of time. For example, in some embodiments, the duration of prolonged MV includes at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50 or 100 hours, up to at least about 1, 10, 20, 50, 75, 100 or more hours, days or years. In another embodiment, the duration of prolonged MV includes at least about 5, 6, 7, 8, 9 or 10 hours, up to at least about 10, 20 or 50 hours. In some embodiments, the prolonged MV is for any time from about at least 10-12 hours to greater than 10-12 hours.

[0068] In some embodiments, the subject requires artificial respiratory support because they suffer from respiratory failure and / or heart failure, which can be exacerbated by sepsis, metabolic disorders, neuromuscular diseases or surgery and postoperative recovery.

[0069] Typically, the subject has a disease with symptom deterioration leading to the subject requiring artificial respiratory support (i.e., mechanical ventilation). For example, some lung 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), generally require some form of ventilation assistance to clinically improve the subject. Most patients with severe coronavirus disease 2019 (COVID-19) are associated with acute respiratory failure and require mechanical ventilation. COVID-19 is a respiratory syndrome, the clinical pathology of which shows similar to mild upper respiratory tract diseases (common cold-like symptoms), and occasionally severe lower respiratory tract diseases and extrapulmonary manifestations, leading to multiple organ failure and death. More than 430 million people worldwide have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), many of whom require mechanical ventilation. It is estimated that 5% of patients infected with SARS-CoV-2 require advanced respiratory support 37 . Thus, in some embodiments, the subject has COVID-19.

[0070] In some embodiments, the subject has suffered trauma. Pulmonary dysfunction in trauma patients is multifactorial and may be the result of direct contusion of lung tissue, lung injury caused by rib fractures, loss of chest wall function, pulmonary fat embolism caused by long bone fractures, aspiration of blood or gastric contents, and activation of the systemic inflammatory response syndrome (SIRS) of shock, reperfusion, and blood transfusion therapy.

[0071] In some embodiments, the compound of formula I is administered before MV, immediately after MV initiation, during MV, and / or immediately after MV. In some embodiments, the administration of the compound of formula I according to the present invention is provided at any time during MV.

[0072] The compound of formula I according to the present invention is also suitable for preventing the risks associated with ventilator-induced diaphragmatic dysfunction. The risks associated with ventilator dependence include increased patient discomfort and the risk of secondary diseases (such as pneumonia, pulmonary fibrosis, aspiration, acute renal failure, arrhythmia, sepsis, vocal cord dysfunction, and acute lung injury secondary to barotrauma or volutrauma), increased morbidity and mortality, high healthcare costs, and prolonged treatment duration. Although patients with chronic ventilator dependence (CVD) account for only 5% to 10% of intensive care unit patients, they consume approximately 50% of ICU resources in terms of staff time and equipment usage. Specifically, it is estimated that among intensive care unit patients, weaned patients consume approximately 41% of the total ventilation time. The economic cost of long-term MV dependence is huge. Long-term MV dependence events may cause economic losses to families and healthcare institutions and result in financial consumption of private insurance companies and government healthcare resources.

[0073] According to the present invention, the compounds of formula I are administered in a therapeutically effective amount. A "therapeutically effective amount" means an amount of a compound of formula I sufficient to treat a target disease at a reasonable benefit / risk ratio applicable to any medical treatment. It is to be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, the route of administration, and the excretion rate of the specific compound employed; the duration of the treatment; drugs used in combination with or concurrently with the specific compound employed; and like factors well known in the medical arts. For example, it is well known to those skilled in the art to start the dosage of the compound at a level below that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. In particular, the composition contains 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 symptom adjustment of the dosage to be administered to the subject to be treated. The medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, particularly from 1 mg to about 100 mg of the active ingredient. The medicament in an effective amount is usually provided at a dosage level of from 0.0002 mg / kg body weight / day to about 20 mg / kg body weight / day, particularly from about 0.001 mg / kg body weight / day to 7 mg / kg body weight / day.

[0074] Compounds of formula I are typically administered in the form of pharmaceutical compositions in combination with pharmaceutically acceptable excipients and optionally a sustained release matrix such as a biodegradable polymer. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse reactions, allergies or other untoward reactions when administered to mammals, especially humans, when appropriate. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. In the pharmaceutical compositions of the present invention, for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient, alone or in combination with another active ingredient, may be administered in unit dosage form as a mixture with conventional pharmaceutical carriers to animals and humans. Suitable unit dosage forms include forms for oral administration such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the carrier contained in the pharmaceutical composition is pharmaceutically acceptable for injectable formulations. These may in particular be isotonic, sterile salt solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride etc. or mixtures of these salts), or dry, especially lyophilized, compositions which, after addition of sterile water or physiological saline as appropriate, can constitute injectable solutions. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions; preparations including 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 a fluid that is easy to inject. It must remain stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. Solutions containing the compounds of the present invention (in free base or pharmaceutically acceptable salts) can be prepared in appropriately mixed water with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms. Antibodies can be formulated in compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins), and these are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid etc. Salts formed with the 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, procaine etc. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols such as glycerol, propylene glycol and liquid polyethylene glycols etc., suitable mixtures thereof and vegetable oils.For example, by using a coating (such as lecithin), in the case of a dispersion, by maintaining the desired particle size and by using surfactants, appropriate fluidity can be maintained. The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride. The absorption time of the injection composition is extended by using agents that extend absorption in the composition, such as aluminum monostearate and gelatin. Sterile injectable solutions are prepared by mixing the required amount of the active antibody with several of the other ingredients described above in a suitable solvent, followed by filtration sterilization as needed. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains a basic dispersion medium and the required other ingredients described 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 other required ingredients from its previously sterile-filtered solution. Upon formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. These formulations are readily administered in various dosage forms, such as the types of injectable solutions described above, but drug-release capsules, etc. may also be used. For parenteral administration of an aqueous solution, for example, if necessary, the solution should be appropriately buffered and first diluted to isotonicity with sufficient saline or glucose with a liquid diluent. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, according to the present disclosure, sterile aqueous media known to those skilled in the art can be used. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous infusion or injected at the proposed infusion site. Depending on the condition of the subject being treated, some variation in the dose will necessarily occur. In any case, the person responsible for administration will determine the appropriate dose for the individual subject.

[0075] Accordingly, 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 thereof and a pharmaceutically acceptable excipient.

[0076] A fourth object of the present invention is a new compound of formula IA or a pharmaceutically acceptable salt thereof:

[0077]

[0078] wherein:

[0079] R1 represents H or OH,

[0080] R2 represents a straight-chain C1-C 10 alkyl, a C2-C7 alkenyl containing 1 double bond or a C2-C 10 alkenyl containing 2 double bonds,

[0081] The condition is that when R2 represents R1 represents H.

[0082] In one embodiment, R2 is a group selected from formulae a, b and c:

[0083]

[0084] The novel compound is particularly selected from:

[0085]

[0086] The compound of formula IA has a structure of formula IIA, formula IIIA, formula IVA, formula S-IVA or formula R-IVA.

[0087] The fifth object of the present invention relates to a compound of formula IA as defined above, which is used in a method for treating a human or animal body.

[0088] The sixth object of the present invention relates to a pharmaceutical composition, which comprises a compound of formula IA as defined above or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0089] The seventh object of the present invention is a method for preparing a compound of formula I or a compound of formula IA as defined above, wherein the method comprises:

[0090] Step A, oxidizing the alcohol of formula V to obtain the aldehyde of formula VI,

[0091]

[0092] R3 is an alcohol protecting group, particularly a silyl protecting group, more particularly a tert-butyldimethylsilyl protecting group,

[0093] R4 is methyl or ethyl,

[0094] R5 is H or OR3, where R3 is as defined above;

[0095] Step B, olefinating the aldehyde of formula VI to obtain the olefin of formula VII,

[0096]

[0097] R3, R4 and R5 are as defined in Step A,

[0098] R2 is as defined for formula I above;

[0099] Step C, deprotecting the hydroxyl group in formula VII to obtain the ester of formula VIII,

[0100]

[0101] R4 and R5 are as defined in step A,

[0102] R1 and R2 are as previously defined for formula I;

[0103] Step D, hydrolyze the ester of formula VIII to obtain the compound of formula I,

[0104]

[0105] R4 is as defined in step A,

[0106] R1 and R2 are as previously defined.

[0107] In the process according to the invention, the protecting group R3 is selected such that it is not removed under the reaction conditions of steps A and B.

[0108] Furthermore, the protecting group R3 is selected such that the group can be selectively removed in the presence of an ester (R4 = methyl or ethyl).

[0109] In a preferred embodiment, the protecting group R3 is selected from silyl protecting groups, preferably tert-butyldimethylsilyl (tBDMS) or triisopropylsilyl (TIPS), particularly tert-butyldimethylsilyl.

[0110] Step A - Oxidation

[0111] In the process according to the invention, the selective oxidation of the hydroxyl group to an aldehyde group can be achieved under conditions that prevent side reactions such as over-oxidation and the removal of undesired R3 and R4 groups.

[0112] Examples of reagents and reactions that can be used in step A are periodane reagents, particularly Dess-Martin periodinane (DMP), pyridinium chlorochromate (PCC), and Swern oxidant ((COCl)2 / DMSO).

[0113] In a specific embodiment, step A is carried out using DMP in dichloromethane at room temperature.

[0114] "Room temperature" should be understood as a temperature of 15 - 30 °C.

[0115] The aldehyde VI obtained after step A can be purified, for example, using column chromatography, or can be used as a crude product in step B.

[0116] The starting material V can be obtained using literature methods (Oger C. et al., Chem. Eur. J., 2010, 16, p. 13976). A typical synthesis is shown in Figure 1 in.

[0117] Step B - Olefination

[0118] The conversion of an aldehyde group to an alkene can be achieved by a reaction capable of forming an alkene with Z-configurational selectivity.

[0119] The selectivity for the Z-isomer can be achieved particularly by the Wittig reaction, in which an aldehyde reacts with a phosphonium ylide, which in turn is formed by deprotonation of a phosphonium salt using a base.

[0120] In particular, at a temperature below -50 °C, especially at -78 °C, a suitable phosphonium salt is deprotonated with NaHMDS in anhydrous THF, and then a compound of formula VI is added.

[0121] Step C - Deprotection

[0122] Deprotection of the hydroxyl group, i.e., removal of the R3 protecting group, can be achieved under conditions depending on the nature of the protecting group.

[0123] In particular, when R3 is a silyl protecting group, such as a tert-butyldimethylsilyl protecting group, a fluoride-based reagent, such as tetrabutylammonium fluoride (TBAF), can be used.

[0124] During the reaction or purification, deprotection of the hydroxyl group can lead to the formation of a lactone by-product (VII-lactone), which is formed by an intramolecular lactone exchange reaction.

[0125] This lactone can also be hydrolyzed in step D to give the compound of formula I.

[0126]

[0127] Step D - Hydrolysis

[0128] Hydrolysis of the ester functional group (and / or lactone group) to obtain a carboxylic acid can be achieved, for example, using a base, such as NaOH, KOH or LiOH, especially LiOH.

[0129] The intermediate product and the compound of formula I can be purified using techniques such as column chromatography.

[0130] The intermediate product and the compound of formula I can be characterized using techniques known to those skilled in the art, such as NMR, elemental analysis and mass spectrometry.

[0131] The purity can be determined using, for example, HPLC and chiral HPLC.

[0132] In one specific embodiment, the starting material V can be used as a mixture of isomers, in which the configuration of the carbon atom at the 4-position is a mixture of R and S.

[0133] In another specific embodiment, the starting material V can be used as a single isomer, in which the configuration of the carbon atom at the 4-position is R or S.

[0134] Accordingly, a seventh object of the present invention relates to a method for preparing a new compound of formula IA, wherein the method is as defined above for the compounds of formula I.

[0135] Despite the complexity of the structures of the compounds disclosed herein, after considerable research, the inventors were able to develop a short and efficient synthesis for obtaining these compounds.

[0136] In addition, the inventors unexpectedly found that these compounds have biological activity against diseases associated with RyR dysfunction.

[0137] The present invention will be further illustrated by the following figures and examples. However, these examples and figures should not be construed in any way as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 : Ca spark frequency recorded in saponin-skinned EDL muscle fibers of control (n = 5), post-myocardial infarction (PMI, n = 3), and post-myocardial infarction with 4F 4t -NeuroP 1 μM (PMI + 4F 4t -NeuroP, n = 3) mice. *, p < 0.05. 2+

[0139] Figure 2: A. 4F 4t -NeuroP prevents diaphragmatic muscle weakness induced after 6 h of mechanical ventilation in anesthetized mice. Force-frequency relationship of the diaphragm under control conditions (n = 8) or after 6 h of mechanical ventilation (n = 6) or without intravenous injection of 4F 4t -NeuroP (n = 6). Average maximum force generation recorded at 120 Hz in control diaphragms after 6 h of mechanical ventilation (VIDD) and after treatment with 4F 4t -NeuroP. Data are expressed as mean ± SEM, *, p < 0.05. B. Dose-response relationship of the protective effect of 4F 4t -NeuroP after 6 h of mechanical ventilation. Mice were treated under induced anesthesia by intravenous injection of 4F 4t -NeuroP at concentrations of 0.01 - 100 μM. Each value corresponds to the maximum force generated by the diaphragm at a frequency of 120 Hz. C / Biochemical remodeling of RyR1 after VIDD: effect of 4F 4t -NeuroP. The mean values of DNP / RyR1, P-RyR1 / RyR1, and Calstabin1 / RyR1 reflect RyR1 oxidation, phosphorylation of Ser2844, and Calstabin1 interaction, respectively. VIDD has a negative impact on all parameters, and is reversed by 4F​4t - NeuroP treatment prevented this post - translational remodeling.

[0140] Figure 3: A. Force - frequency relationship of the diaphragm of mice treated with or without 10 μM 4F after 12 hours of mechanical ventilation 4t - Force - frequency relationship of the diaphragm of NeuroP - treated mice. In addition, 10 μM 4F 4t - NeuroP prevented diaphragm fiber atrophy after 12 hours of mechanical ventilation, independent of fiber phenotype. Representative immunostaining of fast and slow diaphragm fibers in mice. Cryosections of mouse diaphragm were immunostained using antibodies against fast and slow myosin ATPase. The sarcolemma was counterstained with an anti - dystrophin antibody. At 12 hours after MV, with or without 4F 4t - NeuroP was stained in the diaphragm. The mean cross - sectional area was quantified under each condition including all fiber types (B.) or by differentiating slow vs fast fibers (C.). D. Mean distribution of fast - twitch vs slow - twitch fibers under each condition (*p < 0.05 vs control).

[0141] Figure 4: VB558, 4F 4t - A shorter derivative of NeuroP prevented VIDD. A. Structural and synthetic comparison of VB558. B. Effect of VB558 on the force - frequency relationship of 6 - h ventilated mice (control n = 2; MV n = 3; VB558 1 μM n = 6; VB558 10 μM n = 6; *p < 0.05 vs MV).

[0142] Figure 5: VB558 prevented VIDD in piglets after 72 - hour ventilation. Protocol for intravenous injection of VBP58 or methanol (placebo). A. Changes in plasma concentration after bolus injection of VBP58 in 3 piglets. B. Intravenous injection protocol during 72 - hour mechanical ventilation. Transdiaphragmatic pressure was measured at different stimulation frequencies of the phrenic nerve in VBP58 - treated animals (D, n = 6) and placebo group receiving the same volume of solvent (methanol, V) (C, n = 6) at the start of anesthesia (t0) and after 72 hours of mechanical ventilation and before (S). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t72 vs t0).

[0143] Figure 6: Effect of Hexanor on the calcium relaxation level of ventricular cardiomyocytes derived from DMD and control hIPSc. Cardiomyocytes were loaded with indo - 1 to assess intracellular calcium levels in a ratio - metric manner. As previously described, the increase in diastolic calcium reflects RyR2 leak behavior. A. Higher calcium levels in DMD compared to control cells. This was prevented by hexanor (1 μM). B. Repeated experiments under isoproterenol stimulation of the cells (Iso 1 μM) showed complete normalization of resting calcium under this condition.

[0144] Figure 7 The figure shows the changes in intercellular calcium transients and the changes in cell contraction peaks, as analyzed in Example 7. The introduction of 10 nM Iso (isoproterenol) promotes the development of ESV (extrasystole ventricular contraction).

[0145] Figure 8 The figure shows the number of ryanodine-dependent arrhythmia cells in the presence of 10 nM, 100 nM, or 1 μM of compound 20,21,22-trinor-4(RS)-4-F 4t -NeuroP (compound of formula III, VB574), as measured in Example 7. The cell numbers are expressed as a percentage of the total cells and include the control (without VB574).

[0146] Figure 9 The figure shows the number of ryanodine-dependent arrhythmia cells in the presence of 1 μM 4-F 4t -monohydroxylated derivative of NeuroP (compound of formula IV). The amount of cells is expressed as a percentage of the total cells and includes the control (without the compound of formula IV). Detailed Description

[0147] Materials and Methods

[0148] Unless otherwise mentioned, all reactions requiring anhydrous conditions were carried out in dry glassware (oven-dried at 120 °C or flame-dried) under a nitrogen atmosphere with magnetic stirring. All anhydrous solvents and reagents were dried under a nitrogen or argon atmosphere according to standard methods, or using methods obtained from suppliers (Aldrich, Thermo Fischer). Reactions were monitored by TLC using plates pre-coated with silica gel 60 and fluorescent indicator 254 nm (Merck). The reaction components were treated with a 254 nm UV lamp or acidic p-anisaldehyde stain, followed by gentle heating for visualization. Column chromatography was performed using silica gel 40-63 μm, 230-400 mesh, Macherey-Nagel or spherical silica gel Si60, 30 μm, Interchim. Pentane (P), Et2O, EtOAc, MeOH were used for chromatographic separation. Optical rotations were recorded on a JASCO P2000-series instrument, and the concentration c for optical rotation data was g / 100 mL. Infrared spectra were obtained as pure samples on a Spectrum one (Perkin Elmer) using an ATR device, and the absorption was in wave numbers (cm -1)。MS (ESI) and high-resolution MS (HRMS) spectra were measured on a Q-Tof micro spectrometer (resolution 100,000, Waters) or a mass spectrometer Synapt G2-S (Waters). Data were obtained by direct introduction of positive or negative electrospray ionization methods for 100 - 1500 Da. NMR spectra were recorded on a Bruker AMX300 or Bruker Avance 500 MHz spectrometer in CDCl3 or CD3OD. 1 Chemical shifts in 1H NMR were in ppm, using internal references, δ = 7.26 ppm for CDCl3 and δ = 3.31 ppm for CD3OD, and were reported as follows: chemical shift (ppm) [multiplicity shape, coupling constant J (Hz), relative integral, assignment]. Multiplicities were defined as follows: br = broad peak, m = multiplet, s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet or combinations thereof. 13 13C NMR chemical shifts were referenced to the residual solvent central peak (δ = 77.16 ppm for CDCl3 and δ = 49.0 ppm for CD3OD). By 1 H- 1 H COSY experiments were used to determine the connections; carbons were assigned based on HSQC and HMBC NMR experiments.

[0149] Mouse model of heart failure

[0150] 7-week-old male C57Bl / 6 mice (Janvier, France) underwent myocardial infarction after left coronary artery ligation (PMI mice). Briefly, left thoracotomy was performed under anesthesia and cardiac monitoring (2% isoflurane / O2, Baxter, France). An artery protruding 1 - 2 mm from the top of the left atrium was ligated using 8-0 sutures. Subcutaneous injection of 0.01 ml of buprenorphine solution (0.3 mg.ml -1 ) was used for postoperative analgesia. Echocardiography was systematically performed before inclusion of animals to ensure correct ligation. Only animals that survived on the -5th postoperative day and had comparable echocardiographic parameters at this time were included in the study to limit bias due to size differences. All procedures were in accordance with the European Parliament Directive 2010 / 63 / EU and the Animal Protection Council of September 22, 2010, and were approved by the Institutional Animal Research Committee (Departmental Council for the Protection of Populations and Animal Health (Ethics in Animal Welfare and Environmental Protection, N°A 34 - 485) and our Animal Experiment Ethics Committee (Languedoc Roussillon, N°C E-LR-0714)). 10 - 12 weeks after ligation, the mice were euthanized by cervical dislocation, and the EDL muscle was dissected to record calcium sparks.

[0151] Calcium spark measurement

[0152] Diaphragm samples were dissected and stored in HEPES-buffered physiological medium (in mM: 119 NaCl, 5 KCl, 1.25 CaCl2, 1 MgSO4, 10 glucose, 1.1 mannitol, 10 HEPES, pH 7.4). Then, the muscle was quickly placed in the dissection chamber and the solution was exchanged with a relaxation solution (in mM: 140 K-glutamate, 10 HEPES, 10 MgCl2, 0.1 EGTA, pH 7.0). Five to ten EDL fiber bundles were manually dissected, mounted, and permeabilized in the relaxation solution containing 0.01% saponin for 30 seconds. After washing with saponin-free solution, the solution was changed to the internal medium for imaging: (in mM) 140 K-glutamate, 5 Na2ATP, 10 glucose, 10 HEPES, 4.4 MgCl2, 1.1 EGTA, 0.3 CaCl2, fluo-4 0.05 pentapotassium salt (Invitrogen), pH 7.0, for spark acquisition. Potential sparks were identified empirically using an automatic detection algorithm. The average fluorescence (F0) value of the image was calculated by summing and averaging the time F at each spatial position, while ignoring potential spark regions. Then, this F0 value was used to create a smoothing program, visualize potential spark positions, and analyze spatiotemporal properties. Image analysis was performed using IDL (v5.5, Research System, Inc.). Statistical comparisons were made using ANOVA test, and the significance level was set at P<0.05 (Graphpad Prism v8.4).

[0153] Mouse VIDD model

[0154] Thirty-five adult male C57 / BL6 mice (10 - 12 weeks old, 25 - 30 g) were divided into 5 groups. Three groups were intubated with a 22-gauge vascular catheter and continuously mechanically ventilated for 6 hours using a volume-driven small animal ventilator ( Harvard Apparatus, Saint-Laurent, Canada). The tidal volume was 10 μl / mg body weight, the respiratory rate was 150 breaths / minute, the positive end-expiratory pressure (PEEP) level was 2 - 4 cm H2O, and the fraction of inspired oxygen was 0.21. Non-spontaneous ventilation was defined as no diaphragmatic contraction activity, confirmed by repetitive stereotypical deflections observed in the airway pressure curve.

[0155] The mice were divided into two groups. The first group (control) received methanol in a volume equivalent to the equivalent volume brought by 4F 4t -NeuroP, and was intravenously (IV) infused for 5 minutes 20 minutes before the start of MV. The second group of mice was intravenously (IV) infused with 4F within 5 minutes 20 minutes before the start of MV4t -NeuroP. Both groups were intubated and treated in the same manner (see Matecki et al., 2016 15 ).

[0156] Contractile function of mouse muscle samples

[0157] At the end of the MV protocol, the entire diaphragm was surgically excised and the mice were euthanized by exsanguination. Isometric contractile properties were evaluated as previously described (Matecki et al., 2016 15 ). The excised diaphragm strips were mounted in a jacketed tissue bath containing balanced and oxygenated Krebs solution. The muscle was supramaximally stimulated using square-wave pulses (Model S48; Grass Instruments, West Warwick, RI). The force-frequency relationship was determined by stimulating the muscle for 600 ms at 10, 20, 30, 50, 60, 80, 100, and 120 Hz in sequence, with a 1-minute interval between each stimulation series. After measuring the contractile properties, the muscle was measured at L0 (the length at which the muscle generates maximum isometric tension), dried, and weighed. For comparison, the diaphragmatic muscle force production was normalized to the total muscle strip cross-sectional area and expressed in N.cm -2 . The total muscle strip cross-sectional area was determined by dividing the muscle weight by its length and tissue density (1.056 g / cm 3 ).

[0158] The remaining part of the diaphragm was separated, with one part snap-frozen in liquid nitrogen and used again for biochemical analysis, and the other part fresh for Ca 2+ spark measurement.

[0159] Biochemical analysis of RyR1

[0160] Homogenize muscle biopsy tissues in 150 μl of buffer containing 5% SDS, 5% β-mercaptoethanol, 10% glycerol, 10 mM EDTA, and 50 mM Tris / HCl buffer (pH = 8.0). Denature each sample immediately at 90 °C for 4 minutes. After centrifugation at 4 °C (5000 rpm), use the BCA protein assay to repeat the determination of the supernatant protein concentration, dilute it with loading buffer to balance at the same concentration, and aliquot it at 2 μg / μl. At 4 °C, use an anti-RyR antibody (4 μg 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 inhibitor) to immunoprecipitate for 1 hour to precipitate RyR1 from 250 μg of homogenate. Incubate the immune complexes with protein A Sepharose beads (Amersham Pharmacia) at 4 °C for 1 hour, and wash the beads three times with buffer. Separate the proteins on an SDS-PAGE gel (4-20% gradient) and transfer them to a nitrocellulose membrane at 200 mA for 2 hours (SemiDry transfer blot, Bio-Rad). To prevent non-specific antibody binding, incubate the membrane with a blocking solution (LICOR Biosciences) and wash it with Tris-buffered saline containing 0.1% Tween-20.

[0161] Incubate the blots separately with a primary antibody against RyR1 (RyR1-1327, an affinity-purified rabbit polyclonal antibody 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) and affinity-purify it with the uncoupled peptide. The inventors also used an antibody against Calstabin1 (1:2500, in blocking buffer, LICOR Biosciences); a phosphorylation epitope-specific antibody against human RyR2 phosphorylated at Ser2808 (1:5000), which detects PKA-phosphorylated mouse RyR1 (at Ser2844 site) and RyR2 (at Ser2808 site); an antibody against S-nitrosylated cysteine residues (1:1000, Sigma). To determine RyR1 oxidation, treat the immunoprecipitate with 2,4-dinitrophenylhydrazine and detect the derived carbonyl compounds using the OxyBlot Protein Oxidation Detection Kit (Catalog No. S7150, Chemicon International Inc.). After washing three times, incubate the membrane with an infrared-labeled secondary antibody. Analyze control samples on each gel for normalization, and the total levels of RyR1 did not differ between groups.

[0162] Piglet model

[0163] As described above 18,25 , twelve piglets (15 - 20 kg) were divided into two groups of six animals each. In both groups, oxygenation was maintained with an inhaled fraction of oxygen (FIO2) of 25 - 35%, tidal volume of 10 - 12 ml·kg -1 body weight, and a respiratory rate of 15 - 30 cycles per minute to maintain normal oxygen and carbon dioxide concentrations. In the first group (MV group), CMV mechanical ventilation was used for the piglets with a positive end-expiratory pressure level of 5 cm H2O. Absence of spontaneous breathing was verified on the ventilator trend graph, and diaphragmatic electromyographic activity was measured to ensure absence of diaphragmatic electrical activity. In the second group (control group), ASV was used to ventilate the piglets with settings in phase with the piglet body weight (inspiratory flow trigger of 0.3 L·min -1 , mechanical ventilation percentage of 100 - 150%, positive end-expiratory pressure level of 5 cm H2O, and expiratory trigger of 25% of peak inspiratory flow). Diaphragmatic electromyogram activity was evaluated to confirm that the piglets could breathe spontaneously. Except for the MV mode, both groups received the same care.

[0164] Briefly, the piglets were anesthetized with an intravenous injection of 5 - 6 mg / kg -1 sodium pentobarbital, intubated with an endotracheal tube with a cuff and mechanically ventilated ( Hamilton Medical AG, Rhazuns, Switzerland). A continuous intravenous infusion of 15 - 20 mg·kg -1 propofol, 0.1 - 0.3 mg·kg -1 midazolam, and 3 - 4 mg·kg -1 .h -1 ketamine was used to maintain anesthesia. The depth of sedation was monitored using a bispectral index system ( Aspect, Norwood, MA)). Heating pads were used as needed to maintain a normal body temperature of 38.5 - 39.5 °C. A carotid artery catheter was inserted ( Pulsion, Munich, Germany) to monitor heart rate, arterial blood pressure, and cardiac output. Absence of spontaneous breathing in the MV group was verified using the ventilator trend graph and diaphragmatic electromyographic activity measurements.

[0165] Arterial carbon dioxide pressure was monitored using a capnograph ( Datex-Ohmeda, Helsinki, Finland) and verified by arterial blood gas analysis ( Abbott, Abbott Park, IL). Parenteral nutrition was administered starting on day 1 (10% glucose, 20% amino acids, and Hyperamine Braun, Boulogne Billancourt, France) to provide 30 - 35 kcal.kg -1 . per day -1 . All surgeries were performed under sterile conditions. All animals received prophylactic intravenous lactated antibiotic (100 mg.kg -1 . per day -1 amoxicillin - clavulanate).

[0166] Diaphragm contractile function was evaluated in vivo by measuring transdiaphragmatic pressure (Pdi). 18 . A double - lumen balloon - tipped catheter was inserted orally into the stomach and the distal third of the esophagus. A bipolar transvenous pacing catheter was introduced through each internal jugular vein and adjusted to stimulate the phrenic nerve, and then diaphragmatic contractions were induced by supramaximal stimulation in the frequency range of 20 - 120 Hz with a 2 s and 150 ms stimulation train.

[0167] The test solution and placebo solution were injected intravenously before animal intubation at a concentration of 0.250 mg.kg -1 , followed by a maintenance dose of 0.025 mg.kg every 12 hours -1 .

[0168] For these experiments, the surgeons handling the piglets were unaware of whether the injected substance was a placebo or VB558. Analysis was performed by a third - party blind method, and then the piglet assignments were revealed.

[0169] Statistics

[0170] Three statistical analyses were performed: 1 / independent student t - test when comparing a given parameter (cross - sectional area, fiber %) under two conditions (before and after mechanical ventilation), 2 / one - way ANOVA when comparing a given parameter (spark, maximum diaphragmatic muscle force at 120 Hz, co - immunoprecipitation ratio) under three conditions (control, VIDD, VIDD + VB558), followed by a post - hoc Tukey test, 3 / two - way repeated - measures ANOVA when comparing the force or Pdi frequency relationship, followed by a post - hoc Bonferroni test.

[0171] Synthesis

[0172] Synthesis of Compound II - [17,18,19,20,21,22] - hexanor - 4F 4t Neuroprostaglandin - (VB558)

[0173] Compound II was prepared according to Scheme 1:

[0174]

[0175] Scheme 1: a) DMP, CH2Cl2, RT; b) BrPh3P(CH2)2CH3, NaHMDS, THF, -78 °C to RT, 2 steps 81%; c) TBAF, THF, RT, 40%; d) LiOH, THF / H2O (1:1), RT, 36%.

[0176] Example 1a Methyl (E)-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 Synthesis

[0177]

[0178] To a solution of the 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). After stirring at room temperature for 30 minutes, the reaction was quenched by the addition of an aqueous solution of 10% Na2S2O3 / NaHCO3 (1:1, v / v) (ca. 10 mL). The organic phase was separated and the aqueous phase was extracted with Et2O (3 × 10 mL). The combined organic phases were washed with an aqueous solution of 10% Na2S2O3 / NaHCO3 (1:1, v / v) (ca. 10 ml), then with brine, dried over MgSO4 and concentrated in vacuo. The crude product was used in the next step of the synthesis.

[0179] To a solution of propyltriphenylphosphonium bromide (550 mg, 1.42 mmol, 2.3 eq) in anhydrous THF at room temperature was added NaHMDS (682 mL, 2 M in THF, 1.36 mmol, 2.2 eq) and the mixture was stirred at room temperature for 1 hour. The reaction was cooled to -78 °C and added via cannula to a solution of the aldehyde (390 mg, 0.62 mmol, 1 eq) in anhydrous THF (4 mL). The reaction was warmed to room temperature overnight and quenched with an aqueous solution of 10% NH4Cl (ca. 5 mL), extracted with Et2O (3 × 10 mL), and the combined organic phases were washed with an aqueous solution of 10% NH4Cl (ca. 20 ml), washed with brine, dried over MgSO4 and concentrated in vacuo. The crude was purified by SiO2 column chromatography (pentane / Et2O 95:5)

[0180] to give 2 (260 mg, 81%, over 2 steps), as a colorless oil.

[0181] R f= 0.66 (pentane / Et2O 95:5).

[0182] 1 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).

[0183] 13 13C NMR (75 MHz, CDCl3) δ 174.40, 134.94 dia 1, 134.86 dia 2, 132.31 dia 1, 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.

[0184] Example 1b Methyl (E)-6-((1S,2R,3R,5S)-3,5-dihydroxy-2-((Z)-pent-2-en-1-yl)cyclopentyl)-4-hydroxyhex-5-enoate 3 Synthesis

[0185]

[0186] To the compound 2 (260 mg, 0.4 mmol, 1 eq) was added TBAF (4.76 mL, 1 M solution in THF, 4.76 mmol, 12 eq). After stirring at room temperature for 1 h, the reaction was quenched by adding DOWEX-50W (2.5 g), CaCO3 (2.5 g) and MeOH (75 mL), stirred at room temperature for 1 h, filtered through a pad, and concentrated under reduced pressure. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give 3 , as a mixture of ester / lactone (ratio 72:28) (50 mg, 40%), as a colorless oil.

[0187] R f = 0.30 (100% EtOAc).

[0188] Ester:

[0189] 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 = 7.5 Hz, 3H).

[0190] 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.

[0191] Lactone:

[0192]

[0193] 1 H NMR (300 MHz, 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).

[0194] Synthesis of Example 1c (E)-6-((1S,2R,3R,5S)-3,5-dihydroxy-2-((Z)-pent-2-en-1-yl)cyclopentyl)-4-hydroxyhex-5-enoic acid II of

[0195]

[0196] At room temperature, to 2(50 mg, 0.18 mmol, 1 eq) was added to a solution of LiOH (45 mg, 1.07 mmol, 6 eq) in 4 mL of THF / H₂O (1:1, v / v). After stirring for 1 h 15, the reaction was quenched with 1 M NaHSO₄ solution until acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO₄ and concentrated in vacuo. The crude product was purified by SiO₂ column chromatography (100% EtOAc) to give compound II (27 mg, 36%), as a colorless oil.

[0197] R f = 0.13 (100% EtOAc).

[0198] 1 ¹H 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).

[0199] 13 ¹³C NMR (75 MHz, MeOD) δ 176.04, 134.62, 131.93, 129.12, 127.31, 127.16, 74.81, 74.72, 71.07, 52.10, 50.03, 42.17, 32.02, 29.66, 25.76, 20.26, 13.27.

[0200] ESI - : 297.17 [M - H] - .

[0201] HRMS (ESI - ) calcd for C 16 H 25 O₅ [M - H] - 297.1702, found 297.1707.

[0202] Example 2 Synthesis of Compound III - 20,21,22 - trinor - 4(RS) - 4 - F 4t -NeuroP-(VB574)

[0203] Compound III was prepared according to Protocol 2:

[0204]

[0205] Protocol 2: a) DMP, CH2Cl2, RT; b) (Z)-hex-3-en-1-yltriphenylphosphonium iodide, NaHMDS, THF, -78 °C to RT, 83%, 2 steps; c) TBAF, THF, RT, 59%; d) LiOH, THF / H2O (1:1), RT, 69%.

[0206] Example 2a Methyl (E)-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 Synthesis

[0207]

[0208] To the 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). After stirring at room temperature for 30 minutes, the reaction was quenched by the addition of an aqueous solution of 10% Na2S2O3 / NaHCO3 (1:1, v / v) (ca. 10 mL). The organic phase was separated, the aqueous phase was extracted with Et2O (3 x 10 mL), and the combined organic phases were washed with an aqueous solution of 10% Na2S2O3 / NaHCO3 (1:1, v / v) (ca. 10 mL), then with brine, dried over MgSO4 and concentrated in vacuo. The crude product was used in the next step of the synthesis.

[0209] Under RT, NaHMDS (815 μL, 2 M in THF, 1.63 mmol, 2.2 eq) was added to a solution of (Z)-hex-3-en-1-yltriphenylphosphonium iodide (803 mg, 1.7 mmol, 2.3 eq) in anhydrous THF, and the mixture was stirred at -50 °C for 0.5 h. The reaction mixture was cooled to -78 °C and added via cannula to a solution of the aldehyde (465 mg, 0.74 mmol, 1 equiv) in anhydrous THF (5 mL), stirred for 3 h, quenched with 10% aqueous NH4Cl solution (ca. 5 mL), extracted with Et2O (3×10 mL), the combined organic phases were washed with 10% aqueous NH4Cl solution (ca. 20 ml), washed with brine, dried over MgSO4, and concentrated in vacuo. The crude extract was purified by silica gel column chromatography (pentane / Et2O 95:5) to give 5 (172 mg, 83%, over 2 steps), as a colorless oil.

[0210] R f = 0.60 (pentane / Et2O 95:5).

[0211] 1 1H 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 = 7.5 Hz, 3H), 0.89 (s, 27H), 0.00 (s, 18H).

[0212] 13 13C MNR (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.

[0213] HRMS (ESI + ) Calcd for C 38 H 74 O5Si3 [M] + 695.4922, found 695.4918.

[0214] Example 2b 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 Synthesis

[0215]

[0216] To the compound 5 (170 mg, 0.247 mmol, 1 eq) was added TBAF (1.48 mL, 1 M THF solution, 1.48 mmol, 6 eq). After stirring at room temperature for 1 h, the reaction was quenched by the addition of DOWEX-50W resin (1.2 g), CaCO3 (0.4 g) and MeOH (10 mL), stirred at room temperature for 1 h, filtered through a pad and concentrated under reduced pressure. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give 6 (47 mg, 59%) as a colorless oil.

[0217] R f = 0.38 (100% EtOAc).

[0218] 1 1H 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).

[0219] 13 13C NMR (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.

[0220] HRMS (ESI + ) Calcd for C 19 H 29 O4 [M + H] + 321.2066, found 321.2068.

[0221] Example 2 (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 Synthesis

[0222]

[0223] At RT, to 6 (47 mg, 0.15 mmol, 1 eq) in a solution of 3.6 mL of THF / H2O (1:1, v / v) was added LiOH (37 mg, 0.88 mmol, 6 eq). After stirring for 1 h 15, the reaction was quenched with 1 M NaHSO4 solution until acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give compound III (34 mg, 69%) as a colorless oil.

[0224] R f = 0.13 (100% EtOAc).

[0225] 1 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).

[0226] 13 13C NMR (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.

[0227] HRMS (ESI - ) Calcd for C 19 H 30 O5 [M - H]- 337.2015, measured value 337.2007.

[0228] Synthesis of the α-chain synthon of the monohydroxylated derivative IV in Example 3

[0229] Prepare the silyl-protected intermediate 11 according to Reaction Scheme 3:

[0230]

[0231] Scheme 3: α-chain synthon 11 of the monohydroxylated derivative IV

[0232] Example 3a Methyl (E)-6-((1R,2R,5S)-2-(2-acetoxyethyl)-5-((tert-butyldimethylsilyl)oxy)cyclopentyl)-4-oxohex-5-enoate 8 Synthesis

[0233]

[0234] At RT, DMP (12.5 mL, 0.38 M in DCM, 4.74 mmol, 1.5 eq) was added to a solution of alcohol 7 (1 g, 3.16 mmol, 1 eq) in DCM (30 mL). After stirring at room temperature for 30 minutes, the reaction was quenched by adding an aqueous solution of 10% Na2S2O3 / NaHCO3 (1:1, v / v) (ca. 20 mL). The organic phase was separated, the aqueous phase was extracted with Et2O (3 x 200 mL), and the combined organic phases were washed with an aqueous solution of 10% Na2S2O3 / NaHCO3 (1:1, v / v) (ca. 20 mL), washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was used in the next step of the synthesis.

[0235] At 0 °C, NaHMDS (6 mL, 2 M in THF, 12.16 mmol, 3.2 eq) was added to a solution of methyl 5-(dimethoxyphosphoryl)-4-oxopentanoate (3.1 g, 13 mmol, 3.4 eq) in anhydrous THF and the mixture was stirred at 0 °C for 1 h. The reaction was cooled to -78 °C and added via cannula to a solution of the aldehyde (1.2 g, 3.8 mmol, 1 eq) in anhydrous THF (10 mL) and stirred for 14 h and quenched with brine (ca. 20 mL), extracted with Et2O (3 x 20 mL), and the combined organic phases were washed with brine, dried over MgSO4 and concentrated in vacuo. The crude extract was purified by silica gel column chromatography (pentane / Et2O 9:1) to give 8 (1.3 g, 80%, over 2 steps), as a colorless oil.

[0236] R f = 0.64 (pentane / Et2O 9:1).

[0237] 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).

[0238] Synthesis of Methyl (E)-6-((1R,2R,5S)-2-(2-Acetoxyethyl)-5-((tert-butyldimethylsilyl)oxy)cyclopentyl)-4-hydroxyhex-5-enoate 9 in Example 3b

[0239]

[0240] CeCl3 (1.04 g, 2.8 mmol, 1.0 eq) was added to a solution of enone 8 (1.2 g, 2.8 mmol, 1.0 eq) in 30 mL of anhydrous MeOH. After 15 minutes, the mixture was cooled to 0 °C and NaBH4 (53 mg, 1.4 mmol, 0.5 eq) was added. After 20 minutes of addition, the reaction was complete, and 30 mL of EtOAc and 30 mL of brine were added. The aqueous phase was extracted with 3 × 20 mL of EtOAc. The organic layer was extracted with 20 mL of brine, dried over MgSO4, 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%).

[0241] R f = 0.35 (pentane / Et2O 1:1).

[0242] 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).

[0243] Synthesis of Methyl (E)-6-((1R,2R,5S)-2-(2-Acetoxyethyl)-5-((tert-Butyldimethylsilyl)oxy)cyclopentyl)-4-((tert-Butyldimethylsilyl)oxy)hex-5-enoate 10 in Example 3c

[0244]

[0245] To a solution of alcohol 9 (1.1 g, 2.6 mmol, 1.0 eq) in 100 mL of CH2Cl2 were added TBSCl (1.2 g, 7.7 mmol, 3.0 eq), imidazole (0.7 g, 10.3 mmol, 4.0 eq) and 4-DMAP (catalytic amount). After 4.5 h, the reaction was complete. 100 mL of Et2O and 100 mL of brine were added. The aqueous phase was extracted with 3 × 50 mL of Et2O. The organic layer was extracted with 50 mL of brine, dried over MgSO4 and the solvent was removed under reduced pressure. Compound 10 was obtained and used directly in the reaction without further purification (1.3 g, quantitative).

[0246] R f = 0.89 (pentane / Et2O 1:1).

[0247] 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).

[0248] Synthesis of Methyl (E)-4-((tert-Butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-Butyldimethylsilyl)oxy)-5-(2-Hydroxyethyl)cyclopentyl)hex-5-enoate 11 in Example 3d

[0249]

[0250] To a solution of acetate 10 (1.3 g, 2.4 mmol, 1 eq) in 35 mL of anhydrous MeOH was added anhydrous K2CO3 (1.3 g, 9.6 mmol, 4 eq) and the mixture was stirred at room temperature. After 3 h, the reaction was quenched by adding 20 mL of a 1:1 solution of Et2O / H2O and stirred for 1 h. The aqueous phase was extracted with Et2O (3 x 20 mL), and the combined organic phases were washed with brine, dried over MgSO4 and concentrated in vacuo.

[0251] The crude extract was purified by SiO2 column chromatography (20 - 50% Et2O / 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%).

[0252] R f = 0.50 (pentane / Et2O 1:1) for 4S-epimer; R f = 0.40 (pentane / Et2O 1:1) for 4R-epimer

[0253]

[0254] (E)-4S-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)cyclopentyl)hex-5-enoic acid methyl ester (S)-11

[0255] 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).

[0256] 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.

[0257]

[0258] (E)-4R-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-(2-hydroxyethyl)cyclopentyl)hex-5-enoic acid methyl ester (R)-11

[0259] 11H 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).

[0260] 13 13C 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.

[0261] Synthesis of Compound IV in Example 4

[0262] According to Synthesis Scheme 4, the synthon obtained in Example 3 was used to prepare Compound IV ((S)-IV and (R)-IV):

[0263]

[0264] Scheme 4. 4(S)-4F 4t - Monohydroxylated C22 analog of neuroprostaglandin and its C4 epimer 4(R)-4-F 4t - Neuroprostaglandin

[0265] Synthesis of Methyl (S,E)-4-((tert-butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-butyldimethylsilyl)oxy)-5-((2Z,5Z,8Z)-undeca-2,5,8-trien-1-yl)cyclopentyl)hex-5-enoate (S)-12 in Example 4a

[0266]

[0267] 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 stirred at room temperature. After 30 minutes, the reaction was quenched by adding an aqueous solution of 10% NaHCO3 / Na2S2O3 (1 / 1, v / v). 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%).

[0268] Under N2 and at -40 °C, NaHMDS (0.83 mL, 1.66 mmol, 2 M / THF, 2.1 eq) was slowly added to a solution of triphenylphosphine iodide (0.91 g, 1.78 mmol, 2.25 eq) in THF (10 mL) as described above. After stirring for 1 h at -40 °C, the bright orange ylide was cooled to -78 °C and slowly added via cannula to a solution of the previously prepared 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 - 9:1). The solvent was carefully evaporated at room temperature and 700 mbar to give (S)-12 as a colorless oil (126 mg, brsm (based on recovered starting material), 41%, 2 steps).

[0269] R f = 0.80 (pentane / Et2O 9:1).

[0270] 1 1H 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).

[0271] 13 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.

[0272] [α] D 20 (MeOH) = +7.86 (c = 1.26).

[0273] Synthesis of Methyl (R,E)-4-((tert-Butyldimethylsilyl)oxy)-6-((1R,2S,5R)-2-((tert-Butyldimethylsilyl)oxy)-5-((2Z,5Z,8Z)-undeca-2,5,8-trien-1-yl)cyclopentyl)hex-5-enoate (R)-12

[0274]

[0275] 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 stirred at room temperature. After 30 minutes, the reaction was quenched by adding an aqueous solution of 10% NaHCO3 / Na2S2O3 (1 / 1, v / v). 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%).

[0276] Under N2 and at -40 °C, NaHMDS (0.5 mL, 1 mmol, 2 M / THF, 2.1 eq) was slowly added to a solution of triphenylphosphine iodide (0.58 g, 1.12 mmol, 2.25 eq) in THF (10 mL) described above. After stirring at -40 °C for 1 h, the bright orange ylide was cooled to -78 °C and slowly added via cannula to a solution of the previously prepared 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 - 9:1). The solvent was carefully evaporated at room temperature and 700 mbar to give (R)-12 (66 mg, BRSM 30%, 2 steps) as a colorless oil.

[0277] R f = 0.84 (pentane / Et2O 9:1).

[0278] 1 1H 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).

[0279] 1313C 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.

[0280] [α] D 20 (MeOH) = +18.2 (c = 5).

[0281] Synthesis of Example 4c (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

[0282]

[0283] 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 room temperature for 2 h, the reaction was quenched by the addition of DOWEX-50W resin (1.2 g), CaCO3 (0.4 g) and MeOH (15 mL), stirred at room temperature for 1 h, filtered through a pad, and concentrated under reduced pressure. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give (S)-13 (51 mg, 72%) as a colorless oil, which was a 1:1 mixture of lactone / ester.

[0284] R f = 0.34 (EtOAc).

[0285] Synthesis of Example 4d (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

[0286]

[0287] To the compound (R)-12 (60 mg, 0.1 mmol), TBAF (0.4 mL, 1 M in THF, 0.4 mmol) was added. After stirring at room temperature for 2 h, the reaction was quenched by adding DOWEX-50W resin (0.6 g), CaCO3 (0.2 g) and MeOH (10 mL), stirred at room temperature for 1 h, and filtered through a pad, and concentrated under reduced pressure. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give (R)-13 (40 mg, 92%) as a colorless oil, a mixture of ester / lactone 83:17.

[0288] R f = 0.32 (EtOAc).

[0289] Synthesis of Example 4e (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

[0290]

[0291] To a solution of (S)-13 (51 mg, 0.15 mmol, 1 eq) in 4 mL of THF / H2O (1:1, v / v) at room temperature, LiOH (38 mg, 0.89 mmol, 6 eq) was added. After stirring for 1 h 15, the reaction was quenched with 1 M NaHSO4 solution until acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give compound (S)-IV (38 mg, 72%) as a white powder.

[0292] R f = 0.15 (100% EtOAc).

[0293] 1 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).

[0294] 1313C 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。

[0295] HRMS (ESI - ) Calcd for C 22 H 33 O4 [M-H] - 361.2379, found 361.2376。

[0296] [α] D 20 (MeOH) = +22.6 (c = 3.8)。

[0297] Synthesis of Example 4f (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

[0298]

[0299] To a solution of (R)-13 (50 mg, 0.18 mmol) in 4 mL of THF / H2O (1:1, v / v) at room temperature was added LiOH (45 mg, 1.07 mmol, 6 eq). After stirring for 1 h 15, the reaction was quenched with 1 M NaHSO4 solution until acidic pH, extracted with EtOAc (10 mL), washed with brine, dried over MgSO4 and concentrated in vacuo. The crude product was purified by SiO2 column chromatography (100% EtOAc) to give the compound (R)-IV (27 mg, 36%) as a white powder.

[0300] R f = 0.13 (100% EtOAc).

[0301] 1 1H 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).

[0302] HRMS(ESI - ) Calculated value C 22 H 33 O4[M-H] - 361.2379, measured value 361.2376.

[0303] [α] D 20 (MeOH) = -4 (c = 2).

[0304] Example 5 - VIDD prevention study

[0305] Results

[0306] 4F 4t -NeuroP prevents RyR1-mediated Ca leakage

[0307] The term spontaneous Ca 2+ release event (i.e., Ca 2+ spark) refers to a local Ca 2+ release event that occurs spontaneously in resting muscle fibers, which comes from a group of RyRs and is also called a single Ca 2+ release unit (CRU). The measurement of Ca 2+ sparks in muscle fibers provides an in situ direct measurement of the gating behavior of RyRs belonging to a single CRU. In particular, the frequency of Ca 2+ sparks can well estimate the RyR1 opening frequency and represents an index of RyR-mediated SR calcium leakage. The inventors previously reported a defect in RyR1 in peripheral muscles in an animal model of heart failure. In a previous study, the inventors demonstrated that neuroprostaglandins such as 4F 4t -NeuroP, a lipid mediator produced by non-enzymatic free radical peroxidation of docosahexaenoic acid (DHA), can prevent RyR2-mediated Ca 2+ leakage in cardiomyocytes

[30] . To test the efficacy of 4F 4t -NeuroP in preventing RyR1 functional remodeling, the inventors tested the acute application of 1 μM 4F 4t -NeuroP in mouse fast-twitch extensor digitorum longus (EDL) muscle and evaluated the functional effect on RyR1 by measuring the Ca 2+ spark frequency. Thus, the inventors demonstrated that in a mouse model of heart failure obtained after permanent ligation of the left coronary artery (PMI), the function of RyR1 in EDL muscle was impaired, as indicated by a significant increase in spark frequency. When PMI muscle fibers were acutely incubated with 1 μM 4F 4t -NeuroP, complete prevention of this RyR1-dependent leakage behavior was observed ( Figure 1 ). Therefore, this experiment confirmed 4F4t - The ability of -NeuroP to prevent RyR1 dysfunction.

[0308] 4F 4t - -NeuroP prevents VIDD in mice after short MV

[0309] Mice were anesthetized and mechanically ventilated for 6 hours. Mice were randomly assigned to receive or not receive 4F at the start of ventilation 4t - A single IV injection of -NeuroP, at concentrations ranging from 0.01 - 100 μM, was given to establish a dose - response curve for the force - frequency relationship and thus determine the optimal concentration. Under these conditions, complete prevention of VIDD by 4F 4t - -NeuroP was observed, with a maximum effect seen at approximately 0.3 μM ( Figure 2A - B).

[0310] 4F 4t - -NeuroP stabilizes the complex calstabin1 / RyR1

[0311] Diaphragm samples from the above experiments were frozen and solubilized to further study the biochemical properties of the RyR1 macromolecular complex. After 6 hours of mechanical ventilation, RyR1 immunoprecipitation showed that VIDD was responsible for channel oxidation and phosphorylation of ser2844 and depletion of calstabin1. This biochemical signature of leaky RyR1 was completely blocked by 4F 4t - -NeuroP (Figure 2C).

[0312] 4F 4t - -NeuroP prevents VIDD and diaphragm atrophy in mice after long MV

[0313] Six hours of MV is still a rather short ventilation time, which represents a particular interest in studying the proximal pathophysiological mechanisms of VIDD. As previously reported, 6 hours of mechanical ventilation can induce force deficit without histological changes. However, 12 hours of mechanical ventilation can cause muscle atrophy through a RyR1 - dependent SR Ca 2+ - leak - mediated mechanism. Therefore, the inventors here tested the beneficial effects of 4F 4t - -NeuroP after 12 hours of mechanical ventilation. Under these conditions, a single injection of 4F 4t - -NeuroP (10 μM) had a protective effect similar to that observed after 6 hours of ventilation (Figure 3A). Notably, after 12 hours of mechanical ventilation, 10 μM of 4F 4t - -NeuroP also prevented the myofiber remodeling (i.e., atrophy) characteristic of VIDD. 4F 4t-NeuroP prevents cross-sectional area (CSA) reduction, regardless of myofiber phenotype (Figure 3B-C) and does not alter the distribution of fiber types (slow vs fast, Figure 3D).

[0314] VB558, 4F 4t -A shorter analog of NeuroP, prevents VIDD

[0315] Due to its efficiency in stabilizing RyR, 4F 4t -NeuroP (WO2015197562) is a biomolecule of great interest. However, its synthesis is rather long and costly, thus limiting its use for therapeutic purposes. Therefore, the inventors screened 4F 4t -several analogs of NeuroP to identify compounds that could exhibit at least the same efficiency as 4F 4t -NeuroP but with a more feasible synthesis. This screening was carried out in an in vitro model of arrhythmia characterized by RyR dysfunction (as described in WO2014086819). VB558 was selected as the most promising candidate because its synthesis involves only 17 steps, compared to 24 steps for 4F 4t -NeuroP ( Figure 4A ).

[0316] For 4F 4t -NeuroP, VB558 was intravenously injected into mice at concentrations of 1 and 10 μM, and then the mice were ventilated for 6 hours. Both concentrations showed significant protection against mechanical ventilation, with a range similar to that obtained using 4F 4t -NeuroP ( Figure 4B ).

[0317] VB558 prevents VIDD in piglets after 72 hours of ventilation

[0318] To confirm the potential clinical use of neuroprostaglandins in preventing VIDD, the inventors decided to test the efficacy of VB558 in the previously published piglet model of VIDD 17,18 .

[0319] In the first set of experiments, a pharmacokinetic study was conducted on 3 control pigs injected with a single bolus of VB558 (0.250 mg·kg -1 ). Blood samples were collected regularly within 12 hours to assess the plasma concentration of VB558. This demonstrated a rapid plasma peak between the first 5 - 10 minutes, which dissipated rapidly within 1 hour, indicating a rapid biodistribution of the compound ( Figure 5A)。To test the prophylactic effect of VB558 in a piglet model, the intravenous injection protocol was slightly adapted to the duration of mechanical ventilation (i.e., 72 hours). The inventors injected a bolus of 0.250 mg.kg every 12 hours -1 followed by an infusion of 0.025 mg.kg -1 maintenance dose (Figure 5B). The VBP58 group was compared to a group of piglets (placebo) receiving the same amount of solvent (methanol) (Figure 5B).

[0320] By measuring the transdiaphragmatic pressure at different stimulation frequencies of the phrenic nerve, the inventors were able to establish an equivalent in vivo force-frequency relationship, namely the Pdi-frequency relationship at the start of anesthesia (D0). Apparently, in control piglets, 72 hours of mechanical ventilation induced a large and significant decrease in Pdi, which was completely prevented by VB558.

[0321] Interaction of VB558 with prostaglandin receptors

[0322] Prostaglandin (or prostanoid) receptors are a class of cell surface membrane receptors known to mainly bind and respond to polyunsaturated fatty acid metabolites (PUFAs), arachidonic acid. Since some of them are known to regulate cAMP production or intracellular calcium concentration, and based on the chemical structure of neuroprostaglandins (i.e., DHA derivatives), the inventors performed in vitro binding assays to specifically evaluate the potential agonist or antagonist effects of VBP58 on established prostanoid receptors. Results showing more than 50% inhibition (or stimulation assays performed under basal conditions) were considered to represent a significant effect of the test compound.

[0323] No such effects were observed on any of the receptors studied herein, indicating that the effects of VB558 detailed above are independent of prostanoid receptors.

[0324] Discussion :

[0325] Although the vast majority of critically ill patients are easily weaned from mechanical ventilation (MV), approximately 25% of patients experience difficulty in weaning and require an extension of this mechanical ventilation. However, the use of MV leads to severe damage to the respiratory muscles, characterized by progressive muscle weakness and histological remodeling of the respiratory muscles, which further reduces the chances of weaning. This is especially true for patients with aggravating factors such as malnutrition, chronic electrolyte abnormalities, hyperglycemia, excessive resistive and elastic loads, corticosteroids, exposure to muscle relaxants, sepsis, and impaired cardiac function 28 . To date, no treatment strategy has proven its effectiveness in preventing VIDD in critically ill patients. Our team has demonstrated in mouse and piglet models of VIDD, as well as in patients, that the remodeling of RyR1 represents a proximal pathophysiological mechanism 15,19Rather, all VIDD models have shown biochemical features of leaky RyR1 channels (i.e., phosphorylation, oxidation, and dissociation of calcstabin1) and evidence of intracellular Ca 2+ leakage. Thus, RyR1 dysfunction is synergistically driven by β-adrenergic signaling pathways and MV-induced oxidative stress, which has been extensively studied in VIDD 4 . In fact, RyR is highly sensitive to oxidative / nitrosative stress in skeletal muscle and other tissues. Thus, this RyR remodeling occurs in other chronic or genetic diseases, including heart failure, diabetes, Duchenne muscular dystrophy 23-27 . Post-translational modification of RyR1 also develops with aging and partly explains age-related muscle weakness 24 .

[0326] The inventors demonstrated in this work that oxidized derivatives of DHA, including 4F 4t -NeuroP, and its shorter derivative VBP58 efficiently prevent VIDD in both mouse and piglet models of VIDD. This effect is associated with the normalization of RyR function. Although the inventors cannot rule out a direct effect of 4F 4t -NeuroP and VB558 on RyR, in contrast to the action of Rycal 28 , the inventors also observed that both compounds prevent RyR oxidation and phosphorylation, suggesting an upstream mechanism rather than a direct effect on RyR as previously reported by the inventors 21,29 .

[0327] To date, only spontaneous ventilation cycles and possibly phrenic nerve stimulation seem to alleviate the severity of human VIDD, but several pathways are currently being examined in animal models to identify pharmacological options. The inventors provide here proof of concept that VBP58 may be an effective molecule for preventing VIDD in patients

[0328] In the context of the current COVID-19 pandemic, the need for such treatment is even more urgent because the number of patients in intensive care has surged during the peak of contamination

[0329] Finally, this pharmacological strategy may be beneficial in cases of altered RyR function in all of the above pathological conditions (i.e., heart disease, myopathy, sarcopenia, cachexia, diabetes, neurodegenerative diseases)

[0330] Example 6: DMD Study

[0331] Then, the inventors tested the effect of VBP58 on the relaxation level of calcium in ventricular cardiomyocytes derived from Duchenne muscular dystrophy (DMD). The results are shown in Figure 6. These data demonstrate that VB558 can prevent RyR2 dysfunction in DMD, supporting its therapeutic benefit in this pathology (Figure 6A - B).

[0332] Example 7: Study of Calcium Transients and Cell Contraction

[0333] Evaluate the anti - arrhythmic properties of compounds 20,21,22 - trinor - 4(RS)-4 - F 4t -NeuroP (Compound of formula III, VB574) and 4 - F 4t -monohydroxylated derivatives of NeuroP (Compound IV).

[0334] By a photometric system Measure calcium transients and cell contraction.

[0335] a) General Principles of Measurement

[0336] The photometric system can provide real - time, simultaneous acquisition of fluorophotometry and sarcomere length measurement. For the study of isolated muscle cells, the system includes a pacemaker that can fully control the duration, frequency, and voltage of the stimulating pulses. Thus, the photometric system Relates calcium transients to the shortening of electrically stimulated muscle cells (1 hz, 20 v, Figure 7 ) Calcium is studied using a ratiometric fluorescent calcium probe (indo - 1 - AM, excitation wavelength 360 ± 10 nm, emission wavelengths 405 ± 10 nm and 485 ± 10 nm).

[0337] b) Scheme

[0338] To measure intracellular calcium, the first step is to load ventricular cardiomyocytes in contact with a certain amount of indo - 1 - AM. To make it lipophilic, indo - 1 is esterified on its carboxyl functional group to obtain indo - 1 - AM. Once inside the cell, esterases can release the Indo - 1 carboxylic functional group, enabling it to bind intracellular calcium. The mixing time is pre - determined to be 30 minutes. This time was chosen after experiments with different incubation times, and 30 minutes is the time that gives a good ratio / noise ratio and does not significantly perturb cellular calcium homeostasis. The incubation time of the probe is crucial because too low a loading produces a weak signal, while over - exposure will overly increase the cytoplasmic calcium buffering capacity.

[0339] Once the cells are loaded and aligned on the measurement system through a container containing Tyrode's salt solution (450 μL), the probe is excited by a xenon lamp with a wavelength of 360 nm. Through an optical wavelength filter, the device collects fluorescence of two different wavelengths emitted from Indo-1: 405 ± 10 nm and 485 ± 10 nm. The first wavelength represents the fluorescence of the probe bound to calcium, and the second wavelength represents the fluorescence of the free probe. The ratio of these two wavelengths reflects the intracellular calcium concentration. Thus, calcium transients during the contraction phase of ventricular myocytes can be observed, and calcium transients during the relaxation phase when the stimulation stops can also be observed.

[0340] To measure contraction, the striations of cardiomyocytes caused by the presence of sarcomeres are used. These striations can be scanned and acquired by a computer. The recorded signal is similar to a sine function. Then, this function is mathematically processed by Fourier transform, so that the period of the sine or sarcomere length can be obtained.

[0341] The experimental protocol also addresses changes in intracellular calcium transients and changes in the cell contraction peak by analyzing sarcomere shortening during 30 seconds of electrical stimulation at 1 Hz, followed by 30 seconds of scattered pauses ( Figure 7 ).

[0342] Using this protocol, the goal is to identify arrhythmia events, that is, irregularities in the contraction rate, and then study the effect of 4-(RS)-4-F 4t -neuroprostaglandin derivatives on these events. To reproduce the sympathetic tone characterizing the murine model, the cells are placed in 10 nM isoproterenol. This molecule will cause an increase in the contraction amplitude when stimulating the β-adrenergic system, and will also promote the development of extrasystolic ventricular contractions (ESV), as Figure 7 shown.

[0343] These ESVs are related to the abnormal function of the type 2 ryanodine receptor (RyR).

[0344] Therefore, to evaluate the potential antiarrhythmic stabilizing effect of 4-F 4t -neuroprostaglandin derivatives (compounds III and IV), the compounds are administered and the percentage of arrhythmic cells is measured.

[0345] It has been found that compound III ( Figure 8 ) and compound IV ( Figure 9 ) show antiarrhythmic properties, indicating an interaction with RyR.

[0346] References

[0347] Throughout the application, various references describe the situation in the field to which the present invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0348] 1. Kushnir A, Marks AR. Ryanodine receptor patents. Recent Pat Biotechnol. 2012 Dec; 6(3): 157 - 66.

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[0350] 3. Levine S, Nguyen T, Taylor N, Friscia ME, Budak MT, Rothenberg P, Zhu J, Sachdeva R, Sonnad S, Kaiser LR, Rubinstein NA, Powers SK, Shrager JB. Rapid disuse atrophy of diaphragm fibers in mechanically ventilated humans. N Engl J Med. 2008 Mar 27; 358(13): 1327 - 35.

[0351] 4. Jaber S, Jung B, Matecki S, Petrof BJ. Clinical review: ventilator - induced diaphragmatic dysfunction - - human studies confirm animal model findings!Crit Care. 2011 Mar 11; 15(2): 206.

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[0354] 7. Esteban A, Ferguson ND, Meade MO, Frutos-Vivar F, Apezteguia C, Brochard L, Raymondos K, Nin N, Hurtado J, Tomicic V, González M, Elizalde J, Nightingale P, Abroug F, Pelosi P, Arabi Y, Moreno R, Jibaja M, D'Empaire G, Sandi F, Matamis D, AM, Anzueto A; VENTILA Group. Evolution of mechanical ventilation in response to clinical research. Am J Respir Crit Care Med. 2008 Jan 15;177(2):170-7.

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[0356] 9. Falk DJ, Kavazis AN, Whidden MA, et al. Mechanical ventilation-induced oxidative stress in the diaphragm: role of heme oxygenase-1. Chest. 2011;139(4):816-824. doi:10.1378 / chest.09-2787.

[0357] 10. Zergeroglu MA, McKenzie MJ, Shanely RA, Van Gammeren D, DeRuisseau KC, Powers SK. Mechanical ventilation-induced oxidative stress in the diaphragm. J Appl Physiol(1985). 2003 Sep;95(3):1116-24.

[0358] 11. Betters JL, Criswell DS, Shanely RA, Van Gammeren D, Falk D, Deruisseau KC, Deering M, Yimlamai T, Powers SK. Trolox attenuates mechanical ventilation-induced diaphragmatic dysfunction and proteolysis. Am J Respir Crit Care Med. 2004 Dec 1;170(11):1179-84.

[0359] 12. Picard M, Jung B, Liang F, Azuelos I, Hussain S, Goldberg P, Godin R, Danialou G, Chaturvedi R, Rygiel K, Matecki S, Jaber S, Des Rosiers C, Karpati G, Ferri L, Burelle Y, Turnbull DM, Taivassalo T, Petrof BJ. Mitochondrial dysfunction and lipid accumulation in the human diaphragm during mechanical ventilation. Am J Respir Crit Care Med. 2012 Dec 1;186(11):1140 - 9.

[0360] 13. Kavazis AN, Talbert EE, Smuder AJ, Hudson MB, Nelson WB, Powers SK. Mechanical ventilation induces diaphragmatic mitochondrial dysfunction and increased oxidant production. Free Radic Biol Med. 2009 Mar 15;46(6):842 - 50.

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[0362] 15. Matecki S, Dridi H, Jung B, Saint N, Reiken SR, Scheuermann V, Mrozek S, Santulli G, Umanskaya A, Petrof BJ, Jaber S, Marks AR, Lacampagne A. Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation. Proc Natl Acad Sci USA. 2016 Aug 9;113(32):9069 - 74.

[0363] 16. Maes K, Testelmans D, Powers S, Decramer M, Gayan - Ramirez G. Leupeptin inhibits ventilator - induced diaphragm dysfunction in rats. Am J Respir Crit Care Med. 2007 Jun 1;175(11):1134 - 8.

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[0366] 19. Matecki S, Jung B, Saint N, Scheuermann V, Jaber S, Lacampagne A. Respiratory muscle contractile inactivity induced by mechanical ventilation in piglets leads to leaky ryanodine receptors and diaphragm weakness. J Muscle Res Cell Motil. 2017 Feb;38(1):17-24.

[0367] 20. Dridi H, Yehya M, Barsotti R, Reiken S, Angeba ult C, Jung B, Jaber S, Marks AR, Lacampagne A, Matecki S. Mitochondrial oxidative stress induces leaky ryanodine receptor during mechanical ventilation. Free Radic Biol Med. 2020 Jan;146:383-391.

[0368] 21. Roy J, Oger C, Thireau J, Roussel J, Mercier-Touzet O, Faure D, Pinot E, Farah C, Taber DF, Cristol JP, Lee JC, Lacampagne A, Galano JM, Durand T, Le Guennec JY. Nonenzymatic lipid mediators, neuroprostanes, exert the antiarrhythmic properties of docosahexaenoic acid. Free Radic Biol Med. 2015 Sep;86:269-78.

[0369] 22. Oger C, Bultel-Poncé V, Guy A, Balas L, Rossi JC, Durand T, Galano JM. The handy use of Brown's P2-Ni catalyst for a skipped diyne deuteration: application to the synthesis of a [D4]-labeled F4t-neuroprostane. Chemistry. 2010 Dec 17;16(47):13976-80.

[0370] 23. Bellinger AM, Reiken S, Carlson C, Mongillo M, Liu X, Rothman L, Matecki S, Lacampagne A, Marks AR. Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med. 2009 Mar;15(3):325-30.

[0371] 24. Andersson DC, Betzenhauser MJ, Reiken S, Meli AC, Umanskaya A, Xie W, Shiomi T, Zalk R, Lacampagne A, Marks AR. Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging. Cell Metab. 2011 Aug 3;14(2):196 - 207.

[0372] 25. Donoso P, Sanchez G, Bull R, Hidalgo C. Modulation of cardiac ryanodine receptor activity by ROS and RNS. Front Biosci (Landmark Ed). 2011 Jan 1;16:553 - 67.

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Claims

1. A method for treating a disease associated with RyR dysfunction in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof: Wherein: R1 represents H or OH, R2 represents a straight-chain C1-C 10 alkyl or C2-C 10 alkenyl, The condition is that when R2 represents R1 represents H.

2. The method according to claim 1, wherein R2 in the compound of formula I is a group selected from formula a, b and c:

3. The method according to claim 1 or 2, wherein the compound of formula I is selected from: The compound of formula I has the structure of formula II, formula III, formula IV, formula S-IV or formula R-IV.

4. The method according to any one of claims 1-3, wherein the disease associated with RyR dysfunction is ventilator-induced diaphragmatic dysfunction.

5. The method according to claim 4, wherein the subject requires mechanical ventilation support because they suffer from respiratory failure and / or heart failure, which may be exacerbated by sepsis, metabolic disorders, neuromuscular diseases or surgery and postoperative recovery.

6. The method according to claim 4, wherein the subject suffers from a disease that has deteriorated to the point where the subject requires mechanical ventilation, in particular, Wherein 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. The method according to claim 6, wherein the severe acute respiratory syndrome is COVID-19.

8. The method according to claim 4, wherein the subject has suffered trauma.

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

10. The method according to any one of claims 1-3, wherein 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, skeletal disorders and diseases, malignant hyperthermia, diabetes, sudden cardiac death, and sudden infant death syndrome. In particular, wherein the cardiac disorder is selected from the group consisting of: exercise-induced irregular heartbeat disorders and diseases, heart failure, congestive heart failure, chronic heart failure, acute heart failure, systolic heart failure, diastolic heart failure, acute decompensated heart failure, cardiac ischemia / reperfusion (I / R) injury (including I / R injury after coronary angioplasty or thrombolysis during myocardial infarction (MI)), chronic obstructive pulmonary disease, hypertension, and arrhythmia disorders and diseases such as atrial and ventricular arrhythmias, atrial and ventricular fibrillation, atrial and ventricular tachyarrhythmias, atrial and ventricular tachycardia, catecholaminergic polymorphic ventricular tachycardia (CPVT) and its exercise-induced variants. In particular, wherein the musculoskeletal disorders and diseases are selected from the group consisting of: skeletal muscle fatigue, central core disease, exercise-induced skeletal muscle fatigue, bladder disorders, incontinence, age-related muscle fatigue, sarcopenia, congenital myopathy, cancer cachexia, core and rod myopathy, mitochondrial myopathy [e.g., Kearns-Sayre syndrome, MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) syndrome, and MERRF (myoclonic epilepsy with ragged red fibers) syndrome], endocrine myopathy, glycogen storage myopathy [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, neuromyotonia, stiff syndrome, malignant hyperthermia, common muscle cramps, tetany, myasthenia gravis, and muscular dystrophy. In particular, wherein 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 dystrophy, myotonic muscular dystrophy, Emery-Dreifuss muscular dystrophy, and oculopharyngeal muscular dystrophy, or wherein the cognitive dysfunction is selected from the group consisting of: Alzheimer's disease (AD), memory loss, age-related memory loss, post-traumatic stress disorder (PTSD), neuropathy, 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.

11. A compound of formula IA or a pharmaceutically acceptable salt thereof: Wherein: R1 represents H or OH, R2 represents a straight-chain C1-C 10 alkyl group, a C2-C7 alkenyl group containing one double bond, or a C2-C 10 alkenyl group containing two double bonds, The condition is that when R2 represents R1 represents H, In particular, wherein R2 represents a group selected from formulas a, b, and c:

12. The compound according to claim 11, wherein the compound of formula IA is selected from: The compound of formula IA has a structure of formula IIA, formula IIIA, formula IVA, formula S-IVA or formula R-IVA.

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

14. A pharmaceutical composition comprising a compound of formula I or a compound of formula IA or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient, wherein the compound of formula I is as defined in any one of claims 1-3, and the compound of formula IA is as defined in any one of claims 11 or 12.

15. A method for preparing a compound of formula I, wherein the compound of formula I is as defined in any one of claims 1-3, and the method comprises: Step A, oxidizing the alcohol of formula V to obtain the aldehyde of formula VI, wherein R3 is an alcohol protecting group, particularly a silyl protecting group, more particularly a tert-butyldimethylsilyl protecting group, R4 is methyl or ethyl, R5 is H or OR3, where R3 is as defined above; Step B, olefinating the aldehyde of formula VI to obtain the olefin of formula VII, wherein R3, R4 and R5 are as defined in Step A, and R2 is as defined for formula I in claims 1-3; Step C, deprotecting the hydroxyl group in formula VII to obtain the ester of formula VIII wherein R4 and R5 are as defined in Step A, and R1 and R2 are as defined for formula I in claims 1-3; Step D, hydrolyzing the ester of formula VIII to obtain the compound of formula I, wherein R4 is as defined in Step A, and R1 and R2 are as defined for formula I in claims 1-3.

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