Succinic acid prodrug, composition containing the succinic acid prodrug, and use thereof

Abstract

The present invention provides novel isolated succinic acid prodrugs as free compounds or their salts, hydrates, solvates, or complexes that are cell-permeable and intended to increase mitochondrial ATP production, and are useful in the pharmacological treatment of various diseases, nutritional supplements, nutricosmetics, and cosmetics.

Description

Technical Field

[0001] (Field of the Invention) The present invention relates to the fields of chemistry, pharmacologically active compounds, pharmaceutical compositions containing such compounds, and nutrition. Specifically, the present invention relates to cell-permeable precursors of succinic acid useful as pharmaceuticals and nutritional supplements.

Background Art

[0002] (Background of the Invention) Mitochondria are eukaryotic cell organelles that generate most of the cellular supply of adenosine triphosphate (ATP) used as an energy source. Thus, mitochondria are essential for energy production, the survival of eukaryotic cells, and proper cell function. In addition to energy supply, mitochondria are involved in several other processes, such as redox and ion balance, cell signaling, cell differentiation, cell death, and the regulation of metabolic processes, the cell cycle, and cell proliferation. In particular, mitochondria are important regulators of cell apoptosis, which also plays a major role in various forms of non-apoptotic cell death, such as necrosis.

[0003] Mitochondrial dysfunction is a cause of a wide variety of diseases and can be caused by mutations or deletions in the mitochondrial or nuclear genome, primary or secondary impairment of the mitochondrial respiratory system, or other mechanisms related to abnormal mitochondrial function. Currently, there are no available treatments that can cure mitochondrial diseases.

[0004] The oxidation of nutrients to produce usable chemical energy in the form of ATP occurs mostly in the mitochondria through a series of chemical reactions in the tricarboxylic acid cycle and the electron transport system. NADH produced in the tricarboxylic acid cycle is supplied to Complex I of the electron transport system. Succinate is a metabolic intermediate of the tricarboxylic acid cycle in the mitochondria and is unique in that it is directly metabolized by the enzyme succinate dehydrogenase of Complex II of the electron transport system. Succinate can also act as a signaling molecule that reflects the metabolic state of the cell.

[0005] Considering the recognized importance of treating diseases and disorders associated with mitochondrial dysfunction or enhancing mitochondrial function by maintaining or restoring normal mitochondrial function or enhancing cellular energy production (ATP), there is a need for compounds having cell permeability, the ability to liberate intracellular succinate or a precursor of succinate, low toxicity of the compounds and by-products released intracellularly, and physicochemical properties compatible with administration to a subject or patient.

[0006] Succinate compounds have been prepared as prodrugs of other active agents. For example, WO 2002 / 28345 describes succinate bis(2,2-dimethylpropionyloxymethyl) ester, succinate dibutyryloxymethyl ester, and succinate bis-(1-butyryloxy-ethyl) ester. These compounds in their prepared state release formaldehyde and are intended for different medical uses compared to current compounds.

[0007] Various succinate ester compounds are known in the art.

[0008] WO97 / 47584 discloses succinate polyols containing a number of linked succinate moieties.

[0009] WO2015 / 155231 discloses cell-permeable succinate and precursors of succinate.

[0010] The literature of Murli et al. (Appl. Environ. Microbiol. 71:2005:4503-4509) discloses attempts at the chemical synthesis of 6-deoxyerythronolide B analogs by supplying acyl-thioesters to the bacteria Escherichia coli and Streptomyces coelicolor. The structural table discloses various acyl-thioesters including the formal structure of methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate, but the synthesis failed to produce the desired product.

[0011] There is a need for effective and safe new therapeutic options for diseases originating from mitochondrial dysfunction or for enhancing metabolism by supplying metabolic substrates. There is also a need for new nutritional supplements, nutricosmetics, pharmaceuticals, and cosmetics that function as energy stimulants and antioxidants in a subject. Such new therapeutics, nutritional supplements, nutricosmetics, pharmaceuticals, and cosmetics are required to have an attractive combination of properties including high activity for enhancing mitochondrial energy production and / or functioning as an antioxidant, excellent bioavailability, long plasma half-life, stability when formulated into a product, and low toxicity. In particular, there is a need for such new therapeutics, nutritional supplements, nutricosmetics, pharmaceuticals, and cosmetics based on active ingredients having high solubility in water, excellent cell permeability, and, if desired, high blood-brain barrier permeability and / or resulting in a decrease in lactic acid production. SUMMARY OF THE INVENTION

[0012] (Summary of the Invention) In a first aspect, the present invention provides isolated methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in solid form. This may be in free form or its salt, hydrate, solvate, or complex.

[0013] Compound 1 has the structure (Formula 1):

Chem.

[0014] Currently, surprisingly, it has been found that the cell-permeable Compound 1 has a remarkable combination of advantageous properties. It has strong in vivo activity that stimulates energy production in mitochondria, excellent oral bioavailability, blood-brain barrier permeability, excellent plasma stability, reduces lactate production, and restores succinate levels. At the same time, Compound 1 has a significantly high solubility in water and aqueous systems. Solubility evaluation showed a solubility exceeding 500 mg / mL, corresponding to ~2.1 M. This extremely high water solubility is likely due to the low melting point of Compound 1 (less than 55 °C), and when an aqueous solvent is added, it can be miscible with the aqueous solvent. This enables extremely high-concentration aqueous formulations and actually enables high oral administration of compounds that become prodrugs of metabolic substrates.[

[0015] In certain embodiments, the isolated Compound 1 is a solid product having a melting point or melting range in the range of about 35 °C to about 55 °C. In preferred embodiments, the isolated Compound 1 has a purity of at least 80% w / w, for example, at least 85% w / w, at least 90% w / w, for example, at least 95% w / w, although it may have a lower purity, for example, at least 30% w / w, at least 40% w / w, at least 45% w / w, at least 50% w / w, at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, or at least 75% w / w. Depending on the manufacturing method and storage conditions, Compound 1 may contain crystals and / or it may contain amorphous forms such as the amorphous form of Compound 1, and mixtures thereof. As can be seen from the examples herein, all of the methods used yield Compound 1 with a certain degree of crystallinity.[

[0016] It is contemplated that multiple crystalline forms of Compound 1 may exist, and Compound 1 may also exist as an amorphous solid. In this regard, all forms of Compound 1, including mixtures of two or more forms of Compound 1, are within the scope of this application. Thus, the term "Compound 1" means the compound of Formula 1 in solid form, whether the compound is in crystalline form, amorphous form, polymorphic form, powder form, or a mixture thereof.

[0017] In particular, Compound 1 has been found to have several solid forms with different properties. For example, as an amorphous solid or predominantly as an amorphous solid, it exhibits a higher kinetic solubility. It also has a crystalline form (or predominantly crystalline form) that exhibits other improved properties when produced and processed as a solid form. It has also been found that purity affects the properties of the preparation. In particular, the melting point is low, close to body temperature, and is changed by the presence of impurities. The stability of the preparation of Compound 1 is also changed by the presence of impurities that lower the stability of Compound 1, such as impurities in non-purified water.

[0018] In a second aspect, the present invention provides a composition comprising isolated Compound 1.

[0019] In a third aspect, the present invention provides a pharmaceutical cosmetic comprising isolated Compound 1.

[0020] In a fourth aspect, the present invention provides a nutricosmetic comprising isolated Compound 1.

[0021] In a fifth aspect, the present invention is a process for preparing isolated Compound 1, for providing isolated Compound 1, a) reacting N-acetylcysteamine and monomethyl succinate in an organic solvent at 0 °C to 100 °C in the presence of a coupling reagent; b) isolating Compound 1; : comprising a process.

[0022] This method usually includes a purification step to increase the purity of the compound.

[0023] Using the compound of Compound 1 of the present invention, the energy production in mitochondria can be enhanced or restored. In particular, this compound can be used in pharmaceuticals, nutricosmetics, nutritional supplements, medicinal cosmetics, and cosmetics. The compound of Compound 1 can be used in the prevention or treatment of disorders or diseases having components related to mitochondrial dysfunction and / or components lacking energy (ATP), and in order to utilize its anaplerotic effect on the cell signaling properties and metabolic intermediates of succinic acid.

[0024] Furthermore, compared with known succinic acid prodrugs (such as those mentioned in WO 97 / 47584), the isolated Compound 1 of the present invention exhibits improved properties for treatment and for use as nutritional supplements and cosmetics, including better cell permeability, longer plasma half-life, excellent oral bioavailability, reduced toxicity, increased energy release to mitochondria, and improved formulation properties, for example, due to improved solubility in water.

[0025] In another aspect, the present invention provides a pharmaceutical composition containing the compound of Compound 1.

[0026] The pharmaceutical composition may be a solid preparation, or it may be a solid preparation that is reconstituted before use.

[0027] Alternatively, it may be in the form of a liquid, such as an aqueous solution, including, for example, an aqueous phosphate buffered solution (PBS) formulation. Generally, the pharmaceutical composition of the present invention has Compound 1 at a concentration of at least 10% w / w, at least 30% w / w, at least 50% w / w, at least 60%, or at least 70% w / w. In certain embodiments, the pharmaceutical composition is optionally a solution of Compound 1 in purified water isotonic with blood.

[0028] In another aspect, the present invention provides the use of Compound 1 or a composition thereof in the treatment or prevention of metabolic diseases, mitochondrial dysfunction diseases, diseases associated with mitochondrial dysfunction, mitochondrial disorders, mitochondrial energy deficiency, drug-induced mitochondrial side effects, cancer, diabetes, traumatic brain injury, acute liver injury, and atrial fibrillation.

[0029] In one aspect, the present invention provides a process for preparing a pharmaceutical composition comprising Compound 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] (Brief Description of the Drawings)

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Mode for Carrying Out the Invention

[0031] (Description of the Invention) In a first aspect, the present invention provides an isolated methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in solid form, which may be in free form or its salt, hydrate, solvate, or complex.

[0032] Compound 1 has the structure (Formula 1):

Chemical Formula

[0033] As described above, Compound 1 may be in the form of a salt. Suitable salts include pharmaceutically acceptable salts such as hydrochloride, hydrobromide, acetate, citrate, lactate, maleate, malonate, and the like.

[0034] Compound 1 may also be a solvate. Suitable solvates include hydrates and ethanolates.

[0035] Compound 1 may also be in the form of a complex. Examples of suitable complexes may be Compound 1 complexed with cyclodextrin, lipid, triglyceride, carbohydrate, PVA.

[0036] In one embodiment, the isolated compound 1 is a solid product having a melting point or melting range in the range of about 35°C to about 55°C. As can be seen from the examples herein, compound 1 likely has different melting points depending on, for example, the content of different forms of compound 1 such as crystalline form, amorphous form, etc. In particular, melting points in the range of 39 - 51°C, for example, a melting point of 39°C, and melting points in the range of 46 - 51°C, for example, about 46 - 47°C, 48 - 49°C, and 50 - 51°C have been found.

[0037] In a preferred embodiment, the isolated compound 1 has a purity of at least 80% w / w, for example, at least 85% w / w, at least 90% w / w, for example, at least 95% w / w, or at least 97% w / w, although it may have a lower purity, for example, at least 30% w / w, at least 40% w / w, at least 45% w / w, at least 50% w / w, at least 55% w / w, at least 60% w / w, at least 65% w / w, at least 70% w / w, or at least 75% w / w. Depending on the manufacturing method and storage conditions, compound 1 may contain crystals and / or it may contain non-crystals such as the amorphous form of compound 1, and mixtures thereof. As can be seen from the examples herein, all of the methods used yield compound 1 with a certain degree of crystallinity. Compound 1 can also appear as a powder.

[0038] As can be seen from the examples herein, compound 1 has excellent water solubility at room temperature (20 - 25°C). At pH 7.4 and in the aqueous media tested in the examples, compound 1 has a water solubility of at least 300 mg / mL. The water solubility of compound 1 is dependent on the crystallinity of the compound; thus, the lower the crystallinity, the higher the water solubility. As can be seen from Example 10 herein, a predominantly amorphous material can have a water solubility of 850 mg / mL. Therefore, it is assumed that the water solubility of compound 1 is in the range of 300 mg / mL to about 900 mg / mL.

[0039] The dynamic solubility has also been determined, and the rate constant of the dynamic solubility is in the range of 0.005 to 0.2 s -1 For example, in the range of 0.01 to 0.15 s -1 It is known that the dynamic solubility is determined by various factors such as, for example, particle size, crystallinity, and content of amorphous material.

[0040] Regarding the crystallinity of Compound 1, this can have a degree of crystallinity in the range of 0% to 100%, for example, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%. As can be seen from the examples herein, many of the batches prepared by the methods described herein have a crystallinity in the range of at least 50%, for example, about 50% to about 80%.

[0041] As can be seen from the XRPD data herein, the crystals of Compound 1 are characterized by having an X-ray powder diffraction pattern with signals at 21.4, 22.2, 22.8, 23.1, and 23.3 (±0.2 degrees, 2-theta value).

[0042] The crystals of Compound 1 may also have one or more signals, for example, 2 or more, 3 or more, 4 or more, 6 or more, 7 or more, or 8 signals at 10.9, 13.1, 14.9, 16.2, 20.1, 24.0, 24.8, 26.1. As can be seen from the examples, almost all of the compounds tested have signals at these degrees (±0.2 degrees, 2-theta value).

[0043] From the data of the examples, it is assumed that the signals at 11.1 and 16.9 (±0.2 degrees, 2-theta value) are related to the polymorph (Form 1) of Compound 1. Therefore, the crystals of Compound 1 may have an X-ray powder diffraction pattern with signals at 11.1 and 16.9 (±0.2 degrees, 2-theta value) by supplementing one or more of the above signals or in another form.

[0044] As described above, Compound 1 is in a solid form, especially including the crystals of the compound. The melting point is relatively low, but it is advantageous that Compound 1 is not in the form of an oil. First of all, in the manufacture of pharmaceutical / cosmetic compositions, it is easy to process Compound 1 (e.g., grindability, powder flowability, and compressibility). Secondly, the crystalline form is usually the most stable form, and amorphous (less ordered) materials tend to change their form to crystalline (more ordered and lower energy) over time.

[0045] (Definition:) The term "Compound 1" means the compound of Formula 1 in solid form, and this term includes all crystalline forms, all amorphous forms, all polymorphic forms, and mixtures thereof, whether in the same form or different forms. Compound 1 may also be in powder form.

[0046] The term "purity" as used herein with respect to Compound 1 means the extent to which the composition of Compound 1 is methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) relative to the total amount of Compound 1, by-products, abnormal forms (structurally closely related) of Compound 1, and related impurities that are synthetic precursors of Compound 1. Thus, in a composition containing 10% w / w of Compound 1, the purity of the Compound 1 may be, for example, 95% w / w or 50% w / w, meaning that the Compound 1 used to prepare the composition has a purity of 95% w / w or 50% w / w, respectively. Purity can be evaluated by one of several methods including qNMR, HPLC, etc. In qNMR, a known amount of the analyte is dissolved in an NMR solvent together with a known amount of an internal standard. 1The 1H NMR spectrum is obtained using scans sufficient to reduce the signal-to-noise ratio. Integrate exemplary resonances in the internal standard and the analyte. Then, using these integration values and the ratio of the molecular weights of both the analyte and the internal standard, in addition to knowledge of how many protons are included in the signal, determine the purity (units are w / w%). In HPLC, the purity is evaluated as the area under the curve (AUC) of the analyte compared to other signals with different retention times.

[0047] As used herein with respect to Compound 1, the term "isolated" means the Compound 1 product methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate, which is obtained from a synthetic reaction and isolated by purification, for example, from various by-products, synthetic precursors, and abnormal Compound 1 forms.

[0048] As used herein, the term "nutricosmetics" refers to nutritional supplements or cosmetics that are specially formulated to maintain healthy skin, hair, and nails using active ingredients that maintain physiological functions in order to achieve a healthier and more youthful appearance over time. Unlike topical creams or topical treatments, nutricosmetics are taken orally and act internally to promote healthy skin, hair, and nails from the inside.

[0049] As used herein, the term "cosmeceuticals" is intended to mean cosmetics having bioactive ingredients said to have medical benefits. Cosmeceutical products are marketed as cosmetics but reputedly contain at least one bioactive ingredient. Examples of cosmeceuticals include anti-wrinkle skin creams containing ingredients such as alpha lipoic acid and dimethylaminoethanol and creams containing a "cell replenishing serum" labeled as having "anti-aging properties".

[0050] As used herein, the term "treatment" is intended to mean the performance of a treatment with the intention of reducing the severity or frequency of symptoms. As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures.

[0051] As used herein, the term "prevention" is intended to mean preventing, wholly or in part, or ameliorating, reducing, or controlling.

[0052] Compound 1 is comprehensively included by formula (I) of WO2015 / 155231, which discloses cell-permeable succinic acid and precursors of succinic acid. However, with the identification of Compound I, the inventors have made several surprising new discoveries and have revealed that the compound I has an unexpected combination of excellent properties that make it suitable for several therapeutic and non-therapeutic uses. Furthermore, surprising discoveries have been made around the advantages of specific forms and formulations of Compound I.

[0053] (General use of the compounds of the invention) Methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1), which is in free form or its salt, hydrate, solvate, or complex as described in this specification, can be used in medicine, particularly in the medical treatment or prevention of mitochondrial-related diseases, disorders, or impairments, in nutricosmetics, or in cosmetics. Compound I can also be used in the manufacture of a composition for such medical treatment or prevention, nutricosmetics, or cosmetics. Compound 1, or its salt, hydrate, solvate, or complex, can be used in any situation where enhancement or restoration of energy production (ATP) is desired, for example, in the medical treatment of diseases. The medical treatment can be related to metabolic diseases, diseases or disorders of mitochondrial dysfunction, or diseases associated with a decrease in the level of succinic acid or the functional activity of succinic acid, or in the treatment of diseases where the anaplerotic effect of succinic acid or its signal transduction properties are useful, or in treating or suppressing mitochondrial disorders. The compound of Compound 1 can be used in stimulating mitochondrial energy production and in the recovery of drug- or chemical-induced mitochondrial dysfunction, such as sensorineural hearing loss or tinnitus (side effects of certain antibiotics due to mitochondrial toxicity), poisoning by chemicals or gases that affect mitochondrial metabolism, or lactic acidosis. The compound can be used in the treatment of cancer, diabetes, acute starvation, endotoxemia, sepsis, systemic inflammatory response syndrome, multiple organ dysfunction syndrome and subsequent hypoxia, ischemia, stroke, myocardial infarction, acute angina, acute kidney injury, coronary artery occlusion, and atrial fibrillation, or in avoiding or eliminating reperfusion injury. Furthermore, the compounds of the present invention are expected to be beneficial in the treatment of male infertility and female menopausal symptoms.

[0054] The compounds of Compound 1 of the present invention are expected to provide cell-permeable precursors of components of the Krebs cycle and optionally the glycolytic pathway. After entering the cell, succinic acid is expected to be released by enzymatic or chemical hydrolysis. This hydrolysis of Compound 1 is considered particularly advantageous because the released thiol groups have reducing properties. Many diseases have unwanted oxidative stress components that can damage cell structure and cell function. Oxidative stress is also thought to be involved in the aging process. Therefore, the release of components that can act as antioxidants and scavenge free radicals or reduce oxygen reactive species is expected to provide additional benefits in both medical, nutricosmetic, and cosmetic applications.

[0055] Compound 1 can be used to enhance or restore energy production in mitochondria. Compound 1 can also be used as an antioxidant to scavenge free radicals or reduce oxygen reactive species. Compound 1 can be used in the prevention or treatment of disorders or diseases having components related to mitochondrial dysfunction and / or components of energy (ATP) deficiency, and diseases associated with a decrease in the level of succinic acid or its functional activity, or diseases in which the anaplerotic effect of succinic acid or its signaling properties are useful.

[0056] Enhancement of energy production is important, for example, in subjects suffering from mitochondrial defects, disorders, or diseases. Mitochondrial diseases result from the dysfunction of mitochondria, which are specialized compartments present in all cells of the body except red blood cells. When mitochondrial function decreases, the energy produced within the cell decreases, resulting in cell injury or cell death.

[0057] Mitochondrial diseases most often occur in organs that require a great deal of energy, such as the retina, cochlea, brain, heart, liver, skeletal muscle, kidneys, and the endocrine and respiratory systems. Symptoms of mitochondrial diseases can include loss of motor control, muscle weakness, and pain, seizures, visual / auditory problems, heart disease, liver disease, gastrointestinal disorders, dysphagia, fatigue, and many others. Mitochondrial diseases can be hereditary or due to spontaneous mutations that cause changes in the function of proteins or RNA molecules normally present in mitochondria. Many diseases have been found to be related to mitochondrial deficiencies, such as deficiencies in Complex I, II, III, or IV or enzyme deficiencies such as pyruvate dehydrogenase deficiency. However, the reality is complex and many factors may be involved in this disease.

[0058] So far, curative treatments have not been available. The only treatments available are those that can relieve symptoms and slow the progression of the disease.

[0059] Therefore, the discovery described herein by the inventors is very important as it shows the beneficial effects of the cell-permeable compound 1, a thioester prodrug of succinic acid, on energy production in mitochondria.

[0060] Furthermore, in comparison with known succinic acid prodrugs (such as those mentioned in WO 97 / 47584), the compound of the isolated compound 1 of the present invention exhibits improved properties for medical treatment, as well as for use in nutricosmetics, nutritional supplements, pharmaceutical cosmetics, and cosmetics, including better cell permeability, longer plasma half-life, reduced toxicity, increased energy release into mitochondria, and improved formulation (due to improved properties including increased solubility). In some cases, the compound of the isolated compound 1 is also orally bioavailable, which allows for easier administration.

[0061] Accordingly, advantageous properties of the isolated compounds of the present invention can include one or more of the following: - Increased cell permeability - Increased oral bioavailability - Longer half-life in plasma - Reduced toxicity - Increased energy release to mitochondria - Increased antioxidant activity - Improved formulation - Increased solubility

[0062] The present invention particularly provides compound 1 for use as a pharmaceutically active substance in medicaments, etc., especially in the treatment of cellular energy (ATP) deficiency.

[0063] The compounds of the present invention can be used in the treatment of complex I disorders, which can be due to either dysfunction of the complex itself or any disease or disorder that limits the supply of NADH to complex I, such as dysfunction in the Krebs cycle, glycolysis, β-oxidation, pyruvate metabolism, and even the transport of glucose or complex I-related substances.

[0064] The present invention also provides a method for treating mitochondrial complex I-related disorders such as Leigh syndrome, Leber's hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), mitochondrial deletion syndrome, mitochondrial myopathy, and MERRF (myoclonic epilepsy with ragged red fibers), which comprises administering to a subject in need thereof an effective amount of a compound of the present invention.

[0065] The present invention also provides the use of a compound of the present invention for the manufacture of a medicament for the treatment of toxin- or drug-induced lactic acidosis / mitochondrial dysfunction.

[0066] Isolated compound 1 may be useful in any condition where additional energy production is potentially beneficial, such as, but not limited to, in prolonged surgical procedures and intensive care.

[0067] (mitochondria) Mitochondria are organelles of eukaryotic cells and are generally referred to as the "powerhouses" of the cell. One of their main functions is oxidative phosphorylation. The molecule adenosine triphosphate (ATP) functions as the energy "currency" or energy carrier within the cell, and eukaryotic cells obtain most of their ATP from biochemical reactions carried out by mitochondria. These biochemical processes include the citric acid cycle (tricarboxylic acid cycle, or Krebs cycle) that generates reduced nicotinamide adenine dinucleotide (NADH) from oxidized nicotinamide adenine dinucleotide (NAD + ) and reduced flavin adenine dinucleotide (FADH2) from oxidized flavin adenine dinucleotide (FAD), as well as oxidative phosphorylation during which NADH and FADH2 are oxidized back to NAD + and FAD.

[0068] The electrons released by the oxidation of NADH are passed on to a series of protein complexes known as the electron transport system or respiratory chain (Complex I, Complex II, Complex III, and Complex IV). The oxidation of succinate occurs at Complex II (succinate dehydrogenase complex), and FAD is a prosthetic group in the enzyme complex succinate dehydrogenase (Complex II). The respiratory complexes are embedded in the inner membrane of the mitochondria. Complex IV at the end of this chain transfers electrons to oxygen, which is reduced to water. The energy released as these electrons pass through the complexes is used to generate a proton gradient across the inner membrane of the mitochondria, which creates an electrochemical potential across the inner membrane. Another protein complex, Complex V (which is not directly related to Complexes I, II, III, and IV), uses the energy stored by the electrochemical gradient to convert ADP to ATP.

[0069] Before the citric acid / tricarboxylic acid cycle and oxidative phosphorylation, glycolysis occurs in which one molecule of glucose is broken down into two molecules of pyruvate. In glycolysis, two molecules of ATP are net produced per molecule of glucose. Subsequently, the pyruvate molecules enter the mitochondria where they are completely oxidized to CO2 and H2O by oxidative phosphorylation (this entire process is known as aerobic respiration). The complete oxidation of two molecules of pyruvate to carbon dioxide and water yields at least about 28 - 29 molecules of ATP in addition to the two molecules of ATP produced by the conversion of glucose to two molecules of pyruvate. When oxygen is not available, the pyruvate molecules do not enter the mitochondria but rather are converted to lactate in the process of anaerobic respiration.

[0070] Thus, the net total yield per molecule of glucose is at least about 30 - 31 ATP molecules. ATP is used to power almost all other biochemical reactions within the cell, either directly or indirectly. Thus, the additional (approximate) at least 28 or 29 molecules of ATP contributed by oxidative phosphorylation during aerobic respiration are extremely important for the proper functioning of the cell. A lack of oxygen inhibits aerobic respiration and ultimately leads to death in almost all aerobic organisms; a few organisms such as yeast can survive using either aerobic or anaerobic respiration.

[0071] When oxygen is temporarily deprived from the cells of an organism, anaerobic respiration is utilized until oxygen can be utilized again or the cells die. The pyruvate produced during glycolysis is converted to lactate during anaerobic respiration. The accumulation of lactate is thought to be the cause of muscle fatigue during periods of intense activity when oxygen cannot be supplied to muscle cells. Once oxygen can be utilized again, the lactate is converted back to pyruvate for use in oxidative phosphorylation.

[0072] Mitochondrial dysfunction is a contributing factor in various disease states. Some mitochondrial diseases are due to mutations or deletions in the mitochondrial genome or the nucleus. If there are defects in the threshold ratio of mitochondria within a cell, and the threshold ratio of such cells within a tissue has mitochondria with defects, symptoms of tissue or organ dysfunction can occur. In fact, any tissue can be affected, and depending on the degree to which various tissues are involved, a wide variety of symptoms can exist.

[0073] (Use of the compounds of the present invention) The compounds of the present invention can be used in any situation where enhancement or restoration of energy production (ATP) is desirable. Examples are, for example, in all clinical diseases where there is a potential benefit in increasing mitochondrial ATP production or restoring mitochondrial function, such as drug- or chemically-induced mitochondrial dysfunction or lactic acidosis states associated with reduced levels of succinic acid or reduced functional activity of succinic acid, restoration of diseases where the anaplerotic effect of succinic acid or its signaling properties are useful, and in the treatment of inborn errors of metabolism, cancer, diabetes, acute starvation, endotoxemia, sepsis, reduced hearing and vision, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome.

[0074] In particular, Compound 1 can be used, inter alia, in pharmaceuticals for the treatment or prevention of mitochondrial-related diseases, disorders, or impairments, in nutricosmetics, or in cosmetics.

[0075] Mitochondrial dysfunction has also been described in relation to renal tubular acidosis; motor neuron disease; other neurological diseases; epilepsy; genetic diseases; Huntington's disease; mood disorders; schizophrenia; bipolar disorder; age-related diseases; cerebrovascular attack, macular degeneration; diabetes; menopausal symptoms, and cancer.

[0076] (Compound 1 for use in mitochondrial-related disorders or diseases) The compounds according to the present invention can be used in the prevention or treatment of mitochondrial-related diseases selected from the following: · Aging, · Alpers' disease (progressive infantile polyodystrophy), · Alzheimer's disease, · Amyotrophic lateral sclerosis (ALS), · Autism, · Barth syndrome (lethal infantile cardiomyopathy), · β-oxidation defect, bioenergetic metabolism deficiency, · Carnitine-acyl-carnitine deficiency, · Carnitine deficiency, · Creatine deficiency syndrome (Cerebral creatine deficiency syndrome (CCDS) includes guanidinoacetate methyltransferase deficiency (GAMT deficiency), L-arginine:glycine amidinotransferase deficiency (AGAT deficiency), and SLC6A8-related creatine transporter deficiency (SLC6A8 deficiency):), · Coenzyme Q10 deficiency, · Complex I deficiency (NADH dehydrogenase (NADH-CoQ reductase deficiency), · Complex II deficiency (succinate dehydrogenase deficiency), · Complex III deficiency (ubiquinone-cytochrome c oxidoreductase deficiency), · Complex IV deficiency / COX deficiency (Cytochrome c oxidase deficiency is caused by a defect in complex IV of the respiratory chain), · Complex V deficiency (ATP synthase deficiency), · COX deficiency, CPEO (chronic progressive external ophthalmoplegia syndrome), CPT I deficiency, · CPT II deficiency, · Type II diabetes, · Friedreich's ataxia (FRDA or FA), · Glutaric acidemia type II, · KSS (Kearns-Sayre syndrome), · Lactic acidosis, · LCAD (long-chain acyl-CoA dehydrogenase deficiency), ·LC-FAOD (Long-chain fatty acid oxidation disorder), ·LCHAD, Leigh disease or Leigh syndrome (subacute necrotizing encephalomyelopathy), ·LHON (Leber hereditary optic neuropathy), · Luft disease, ·MCAD (Medium-chain acyl-CoA dehydrogenase deficiency), ·MELAS (Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), ·MERRF (Myoclonic epilepsy and ragged-red fibers), · Methylmalonyl-CoA epimerase deficiency, · Methylmalonyl-CoA mutase deficiency, · Mitochondrial DNA depletion syndrome 5, · Mitochondrial DNA depletion syndrome 9, · Mitochondrial DNA depletion syndrome 15 (hepatoencephalic type) (1 family), · Maternal hereditary diabetes and deafness, ·MIRAS (Mitochondrial recessive ataxia syndrome), · Mitochondrial cytopathy, · Mitochondrial DNA depletion, · Encephalomyopathy and cerebrospinal disorder, mitochondrial myopathy: including mitochondrial encephalopathy, ·MNGIE (Myoneurogastrointestinal and encephalopathy, ·NARP (Neuropathy, ataxia, and retinitis pigmentosa), · Neurodegenerative disorders associated with Parkinson's disease, Alzheimer's disease, or Huntington's disease, · Parkinson's disease, · Pearson syndrome, · Progressive external ophthalmoplegia, · Propionic academia, · Pyruvate dehydrogenase deficiency, ·POLG mutation, · Respiratory chain deficiency, ·SCAD (Short-chain acyl-CoA dehydrogenase deficiency), ·SCHAD, and ·VLCAD (Very-long-chain acyl-CoA dehydrogenase deficiency).

[0077] Particularly interesting is the use of Compound 1 in the treatment of Leigh syndrome, LHON, MELAS, MERRF (myoclonic epilepsy with ragged red fibers), and other diseases / illnesses related to Complex I deficiency.

[0078] (Use of the compounds of the present invention in cosmetics) The compounds according to the present invention can be used in cosmetics for the following purposes: · Improvement of the metabolic function of skin cells (aging skin) · Astringents (acne)

[0079] (Use of the compounds of the present invention as nutritional supplements) The compounds according to the present invention can be used as nutritional supplements for the following purposes: · Increased energy demand due to intense physical activity · Increased energy demand due to metabolic decompensation during infection and surgery · Enhanced muscle recovery by rapid distribution to tissues and bypassing glycolysis

[0080] (Pharmaceutical compositions containing the compounds of the present invention) The present invention also provides a pharmaceutical composition comprising the isolated Compound 1 of the present invention together with one or more pharmaceutically acceptable diluents or carriers.

[0081] The compounds or their formulations of the present invention can be administered by any conventional method, for example, but not limited to, parenterally, orally, topically (including to mucous membranes, intraoral, sublingual, transdermal, or to the skin), by medical devices (such as stents), by inhalation, or by injection or infusion (intravenous, subcutaneous, intramuscular, etc.). Treatment can consist of a single dose or multiple doses over a certain period.

[0082] The treatment can be by administration once a day, twice a day, three times a day, four times a day, etc. The treatment can also be by continuous administration, such as intravenous administration by infusion.

[0083] Although it is possible to administer the compounds of the present invention alone, it is preferred to provide them as pharmaceutical formulations together with one or more acceptable carriers. A carrier must be "acceptable" in the sense of being compatible with the compounds of the present invention and not injurious to its recipient. Examples of suitable carriers are described in more detail below.

[0084] The formulations can conveniently be provided in dosage unit form and can be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active ingredient (the compound of the present invention) into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.

[0085] The compounds of the present invention are usually administered as a medicament in the form of pharmaceutical formulations containing the active ingredient, by intravenous, oral, or any parenteral route, optionally in the form of non-toxic organic or inorganic acids or addition salts, in a pharmaceutically acceptable dosage form. Depending on the disorder to be treated, the patient, and the route of administration, the composition can be administered in various dosages.

[0086] The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably, they should be protected from the contaminating action of microorganisms such as bacteria and fungi. Depending on the type of formulation selected and the route of administration, the carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0087] For example, the compounds of the present invention can be administered orally, buccally, or sublingually in the form of tablets, capsules, ovules, elixirs, gels, solutions, emulsions, or suspensions, which may contain a perfume or a colorant for immediate, delayed, or controlled release applications.

[0088] Formulations according to the present invention suitable for oral administration can be presented as individual units, such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion. The active ingredient can also be presented as a bolus, a lozenge, or a paste.

[0089] Solutions or suspensions of the compounds of the present invention suitable for oral administration may also contain excipients, such as solvents, such as water, ethanol, etc., N,N-dimethylacetamide, dispersants, such as polysorbate 80, surfactants, and solubilizers, such as polyethylene glycol, Phosal 50 PG (which consists of phosphatidylcholine, soybean fatty acids, ethanol, mono / diglycerides, propylene glycol, and ascorbyl palmitate). The formulations according to the present invention can also be in the form of an emulsion, where the compound of Compound 1 can be present in an oil-in-water or water-in-oil emulsion. The oil can be any oily substance, such as soybean oil, safflower oil, etc., triglycerides, such as medium-chain triglycerides (MCT oil), such as coconut oil, palm oil, etc., or a combination thereof.

[0090] The tablets may contain pharmaceutically acceptable excipients, such as bulking agents, binders, dispersants, disintegrants, lubricants, pH adjusters, stabilizers, flavoring agents, etc. Specific examples include microcrystalline cellulose, lactose (e.g., lactose monohydrate or anhydrous lactose), sodium citrate, calcium carbonate, dibasic calcium phosphate, and glycine, butylated hydroxytoluene (E321), crospovidone, hypromellose, disintegrants such as starch (preferably corn, potato, or tapioca starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates, and granulating binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy - propylcellulose (HPC), macrogol 8000, sucrose, gelatin, and gum arabic. Further, lubricants such as magnesium stearate, stearic acid, glyceryl behenate, and talc may be included.

[0091] The tablets can be prepared by compression or molding, optionally using one or more pharmaceutically acceptable excipients. Compressed tablets can be prepared by compressing, in a suitable machine, free - flowing forms of the active ingredient, such as powders or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate, cross - linked povidone, cross - linked sodium carboxymethylcellulose), a surfactant, or a dispersant. Molded tablets can be prepared by molding, in a suitable machine, a mixture of a powdered compound moistened with an inert liquid diluent. The tablets can optionally be coated or scored and formulated, for example, using hydroxypropylmethylcellulose in various ratios to provide a slow or controlled release of the active ingredient therein to provide a desired release profile.

[0092] Solid compositions of the same type can also be used as bulking agents in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactitol, or high molecular weight polyethylene glycol. For aqueous suspensions and / or elixirs, the compounds of the invention can be combined with various sweetening or flavoring agents, coloring substances or dyes, emulsifying and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, and combinations thereof.

[0093] Formulations suitable for topical administration in the mouth include flavored bases, usually film formulations or lozenges containing the active ingredient in sucrose and gum arabic or tragacanth; troches containing the active ingredient in an inert base such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid carrier.

[0094] Pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, emulsions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols, or oils, transdermal devices, dusting powders, etc. These compositions can be prepared by conventional methods containing the active agent. Thus, they can also contain suitable conventional carriers and additives, such as preservatives, solvents to aid drug penetration, emollients in creams or ointments, and ethanol or oleyl alcohol for lotions. Such carriers can be present as about 1% to up to about 98% of the composition. More generally, they form up to about 80% of the composition. By way of mere example, a cream or ointment is prepared by mixing a sufficient amount of a hydrophilic material and water containing about 5 to 10% by weight of the compound to yield a cream or ointment having the desired consistency.

[0095] A pharmaceutical composition adapted for transdermal administration can be presented as an individual patch that is intended to remain in close contact with the recipient's epidermis for an extended period. For example, the active agent can be delivered from the patch by iontophoresis.

[0096] For application to external tissues, such as the mouth and skin, the composition is preferably applied as a topical ointment or cream. When formulated in an ointment, the active agent can be utilized with either a paraffin-based ointment base or a water-miscible ointment base.

[0097] Alternatively, the active agent can be formulated in a cream having an oil-in-water cream base or a water-in-oil base.

[0098] For parenteral administration, fluid unit dosage forms or infusion solutions are prepared using the active ingredient and a sterile vehicle, such as, but not limited to, water, alcohol, polyols, glycerin, and vegetable oils, with water being preferred. The active ingredient can be in the form of being colloidal, suspended, or dissolved in the vehicle, depending on the vehicle and concentration used. When preparing a solution, the active ingredient is dissolved in water for injection and can be sterilized, for example, by filter sterilization, and then filled and sealed into a suitable vial or ampoule.

[0099] Advantageously, agents such as local anesthetics, preservatives, and buffering agents can be dissolved in the vehicle. To enhance stability, after filling the composition into, for example, a vial, it can be frozen by freeze-drying and the water can be removed under vacuum. The dried lyophilized powder can then be sealed in the vial, and a vial of the accompanying water for injection can be provided to reconstitute the liquid before use.

[0100] The pharmaceutical composition of the present invention suitable for injection use comprises a sterile aqueous solution or dispersion. Further, the composition can be in the form of a sterile powder for the immediate preparation of such sterile injection solution or dispersion. In all cases, the final injection form must be sterile and must be an efficient liquid for ease of injection.

[0101] The pharmaceutical composition of the present invention includes formulations suitable for intravitreal administration. These consist of a therapeutically effective amount of Compound 1, one or more pharmaceutically acceptable excipients, or a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be in the conventional dosage forms of eye drops or other compositions having better bioavailability. Such compositions that overcome the intravitreal drug delivery barrier and have improved intravitreal bioavailability are, for example, emulsions, ointments, suspensions, aqueous gels, nanomicelles, nanoparticles, liposomes, dendrimers, nanosuspensions, microneedles, and in situ thermosensitive gels.

[0102] Parenteral suspensions are prepared in substantially the same manner as solutions, except that the active ingredient is suspended in the vehicle rather than dissolved and sterilization cannot be achieved by filtration. The active ingredient can be sterilized by exposure to ethylene oxide and then suspended in a sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient.

[0103] As can be seen from the examples herein, excipients containing carbonates should be avoided, especially in liquid or semi-solid formulations. Preferably, the carbonate concentration should be less than 0.85 mM.

[0104] In addition to the components specifically mentioned above, the formulations of the present invention can include other agents customary in the art, taking into account the type of the formulation in question. For example, it should be understood that formulations suitable for oral administration can include flavoring agents. Those skilled in the art will know how to select a suitable formulation and how to prepare it (see, for example, Remington’s Pharmaceutical Sciences, 18th edition and later). Those skilled in the art will also know how to select a suitable route of administration and dosage.

[0105] The present invention provides a process for preparing a liquid pharmaceutical composition according to any of the preceding claims, the process comprising, to obtain the pharmaceutical composition: a) obtaining methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in free form or its salt, hydrate, solvate, or complex; b) optionally heating to below 90 °C, for example up to 60 °C or holding at room temperature; c) adding an aqueous liquid (e.g., phosphate buffered saline at pH 7.4), an aqueous saline solution, or pure water; d) optionally assisting dissolution by sonication; e) mixing at room temperature. comprises.

[0106] It will be recognized by those skilled in the art that the optimal amounts and intervals of individual dosages of the compounds of the present invention are determined by the nature and severity of the disease being treated, the form, route, and site of administration, and the age and condition of the particular subject being treated, and that the appropriate dosage to be used by the physician will ultimately be determined. This dosage can be repeated as often as appropriate. If side effects occur, the amount and / or frequency of the dosage can be altered or reduced according to normal clinical practice.

[0107] All % values referred to herein are % w / w, unless the context dictates otherwise.

[0108] (Neutricosmetic composition containing the compound of the present invention) Neutricosmetics are oral administration products. The present invention also provides a neutricosmetic composition containing the compound of Compound 1. The neutricosmetic composition contains the free form of Compound 1 or a salt, hydrate, solvate, or complex thereof, together with one or more orally acceptable diluents or carriers. The neutricosmetic composition closely resembles a pharmaceutical composition for oral administration.

[0109] Thus, typical compositions are tablets, capsules, ovules, elixirs, gels, solutions, or suspensions.

[0110] (Cosmeceutical composition containing the compound of the present invention) Cosmeceutical compositions are usually administered to the skin or mucous membranes. Sometimes, this can also be administered by injection. The present invention also provides a cosmeceutical composition containing the compound of Compound 1. The cosmeceutical composition contains the free form of Compound 1 or a salt, hydrate, solvate, or complex thereof, together with one or more orally acceptable diluents or carriers.

[0111] Typical cosmeceutical compositions include those mentioned above in this specification suitable for application to the skin, to the mucous membranes, or by injection.

[0112] (Other aspects of the present invention) The present invention relates to a compound of formula (I) as defined above (for example, for the treatment of mitochondrial dysfunction) or a pharmaceutically acceptable form thereof and · Quinone derivatives, such as ubiquinone, idebenone, MitoQ · Vitamins, such as tocopherol, tocotrienol, and trolox (vitamin E), ascorbic acid (C), thiamine (B1), riboflavin (B2), nicotinamide (B3), menadione (K3), · Antioxidants in addition to vitamins, such as TPP-compounds (MitoQ), Sk-compounds, epicatechin, catechin, lipoic acid, uric acid, melatonin · Dichloroacetic acid · Methylene blue · L-arginine · Szeto-Schiller peptide, elamipretide, and elamipretide analogs · Creatine · Benzodiazepine · Modulators of PGC-1α · Modulators of AMPK · Modulators of mitochondrial fission and fusion · PPARα / β / γ-agonists · Trolox analogs, carboxamide derivatives · Nrf-2 activators · NAD + Modulators · NAD + Precursors · Ketogenic diet : also provides a combination of one or more agents independently selected from.

[0113] Another aspect of the present invention is that any of the compounds of Compound 1 disclosed herein can be administered together with any other compound, such as sodium bicarbonate (as a bolus (e.g., 1 mEq / kg) followed by continuous infusion), as a co-drug to the compounds disclosed herein.

[0114] (Lactic acidosis or drug-induced side effects due to Complex I-related disorders of mitochondrial oxidative phosphorylation) The present invention also relates to the prevention or treatment of lactic acidosis and mitochondrial-related drug-induced side effects. In particular, the compounds of Compound 1 according to the present invention are used in the prevention or treatment of mitochondrial-related drug or toxin-induced side effects at Complex I or upstream thereof, or in another form of expression, the present invention, according to the present invention, prevents or treats the drug-induced direct inhibition of Complex I or any drug-induced action that limits the supply of NADH to Complex I (e.g., but not limited to, Krebs cycle, glycolysis, β-oxidation, pyruvate metabolism, and even the transport or level of glucose or other Complex I-related substrates).

[0115] Drug-induced mitochondrial toxicity may be part of the desired therapeutic effect (e.g., mitochondrial toxicity induced by anticancer agents), but in most cases, drug-induced mitochondrial toxicity is an undesirable effect. Mitochondrial toxicity can significantly increase glycolysis and offset the intracellular loss of mitochondrial ATP formation by oxidative phosphorylation. This can lead to an increase in the plasma level of lactate, and in excess, this results in the induction of lactic acidosis, which can be fatal. Type A lactic acidosis is mainly associated with tissue hypoxia, while type B aerobic lactic acidosis is associated with drugs, toxins, or systemic disorders such as liver disease, diabetes, cancer, and congenital abnormalities of metabolism (e.g., genetic defects in mitochondria).

[0116] Many known drug substances have a negative impact on mitochondrial respiration (e.g., antipsychotics, local anesthetics, and antidiabetic drugs), and therefore, there is a need to identify or develop means that can be used to avoid or reduce the negative mitochondrial effects induced by the use of such drug substances. Furthermore, some chemical agents and gases have a negative impact on mitochondrial metabolism and function.

[0117] The present invention provides a compound of compound 1 for use in the prevention or treatment of lactic acidosis and the prevention or treatment of mitochondrial-related drug or toxin-induced side effects. In particular, the succinate prodrug is used in the prevention or treatment of mitochondrial-related drug-induced side effects at complex I or upstream thereof, or in other words, the present invention also provides for the prevention or treatment of the direct inhibition of complex I, the drug-induced inhibition of other respiratory complexes, or any drug-induced effect that limits the supply of NADH to complex I (e.g., but not limited to, effects on the Krebs cycle, glycolysis, β-oxidation, pyruvate metabolism, and even the transport or levels of glucose or other complex I-related substrates). The present invention provides a succinate prodrug for the prevention or treatment of drugs).

[0118] As described above, an increase in plasma levels of lactic acid is often observed in patients treated with drugs that can have mitochondrial-related side effects. The present invention is based on experimental results showing that metformin, the first choice treatment for type 2 diabetes and associated with lactic acidosis as a rare side effect, inhibits mitochondrial function in human peripheral blood cells at complex I in a time- and dose-dependent manner at concentrations relevant to metformin intoxication. Metformin also causes a significant increase in lactic acid production over time by intact platelets.

[0119] Accordingly, the present invention provides a compound of formula (I) for use in the prevention or treatment of lactic acidosis. However, since the results reported herein are based on lactic acidosis associated with direct inhibition of Complex I or defects in Complex I or upstream thereof, it is contemplated that the compounds according to the invention are suitable for use in the prevention or treatment of mitochondrial-related drug-induced side effects in Complex I or upstream thereof. The compounds according to the invention will neutralize drug actions that disrupt metabolism upstream of Complex I (any drug action that limits the supply of NADH to Complex I, e.g., drug actions on the Krebs cycle, glycolysis, β-oxidation, pyruvate metabolism, and even drugs that affect the levels of glucose or other Complex I-related substrates, i.e., indirect inhibition of Complex I). The compounds can also neutralize defects downstream of Complex I (Complexes III, IV, and V) by increasing the proton motive force.

[0120] It is contemplated that Compound 1 can be used in industrial applications, e.g., in vitro, to reduce or inhibit the formation of lactic acid or to increase the ATP availability of commercial or industrial cell lines. Examples include use in cell culture, organ preservation, etc.

[0121] The compounds according to the invention are used in the treatment or prevention of drug-induced mitochondrial-related side effects or to increase or restore cellular levels of energy (ATP) or succinate in the treatment. In particular, it is used in the treatment or prevention of direct or indirect drug-induced Complex I mitochondrial-related side effects. In particular, it is used in the treatment or prevention of lactic acidosis, e.g., lactic acidosis induced by drug substances.

[0122] The present invention also relates to a combination of a compound 1 and a drug substance that can induce mitochondrial-related side effects, in particular side effects caused by direct or indirect impairment of complex I by a drug substance. Such a combination can be used for preventive prophylaxis of mitochondrial-related side effects or, when side effects appear, for reduction and / or treatment of mitochondrial-related side effects.

[0123] Compound 1 is assumed to be effective in the treatment or prevention of drug-induced side effects, in particular side effects related to direct or indirect inhibition of complex I.

[0124] Drug substances known to cause defects, dysfunctions, or impairments of complex I and / or known to have lactic acidosis as a side effect are as follows: Analgesics including acetaminophen, capsaicin Antianginal drugs including amiodarone, perhexiline Antibiotics including linezolid, trovafloxacin, gentamicin Anticancer drugs including quinones containing mitomycin C, adriamycin Anticonvulsants including valproic acid Antidiabetic drugs including metformin, phenformin, butylbiguanide, troglitazone, and rosiglitazone, pioglitazone Antiviral drugs for hepatitis B including fialuridine Antihistamines Antiparkinson drugs including tolcapone Antipsychotic drug risperidone, Antischizophrenic drugs zotepine, clozapine Disinfectants, quaternary ammonium compounds (QAC) Antituberculosis drugs including isoniazid Fibrates including clofibrate, ciprofibrate, simvastatin Hypnotics including propofol Immunosuppressive disease-modifying antirheumatic drug (DMARD) leflunomide Local anesthetics including bupivacaine, diclofenac, indomethacin, and lidocaine Muscle relaxants containing dantrolene Neuroleptics containing antipsychotic neuroleptics such as chlorpromazine, fluphenazine, and haloperidol NRTIs (nucleotide reverse transcriptase inhibitors) containing efavirenz, tenofovir, emtricitabine, zidovudine, lamivudine, rilpivirine, abacavir, didanosine Nimes Li NSAIDs containing mefenamic acid, sulindac Barbituric acid.

[0125] Other drug substances known to have lactic acidosis as a side effect include β2 - agonists, epinephrine, theophylline, or other herbicides. Alcohol and cocaine can also cause lactic acidosis.

[0126] Furthermore, it is assumed that the compounds of the present invention may also be effective in the treatment or prevention of lactic acidosis even if not related to complex I deficiency.

[0127] (Combination of a drug and a compound of the present invention) The present invention also relates to a combination of a drug substance and a compound of the present invention for use in the treatment and / or prevention of drug - induced side effects selected from lactic acidosis and side effects associated with defects, inhibition, or dysfunction of complex I, i) the drug substance is used in the treatment of a disease for which the drug substance is indicated, and ii) the compound of the present invention is used in the prevention or reduction of side effects induced or inducible by the drug substance, where the side effects are selected from lactic acidosis and side effects associated with defects, inhibition, or dysfunction of complex I, relating to the combination.

[0128] Any combination of such drug substances with any compound of the invention is within the scope of the invention. Thus, based on the disclosure herein, one of ordinary skill in the art will understand that the gist of the invention is the discovery of valuable properties of the compounds of the invention that avoid or reduce the side effects described herein. Thus, the potential use of the compounds of the invention that can enter cells and supply succinic acid and optionally other active moieties in combination with any drug substance having or potentially having the side effects described herein is apparent from this disclosure.

[0129] The invention further relates to i) a composition comprising a drug substance and a compound of the invention, wherein the drug substance has potential drug-induced side effects selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of complex I ii) the composition described in i) above, wherein the compound of the invention is used for the prevention or alleviation of side effects induced or inducible by the drug substance, wherein the side effects are selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of complex I relates to.

[0130] The composition may be in the form of two separate packages: a first package containing the drug substance or a composition containing the drug substance and a second package containing the compound of the invention or a composition containing the compound of the invention It may be in the form. The composition may also be a single composition containing both the drug substance and the compound of the invention.

[0131] If the composition comprises two separate packages, the drug substance and the compound of the invention may be administered by different routes of administration (e.g., the drug substance by oral administration and the compound of the invention by parenteral or mucosal administration), and / or they may be administered essentially simultaneously, or the drug substance may be administered before the compound of the invention, or vice versa.

[0132] (Kit) The present invention relates to i) a first container containing a drug substance having a potential drug-induced side effect selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of Complex I, and ii) a second container containing a compound of Compound 1 of the present invention that has the potential to prevent or reduce side effects induced by or that can be induced by the drug substance, where the side effects are selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of Complex I also provides a kit comprising the same.

[0133] (Method for treating / preventing side effects) The present invention also relates to a method for treating a subject suffering from a drug-induced side effect selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of Complex I, the method comprising administering to the subject an effective amount of a compound of Compound 1 of the present invention, and a method for preventing or reducing a drug-induced side effect selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of Complex I in a subject suffering from a disease treated with a drug substance that potentially induces a side effect selected from side effects associated with lactic acidosis and defects, inhibitions, or dysfunctions of Complex I, the method comprising administering to the subject an effective amount of a compound of Compound 1 of the present invention before, during, or after treatment with the drug substance.

[0134] (Metformin) Metformin is an antidiabetic drug belonging to the biguanide class. It is the first-choice treatment for type 2 diabetes, which accounts for approximately 90% of diabetes cases in the United States. The antidiabetic effect is due to a decrease in hepatic glucose production, an increase in the biological effect of insulin due to increased glucose uptake in peripheral tissues, and a decrease in glucose uptake in the intestine, although the exact mechanism of action is not fully understood. Despite its advantages over other antidiabetic drugs, it is associated with rare cases of lactic acidosis (LA) as a side effect. LA is defined as an increase in the anion gap, an arterial blood lactate level above 5 mM, and a pH ≤ 7.35.

[0135] The following list of non-limiting embodiments further illustrates the present invention: 1. Isolated methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in free form or its salt, hydrate, solvate, or complex. 2. The isolated Compound 1 according to Embodiment 1, which is a solid product. 3. The isolated Compound 1 according to any of the foregoing embodiments, which is a crystalline product having the XRPD pattern of Compound 1 Batch 12 (Figure 7) or having the XRPD pattern of Compound 1 Batch 15 (Figure 8), or having polymorphs with positions (°2θ) of 11.2 (±0.2) and 16.9 (±0.2), or comprising such a crystalline product. 4. The isolated Compound 1 according to any of Embodiments 1 to 2, which is an amorphous product or comprises an amorphous product. 5. The isolated Compound 1 according to any of the foregoing embodiments, having a purity of at least 20% w / w, at least 30% w / w, at least 40% w / w, at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 75% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, at least 97% w / w, at least 98% w / w, or at least 99% w / w. 6. An isolated compound 1 according to any of the foregoing embodiments, having a related impurity content of less than 75% w / w, less than 70% w / w, less than 65% w / w, less than 60% w / w, less than 55% w / w, less than 50% w / w, less than 45% w / w, less than 40% w / w, less than 35% w / w, less than 30% w / w, less than 25% w / w, less than 20% w / w, less than 15% w / w, less than 10% w / w, less than 5% w / w, less than 3% w / w, less than 2% w / w, or less than 1% w / w. 7. An isolated compound 1 according to any of the foregoing embodiments, having a synthetic precursor content of less than 50% w / w, less than 40% w / w, less than 30% w / w, less than 25% w / w, less than 20% w / w, less than 15% w / w, less than 10% w / w, less than 5% w / w, less than 3% w / w, less than 2% w / w, or less than 1% w / w. 8. An isolated compound 1 according to any of the foregoing embodiments, having a purity sufficient for pharmaceutical use. 9. An isolated compound 1 according to any of the foregoing embodiments, in free form. 10. An isolated compound 1 according to any of embodiments 1 to 8, which is a salt. 11. An isolated compound 1 according to embodiment 10, which is a hydrochloride, hydrobromide, acetate, citrate, lactate, maleate, or malonate. 12. An isolated compound 1 according to any of embodiments 1 to 8, which is a hydrate such as a monohydrate. 13. An isolated compound 1 according to any of the foregoing embodiments, for use in humans or animals. 14. An isolated compound 1 according to any of the foregoing embodiments, for use in humans. 15. An isolated compound 1 according to any of the foregoing embodiments, for use in a medicament. 16. An isolated compound 1 according to any of the foregoing embodiments, for use as an active pharmaceutical ingredient in a pharmaceutical product. 17. An isolated compound 1 according to any of the foregoing embodiments for use in the treatment or prevention of metabolic diseases, diseases of mitochondrial dysfunction, diseases associated with mitochondrial dysfunction, mitochondrial disorders, mitochondrial energy deficiency, drug-induced mitochondrial side effects, cancer, diabetes, traumatic brain injury, cardiac arrest, hypoxia, ischemia, stroke, myocardial infarction, acute angina, acute liver injury, coronary artery occlusion, atrial fibrillation, male infertility, and menopausal symptoms in women. 18. The disease of mitochondrial dysfunction or the disease associated with mitochondrial dysfunction is aging Alpers disease (progressive infantile poliodystrophy), Alzheimer's disease, amyotrophic lateral sclerosis (ALS) autism, Barth syndrome (lethal infantile cardiomyopathy), β-oxidation defect, bioenergetic metabolism deficiency disorder, carnitine-acyl-carnitine deficiency, carnitine deficiency, creatine deficiency syndrome (guanidinoacetate methyltransferase deficiency (GAMT deficiency), L-arginine: glycine amidinotransferase deficiency (AGAT deficiency), and SLC6A8-related creatine transporter deficiency (SLC6A8 deficiency): brain creatine deficiency syndrome (CCDS) including, coenzyme Q10 deficiency, complex I deficiency (NADH dehydrogenase (NADH-CoQ reductase deficiency), complex II deficiency (succinate dehydrogenase deficiency), complex III deficiency (ubiquinone-cytochrome c oxidoreductase deficiency), complex IV deficiency / COX deficiency (cytochrome c oxidase deficiency is caused by a defect in complex IV of the respiratory chain), complex V deficiency (ATP synthase deficiency), COX deficiency, CPEO (chronic progressive external ophthalmoplegia syndrome), CPT I deficiency, CPT II deficiency, type II diabetes, Friedreich's ataxia (FRDA or FA), Glutaric acidemia type II, KSS (Kearns–Sayre syndrome), Lactic acidosis, LCAD (long-chain acyl-CoA dehydrogenase deficiency), LC-FAOD (long-chain fatty acid oxidation disorder), LCHAD, Leigh disease or Leigh syndrome (subacute necrotizing encephalomyelopathy), LHON (Leber hereditary optic neuropathy), Luft disease, MCAD (medium-chain acyl-CoA dehydrogenase deficiency), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), MERRF (myoclonic epilepsy and ragged-red fibers), Methylmalonyl-CoA epimerase deficiency, Methylmalonyl-CoA mutase deficiency, Mitochondrial DNA depletion syndrome 5, Mitochondrial DNA depletion syndrome 9, Mitochondrial DNA depletion syndrome 15 (hepatoencephalic type) (1 family), Maternally inherited diabetes and deafness, MIRAS (mitochondrial recessive ataxia syndrome), Mitochondrial cytopathy, Mitochondrial DNA depletion, Encephalopathy and cerebrospinal disorder, mitochondrial encephalopathy including mitochondrial myopathy, MNGIE (myoneurogastrointestinal encephalopathy), NARP (neuropathy, ataxia, and retinitis pigmentosa), Neurodegenerative disorders associated with Parkinson's disease, Alzheimer's disease, or Huntington's disease, Pearson syndrome, Parkinson's disease, Progressive external ophthalmoplegia, Propionic acidemia, Pyruvate dehydrogenase deficiency, POLG mutation, Respiratory chain deficiency SCAD (short-chain acyl-CoA dehydrogenase deficiency), SCHAD VLCAD (very long-chain acyl-CoA dehydrogenase deficiency) Isolated compound 1 according to embodiment 17, selected from 19. Isolated compound 1 according to embodiment 18, wherein the disease of mitochondrial dysfunction or the disease associated with mitochondrial dysfunction is caused by complex I dysfunction and is selected from Leigh syndrome, Leber hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), and MERRF (myoclonic epilepsy with ragged red fibers). 20. Isolated compound 1 according to any one of embodiments 1 to 17 for use in the treatment or prevention of metabolic dysfunction. 21. Isolated compound 1 according to embodiment 20, wherein the metabolic dysfunction is diabetes (type 2 diabetes) such as a defect in insulin secretion. 22. Isolated compound 1 according to embodiment 20, wherein the metabolic dysfunction is a drug-induced side effect on mitochondria. 23. Isolated compound 1 according to embodiment 22, wherein the drug-induced side effect on mitochondria is selected from metformin-induced complex I inhibition (lactic acidosis), paracetamol / acetaminophen-induced complex I inhibition (liver failure), or drug-induced mitochondrial deletion. 24. Isolated compound 1 according to embodiment 20, wherein the metabolic dysfunction is a chemically induced side effect on mitochondria. 25. Isolated compound 1 according to embodiment 24, wherein the chemically induced side effect on mitochondria is selected from rotenone inhibition of complex I (Parkinson-like symptoms), insecticide-induced inhibition of respiratory complexes and mitochondrial enzymes, chemical warfare agent-induced inhibition of respiratory complexes and mitochondrial enzymes, and gas poisoning of respiratory complexes and mitochondrial enzymes, for example, carbon monoxide poisoning. 26. Isolated compound 1 according to embodiment 20, wherein the metabolic dysfunction is hereditary mitochondrial dysfunction. 27. The isolated compound 1 according to embodiment 26, wherein the hereditary mitochondrial dysfunction is selected from a decrease in the number of mitochondria resulting in energy production dysfunction, mitochondrial transcription factor dysfunction, mitochondrial protein encoded by nuclear DNA, mitochondrial membrane protein transcription factor dysfunction contributing to the stabilization of a large mitochondrial DNA (mtDNA)-protein complex called a nucleoid, energy production dysfunction, pyruvate dehydrogenase deficiency, deficiency of complex I, II, III, or IV, or enzyme deficiency such as pyruvate dehydrogenase deficiency, and dysfunction of enzymes involved in succinic acid synthesis such as propionyl-CoA carboxylase, methylmalonyl-CoA mutase, and succinyl-CoA synthetase. 28. The isolated compound 1 according to embodiment 27, wherein the effective amount is compound 1 or a salt, hydrate, solvate, or complex thereof in the range of 1 mg to 5.0 g per day, 10 mg to 2.0 g per day, 25 mg to 1 g per day, 50 mg to 500 mg per day, 100 mg to 1000 mg per day, 250 mg to 1000 mg per day, or 50 mg to 500 mg per day. 29. The isolated compound 1 according to any one of embodiments 15 to 28, wherein the compound 1 or a salt, hydrate, solvate, or complex thereof is administered to the subject once a day to 10 times a day, or once a day to 4 times a day. 30. The isolated compound 1 according to any one of embodiments 15 to 29, wherein the treatment or prevention is for pretreatment, such as use before surgery, use before a planned medical intervention with high metabolic requirements, and use before the subject enters a war zone or other dangerous environment. 31. The isolated compound 1 according to any one of embodiments 15 to 30, wherein the treatment or prevention is a long-term treatment. 32. The isolated compound 1 according to any one of embodiments 1 to 14 for non-medical use in humans or animals. 33. The isolated compound 1 according to embodiment 19 for use as a pharmaceutical cosmetic or nutricosmetic. 34. Isolated compound 1 according to any of embodiments 28 - 29 for use as an energy drink or a cream. 35. A composition comprising isolated compound 1 according to any of the foregoing embodiments. 36. A pharmaceutical cosmetic comprising isolated compound 1 according to any of embodiments 1 - 14. 37. A nutricosmetic comprising isolated compound 1 according to any of embodiments 1 - 14. 38. An energy drink comprising isolated compound 1 according to any of embodiments 1 - 14. 39. A pharmaceutical composition comprising isolated compound 1 according to any of embodiments 1 - 33. 40. A process for preparing isolated compound 1 according to any of embodiments 1 - 33, for providing isolated compound 1, a) reacting N - acetylcysteamine and monomethyl succinate in an organic solvent in the presence of a coupling reagent at 0 °C - 100 °C b) isolating compound 1 : comprising the process. 41. The process according to embodiment 40, wherein step a) is carried out, and in that case, independently, the solvent is dichloromethane, the coupling agent is carbonyldiimidazole, and the temperature is 15 - 30 °C. 42. The process according to any of embodiments 40 - 41, wherein step b) comprises extracting with an aqueous acid solution (optionally 20% ammonium chloride) and then extracting the organic layer with another aqueous medium (suitable brine or water). 43. The process according to embodiment 42, wherein the organic layer is removed in vacuo and the residue is dissolved in an organic solvent having dissolution properties suitable for crystallization, such as methyl - tert - butyl ether (MTBE). 44. The process according to embodiment 42, wherein the solution is preferably cooled to about 5 °C, a poor solvent, such as n - heptane, is added, stirred for a certain period, preferably about 24 hours, and then compound 1 is recovered by filtration and washed with the poor solvent. Isolated compound 1 according to any of embodiments 1 to 3, wherein the position (°2θ) is 11.2 (±0.2) and 16.9 (±0.2). The pharmaceutical composition according to embodiment 39, which is a solid preparation. The pharmaceutical composition according to embodiment 46, which is a solid preparation for reconstitution before use. The pharmaceutical composition according to embodiment 46, which is an aqueous preparation. The pharmaceutical composition according to embodiment 48, which is an aqueous phosphate buffered saline (PBS) preparation. The pharmaceutical composition according to any of embodiments 46 to 49, having compound 1 at a concentration of at least 10% w / w, at least 30% w / w, at least 50% w / w, at least 60%, or at least 70% w / w. The pharmaceutical composition according to any of embodiments 46 to 50, which is for oral administration, subcutaneous administration, intravenous administration, parenteral administration, ocular administration, or topical administration. The pharmaceutical composition according to embodiment 51, which is a beverage or a gel. The pharmaceutical composition according to any of embodiments 46 to 50, comprising 1 mg to 5.0 g, 10 mg to 2.0 g, 25 mg to 1 g, 50 mg to 500 mg, 100 mg to 1000 mg, 250 mg to 1000 mg, or 50 mg to 500 mg of compound 1 or a salt, hydrate, solvate, or complex thereof. The pharmaceutical composition according to any of embodiments 46 to 53, which is an immediate release preparation.

Examples

[0136] (Example) General methods, materials, and assays HPLC method for purity analysis (HPLC method 1) Solvent A is water + 0.1% NH4OH. Solvent B is 2.5 L acetonitrile + 130 ml H2O + 0.1% NH4OH. Gradient: T = 0 min, B% = 5, flow rate = 1 ml / min; T = 0.1 min, B% = 5, flow rate = 1 ml / min; T = 9.5 min, B% = 95, flow rate = 1 ml / min; T = 10.2 min, B% = 95, flow rate = 1 ml / min; T = 10.3 min, B% = 95, flow rate = 1.5 ml / min; T = 11.1 min, B% = 95, flow rate = 1.5 ml / min; T = 11.15 min, B% = 5, flow rate = 1.5 ml / min; T = 11.5 min, B% = 5, flow rate = 1.5 ml / min; The column is Waters XSelect CSH C18 3.5um, 2.1mm×50mm. Absorbance is monitored at 234 nm with a diode array detector. Sample concentration of 1 mg / ml, injection volume of 1 γl.

[0137] (HPLC method 2) Solvent A is water + 1.57 g NH4HCO2 + 5 ml formic acid. Solvent B is 2.5 L acetonitrile + 130 ml H2O + 4.5 ml formic acid. Gradient: T = 0 min, B% = 0, flow rate = 1 ml / min; T = 1 min, B% = 0, flow rate = 1 ml / min; T = 9.5 min, B% = 20, flow rate = 1 ml / min; T = 10.3 min, B% = 95, flow rate = 1 ml / min; T = 10.5 min, B% = 95, flow rate = 1.5 ml / min; T = 11.0 min, B% = 95, flow rate = 1.5 ml / min; T = 11.05 min, B% = 0, flow rate = 1.5 ml / min; T = 11.5 min, B% = 0, flow rate = 1.5 ml / min; The column is Waters XSelect CSH C18 3.5 um, 2.1mm×50mm. Absorbance is monitored at 230 nm with a diode array detector. Sample concentration of 1 mg / ml, injection volume of 1 γl.

[0138] (Synthesis of Example 1 - Methyl 3 - [(2 - acetylaminoethylthio)carbonyl]propionate (Compound 1)) Detailed description of the synthesis and isolation of Compound 1:

Chemical formula

[0139] (Method A) Acetic anhydride (204 g, 2 mol) was added dropwise to an aqueous solution (4 L) of 2-aminoethanethiol hydrochloride (226 g, 2 mol), KOH (114 g, 2 mol), and NaHCO3 (168 g, 2 mol). The mixture was stirred at room temperature for 45 minutes. The reaction mixture was extracted with EtOAc (8 × 2 L), dried over MgSO4, and the solvent was removed to obtain Intermediate 1 (190 g, 80% yield) as a pale yellow oil under reduced pressure.

[0140] N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (304 g, 1.583 mol) was added to a dichloromethane (4 L) solution of 4-methoxy-4-oxobutanoic acid (209 g, 1.583 mol) and HOBT (214 g, 1.583 mol). The mixture was stirred at room temperature for 2 hours. Intermediate 1 (189 g, 1.583 mol) was added dropwise. The mixture was stirred at room temperature for 2 hours. Triethylamine (160 g, 1.583 mol) was added dropwise. The mixture was stirred at room temperature overnight. The resulting mixture was washed with water (2 L) and saturated NaHCO3 solution (2 × 2 L), dried over Na2SO4, and concentrated under reduced pressure to obtain crude Compound 1 (350 g) as a yellow oil. The crude Compound 1 was purified by silica gel column chromatography (2000 g of silica gel, eluting with CH2Cl2 / MeOH = 100 / 1 to 80 / 1) to obtain Compound 1 (201 g, 94.9% by LCMS) as a white solid. The crude side cut (110 g) obtained from the purification was purified by silica gel column chromatography (1200 g of silica gel, eluting with CH2Cl2 / MeOH = 100 / 1 to 80 / 1) to obtain Compound 1 (40 g, 96.7% by LCMS) as a white solid.

[0141] (Method B) Acetic anhydride (7.14 g, 0.07 mol) was added dropwise to a solution of 2-aminoethanethiol hydrochloride (11.3 g, 0.1 mol), KOH (5.6 g, 0.1 mol), and NaHCO3 (5.88 g, 0.07 mol) in water (200 mL) at room temperature. The mixture was stirred at room temperature for 45 minutes. The reaction mixture was extracted with EtOAc (8 × 200 mL), dried over MgSO4 (1 hour), and then the solvent was removed in vacuo at 50 °C to give crude intermediate 1 (7 g, 84% yield) as a pale yellow liquid.

[0142] 1,1'-Carbonyldiimidazole (11.34 g, 0.07 mol) was added portionwise to a solution of 4-methoxy-4-oxobutanoic acid (9.24 g, 0.07 mol) in dichloromethane (200 mL). The mixture was stirred at room temperature for 1 hour. Intermediate 1 (7 g, 0.059 mol) was added dropwise, and then the mixture was stirred at room temperature for 3 hours. The resulting mixture was washed with HCl (1 N, 3 × 150 mL) and saturated solution of NaHCO3 (3 × 150 mL), dried over Na2SO4 (1 hour), and then the solvent was removed in vacuo at 50 °C to give 9.5 g of compound 1 as a yellow solid.

[0143] (Method C) To a solution of KOH (0.71 kg, 13.2 mol) and Na2CO3 (1.00 kg, 9.43 mol) in water (15 L) was added 2-aminoethanethiol hydrochloride (1.5 kg, 13.2 mol). Acetic anhydride (0.96 kg, 9.43 mol) was added dropwise to the resulting clear dark purple solution at +22 °C, maintaining the internal temperature below +30 °C during the addition (the addition time was 24 minutes). The reaction mixture was stirred at +20 °C for 1 hour 35 minutes. Dichloromethane (23 L) was added, and the mixture was stirred at +28 ± 2 °C for 20 minutes. The layers were separated. The aqueous phase was extracted with dichloromethane (2 × 15 L), adjusting the internal temperature to +28 ± 2 °C during the extraction. The organic phases were combined and the solvent was removed in vacuo. Then, intermediate 1 was dried under vacuum at +40 °C for 18 hours. Yield 996 g and purity >97 area-% (GC). Crude intermediate 1 was obtained as a brown oil.

[0144] Distillation: Under the following conditions: T = +110 °C, P = 1 mbar, 933 g of Intermediate 1 was distilled using a thin-film distillation unit at a rate of 202 g / h. Intermediate 1 was obtained as a clear, colorless oil.

[0145] To a solution of 4-methoxy-4-oxobutanoic acid (1.33 kg, 10.07 mol) in DCM (10 L), 1,1'-carbonyldiimidazole (CDI) (1.63 kg, 10.07 mol) was added portionwise. Strong foaming and gas evolution were observed during the addition. After the addition was complete, the mixture was stirred at +20 to +25 °C for 1 h. While maintaining the internal temperature below +30 °C, a solution of Intermediate 1 (1.00 kg, 8.39 mol) in dichloromethane (5 L) was added. The reaction mixture was stirred at +20 to +25 °C for 2 h. 20% aqueous NH4Cl solution (10 L) was added and the mixture was stirred for 20 min. The layers were separated. Subsequently, the organic phase was extracted with 13% aqueous NaCl solution and water (10 L and 5 L, respectively). Then, the solvent (DCM) was exchanged for MTBE by distillation. The MTBE (ca. 6 L) solution of Compound 1 was gradually cooled to +5 °C. Crystallization started when the internal temperature reached +12 °C. n-Heptane (15 L) was added to the slurry and the mixture was stirred at 0 to +5 °C for 20 h (overnight). The slurry was filtered and the filter cake was washed with n-heptane (2 × 3 L). The product was dried by pulling air into the product for 42 h. The yield was 1.26 kg (64%) and the purity was 98.5 area-% (HPLC).

[0146] Several batches of Compound 1 were prepared by the above synthesis. These batches were purified by different purification methods shown in Methods A, B, and C.

[0147] Batch 3 (or Compound 1-s3) was prepared by Method A.

[0148] Batches 12 (or Compound 1-s12), 13 (or Compound 1-s13), and 14 (or Compound 1-s14) were prepared by Method B.

[0149] Batches 15 (or Compound 1-s15), 16, and 17 were prepared by Method C.

[0150] (Characterization of Compound 1 from Different Batches - Example 2) [Table 1] It should be mentioned that the temperature rises during the XRPD analysis. Since Compound 1 has a low melting point (and the amorphous form is thought to have a lower melting point than the crystalline form), the degree of crystallinity shown in the above table can be regarded as a minimum value.

[0151] (Batch 3) In the TGA analysis, Batch 3 had a loss of 0.04 wt% at a temperature of 20 - 150 °C. Figures 1 - 3 show the spectra obtained from the LCMS analysis of Batch 3. When determined by differential scanning calorimetry (DSC), the melting point of Batch 3 was 50.4 °C.

[0152] (Batch 12) Batch 12 was analyzed by the same method as used for the analysis of Batch 3 above. The loss in the TGA analysis was 0.12 wt% at a temperature of 20 - 150 °C. The LCMS results are shown in Figures 4 - 6, the purity by qNMR was 96.1%, and the melting point was 48.6 °C.

[0153] (Batch 13) Batch 13 was analyzed by the same method as used for the analysis of the above batches. The loss in the TGA analysis was 0.18 wt% at a temperature of 20 - 150 °C. The spectra obtained from LCMS are not shown, but the results are summarized in Table 5. The purity by qNMR was 96.3%, and the melting point was 49.0 °C.

[0154] (Batch 14) Batch 14 was analyzed by the same method as used for the analysis of the above batches. The loss in TGA analysis was 0.45 wt% at a temperature of 20 - 150 °C. Although the spectra obtained from LCMS are not shown, the results are summarized in Table 5. The purity by qNMR was 91.6% and the melting point was 46.9 °C.

[0155] (Batch 15) Batch 15 was analyzed by some of the same methods used for the analysis of the above batches. Although the spectra obtained from LCMS are not shown, the results are summarized in Table 5. The purity by qNMR was 98.9% and the melting point was 39 °C.

[0156] (Comparison of batch characteristics: Batches 3, 12, 13, 14, 15, and 16) Table 5 also summarizes the purity, melting point, and visual description of solid Compound 1. The batches of solid Compound 1 occurred as a white free-flowing powder. See Table 5.

[0157] Furthermore, Table 5 shows that the solubility of all batches of Compound 1 prepared was at least 366 - 398 mg / ml, and the appearance of such formulations in water was as a clear transparent or translucent solution. Table 5. Summary of results from the analysis of various batches of Compound 1.

Table 2

Table 3

[0158] (Example 3 - Preparation of an aqueous formulation of Compound 1) (Formulation protocol) 1. Weigh out the required amount of Compound 1, and then add the required amount of excipients (0.9% w / v normal saline, 100 mM PBS pH 7.4, water) to the solid Compound 1 to produce Compound 1 at the required mg / ml concentration. For example, for a 400 mg / ml formulation of Compound 1 in water, weigh out 400 mg of Compound 1 and add 0.7 ml of water. 2. Sonicate the solution for 10 minutes and then shake it for 20 minutes to ensure that Compound 1 is completely dissolved. 3. If necessary, centrifuge the solution (13000 rpm, 10 minutes) to remove fine particles. 4. If necessary, the solution can be sterile filtered. Table 7. Data of formulations of various batches of Compound 1 in water prepared according to the above protocol

Table 4

[0159] (Formulation 50% w / v) Four batches of Compound 1 were formulated in PBS at 50% w / v.

[0160] Weigh out ~500 mg of the compound, place it in a vial, add ~500 μl of 100 mM PBS pH 7.4, sonicate the vial for 10 minutes, and then shake it for 20 minutes. Then, take out the sample, dilute it 1 / 2000, and calculate the concentration by HPLC analysis. Table 8. Preparation of Compound 1 formulation in PBS at pH 7.4. The concentration of Compound 1 was measured in the soluble formulation by HPLC.

Table 5

[0161] (Formulation Liquid Compound 1) Batch 3 of Compound 1 was heated in an oven to 60 °C for 20 minutes, at which point it became a translucent pale yellow liquid. 100 mM PBS pH 7.4 (20% v / v) was added and the solution was mixed on a shaker for 20 minutes. After this, the solution had cooled to room temperature and remained a translucent solution. The solution was left at 4 °C for 72 hours. Subsequent observation confirmed that it remained a translucent solution.

[0162] (Summary of Formulations) The amount of Compound 1 that can be formulated in an aqueous solution such as 0.9% w / v saline, 100 mM PBS pH 7.4, or water does not appear to have a reachable limit. This may be explained by the melting point of Compound 1 measured up to about 47 - 50 °C in several batches. When an aqueous solution is added to the solid, it disrupts the intramolecular interactions of the Compound 1 molecules and becomes miscible with water.

[0163] (Example 4 - Gel Formulation) Compound 1 is formulated by placing it in a gel pack at 2.25 mg / ml.

[0164] (Details of Experiments) One HydroGel gel pack (transparent H20 hydrogel, 8 ounce portion, HydroGel, Portland, ME) was taken and dispensed into various Falcon tubes for experiments.

[0165] First, using blue food coloring, it was investigated how easily an aqueous solution could be mixed into the gel. Two gel samples were taken, one held at RT and the other melted in the microwave (1 minute). Blue food coloring (1% v / v) was added and the solution was mixed. Mixing is much more efficient when the gel is melted. The coloring can be fully incorporated after mixing for less than 10 seconds.

[0166] Next, the coloring solution was replaced with a solution of Compound 1.

[0167] Water was added to Compound 1 (225 mg / ml, 100× the required concentration), sonicated for 20 minutes, and then shaken for 30 minutes. Samples were taken for HPLC analysis and the concentration was checked. Table 9. Measured Concentration of Compound 1 in the Water Stock [Table 6]

[0168] This analysis showed that Compound 1 was completely solubilized.

[0169] Another two gels were dissolved and an aqueous solution of Compound 1 was added (1% v / v). The gels were shaken for the same length of time as when the dye was added (10 seconds). The gels were allowed to solidify.

[0170] Once the gels had solidified, samples were taken for HPLC analysis to check that Compound 1 was uniformly distributed.

[0171] (Sampling Procedure) 1. Add a gel sample (100 mg) to an Eppendorf and add MeOH (0.9 ml, 1 / 10 dilution) 2. Shake the sample on a vibrax for 30 minutes 3. Centrifuge the sample (13000 rpm, 10 minutes) 4. Take the supernatant for HPLC analysis Table 10. Measured Concentration of Compound 1 in the Gel Sample [Table 7]

[0172] The concentration of Compound 1 was slightly lower than the expected 2.25 mg / ml, but the two samples were in good agreement, suggesting that Compound 1 was uniformly distributed and that the dilution factor was slightly off.

[0173] One of the gel samples was kept at RT (room temperature) and the other at 4 °C to test the stability of Compound 1 in the gel.

[0174] (Stability of the gel formulation) The gel was sampled at regular intervals as described above to check its stability at both 4 °C and RT. The data obtained from these experiments are shown in Tables 11 - 12. Table 11. Concentration of Compound 1 in gel samples taken after 20 days of storage at 4 °C [Table 8] Table 12. Concentration of Compound 1 in gel samples taken after 20 days of storage at RT (about 20 °C) [Table 9]

[0175] From the data, especially the general trend in the stability data at 230 nm, it is suggested that Compound 1 is stable in the gel formulation for at least 20 days. The AUC data have more errors due to inaccuracies in weighing the gel samples for extraction and, in some cases, differences in the localized concentration of Compound 1 in the gel.

[0176] (Protocol for the preparation of the gel formulation) A 100× concentrated aqueous solution of Compound 1 can be prepared and added to the gel at 1 / 100 of the gel's volume. The example given is for a final concentration of 2.25 mg / ml in a 200 ml gel pack, and thus 2 ml of 100× Compound 1 (225 mg / ml) in water is required. It is also proposed to add food coloring to the stock solution and inject the combined solution into the gel pack (under the condition that this has no harmful effect on the test). This gives a visual inspection that the Compound 1 solution is uniformly distributed in the gel. When including food coloring, it is recommended to add food coloring to the gel in the control group as well. 1. Weigh 500 mg of Compound 1 and place it in a 3 ml vial (or similar). 2. Add 2 ml of water (the addition of 2 ml of water to 500 mg of Compound 1 corresponds to the volume of the solid Compound 1, and it has been confirmed by the HPLC calibration curve that it is 225 mg / ml), sonicate for 20 minutes, and then shake for 30 minutes (the solution may remain slightly cloudy). Add 1 ml of natural food coloring (the food coloring helps to indicate that a uniform distribution has been achieved). 3. If necessary, this solution can be sterile filtered. 4. When the unopened gel pack is heated by immersion in water at 70 °C for 10 minutes, the gel becomes a fluid liquid. 5. Aspirate the Compound 1 solution and place it into a needle / syringe (with / without food coloring). 6. Inject the Compound 1 solution (and food coloring) into the gel pack by making small holes in the gel pack with a needle. 7. Use tape to close the gel pack again at the injection site. 8. Vigorously shake the gel pack for 5 minutes to obtain a uniform distribution of Compound 1 (when food coloring is included, it becomes apparent when the injected solution is uniformly distributed by the color change of the gel). 9. Let the gel solidify, which takes about 45 minutes at room temperature. 10. Use the gel or seal and store it at 4 °C (if opened, it is recommended to store the gel at 4 °C for a maximum of 14 days). 11. Compound 1 is stable in the gel at 4 °C and room temperature for at least 14 days.

[0177] (Protocol Test) The gel formulation was prepared according to the protocol, and HPLC analysis showed that the accurate concentration of Compound 1 was achieved in the water stock and the gel formulation. Table 13. Concentrations of Compound 1 in the water stock solution and the gel formulation

Table 10

[0178] 2 ml of water stock was added to 1 ml of food coloring. This was diluted and analyzed by HPLC. The AUC of the water stock diluted with food coloring was 0.63 times that of the water stock before dilution, indicating that Compound 1 remained soluble when diluted with food coloring.

[0179] (Results / Conclusions) Compound 1 can be formulated at 2.25 mg / ml in an aqueous gel pack and is stable at both 4 °C and RT for at least 20 days.

[0180] (Example 5 - Physiological Saline Formulation of Compound 1) 1. Weigh 400 mg of Compound 1 and add 0.7 ml of physiological saline (0.9% w / v). 2. Sonicate for 20 minutes and then shake for 30 minutes or until all the compound is visibly dissolved.

[0181] (Freeze / Thaw Stability of Compound 1) Method: 1. Add physiological saline (0.9% w / v) to Compound 1 (1 mg / ml) and then sonicate for 10 minutes until completely dissolved. 2. Collect samples for HPLC analysis (F / T 0). 3. Freeze the solution at -80 °C overnight. 4. Thaw the solution and collect samples for HPLC analysis (F / T 1). 5. Repeat steps 3 and 4 for 3 cycles. Table 14. Purity of Compound 1 during Freeze / Thaw (FT) Cycles [Table 11]

[0182] The results (Table 14) show no significant change in the assay or purity of Compound 1 in physiological saline, and thus indicate that Compound 1 is stable during at least 3 freeze / thaw cycles.

[0183] (RT Stability of Compound 1) Method: 1. Add physiological saline (0.9% w / v) to Compound 1 (1 mg / ml), and then sonicate for 10 minutes until completely dissolved. 2. Samples were taken for HPLC analysis (T = 0). 3. The solution was stored at RT. 4. Samples were taken periodically for HPLC analysis regarding stability.

[0184] Results: The results showed no significant change in the assay or purity of Compound 1 in physiological saline (data not shown), thus indicating that Compound 1 is stable at room temperature for at least 14 days.

[0185] (Compound 1 formulation at 200 - 500 mg / ml) Method: 1. Add physiological saline (0.9% w / v) to Compound 1 Batch 11 in various amounts, and then sonicate for 10 minutes - the samples remained slightly turbid. 2. Shake the samples for 30 minutes, at which point the solution became translucent. 3. Dilute the solution for HPLC analysis.

[0186] Results: Table 15. Purity of Compound 1 at increasing concentrations in physiological saline solution

Table 12

[0187] (Protocol for transfer (400 mg / ml formulation)) 3. Weigh out 400 mg of Compound 1 Batch 11 and add 0.7 ml of physiological saline (0.9% w / v). 4. Sonicate for 20 minutes and then shake for 30 minutes or until all the compound is visually dissolved.

[0188] (Results / Conclusions) (Freeze / Thaw Stability of Compound 1) Compound 1 is stable during at least 3 freeze / thaw cycles.

[0189] (RT Stability of Compound 1) Compound 1 is stable at room temperature for at least 14 days (data collection is to be continued).

[0190] (Compound 1 400 mg / ml Formulation) It is possible to reach Compound 1 exceeding 500 mg / ml in physiological saline.

[0191] (Example 6 - Stability of Compound 1 in Water and DMSO) Compound 1 was separately dissolved in water and DMSO at a concentration of 1 mg / ml and stored at RT, 37 °C, and 65 °C for stability over 40 days. Table 16. Compound 1 in water (concentration 1 mg / ml) at RT in the dark using purity measurement

Table 13

[0192] Slight losses in purity and assay are observed for Compound 1 in water at room temperature over 38 days. Table 17. Compound 1 in water (concentration 1 mg / ml) at 37 °C in the dark using purity measurement

Table 14

[0193] Losses in purity and assay are observed for Compound 1 in water at 37 °C over 38 days. The loss in assay is significantly greater than the loss observed in water at room temperature. Table 18. Compound 1 in water (concentration 1 mg / ml) at 65 °C in the dark using purity measurement

Table 15

[0194] Losses in purity and assay are observed with Compound 1 in water over 29 days. The losses in purity and assay are significantly greater than those observed in water at 37 °C. Table 19. Compound 1 (concentration 1 mg / ml) in DMSO at 37 °C in the dark using purity measurement [Table 16]

[0195] Neither significant loss in purity nor significant loss in assay is observed with Compound 1 in DMSO at 37 °C over 37 days. Table 20. Compound 1 (concentration 1 mg / ml) in DMSO at 65 °C in the dark using purity measurement [Table 17]

[0196] Neither significant loss in purity nor significant loss in assay is observed with Compound 1 in DMSO at 65 °C over 29 days.

[0197] (Example 7 - Analysis of Compound 1) The material obtained from Method C of Example 1 (Batch 15) was analyzed by XRPD. The data is shown in Figure 12, which indicates a crystalline material.

[0198] The material obtained from Method A (Batch 12) was also analyzed by XRPD and appeared to show a crystalline material with the same polymorph (Figure 11).

[0199] (Example 8 - Comparison of Solubilities of Compound 1 and Other Succinic Acid Prodrugs) The solubility of Compound 1 in aqueous formulations was evaluated by dissolving the solid material in the aqueous formulation and measuring the amount in solution by HPLC(-MS), and compared with other succinic acid prodrugs. It was found that Compound 1 dissolved in water above 350 mg / mL, in PBS (pH 7.4) at 190 mg / mL, and in 0.9% saline above 500 mg / mL, whereas the other succinic acid prodrugs evaluated had much lower solubilities. In many cases, the maximum solubility of the other prodrugs was lower than 100 μM. For example, see Table 21 for examples of solubility data of other succinic acid prodrugs in PBS pH 7.4. Table 21: Solubility of exemplary succinic acid prodrugs

Table 18

[0200] (Example 9 - Comparison of Bioavailability of Compound 1 and Other Succinic Acid Prodrugs) The cell permeability and potential oral bioavailability of Compound 1 were tested using a standard caco-2 bioavailability in vitro assay (Briefly, confluent Caco-2 cells (L1, A. P., 1992; Grass, G. M; et al., 1992, Volpe, D. A. et al., 2001) in a 24-well Corning Costar Transwell format were provided by In Vitro Technologies (IVT, Baltimore, Md., USA). The apical chamber contained 0.15 mL of Hank's balanced buffer solution (HBBS) pH 7.4, 1% DMSO, and 0.1 mM Lucifer Yellow. The basal chamber contained 0.6 mL HBBS pH 7.4, 1% DMSO. The control and test substances were incubated at 37 °C in a humidified incubator and shaken at 130 rpm for 1 hour. Lucifer Yellow permeates only through the paracellular (between tight junctions) pathway, and a high apparent permeability (Papp) of Lucifer Yellow indicates cell damage during the assay. All such wells were rejected. Propranolol (good passive permeation with no known transporter effect) and acebutolol (poor passive permeation attenuated by active efflux by P-glycoprotein) were used as reference compounds. The compound was tested in unidirectional and bidirectional formats by applying the compound (0.01 mM) to the apical chamber or the basal chamber. The compound in the apical chamber or the basal chamber was analyzed by HPLC-MS. The results were expressed as the apparent permeability Papp (nm / s) compared to other succinate prodrugs including the numbers from WO2015 / 155231. The data are shown in Table 22 below, which shows that the movement from the apical side to the basal outside is the highest for Compound 1, indicating an improvement in cell permeability and bioavailability. This was confirmed by in vivo pharmacokinetic studies, in which Compound 1 was also shown to have high oral bioavailability and, unlike the other prodrugs tested, to have brain permeability. Tr ​Table 22. Caco-2 Bioavailability of Compound 1 Compared with Other Succinic Acid Prodrugs [Table 19] TIFF0007699551000024.tif111170

[0201] (Example 10 - Comparison of the Thermodynamic Solubilities of Various Batches of Compound 1) Two batches of Compound 1 with varying degrees of crystallinity were prepared. Batch 2 had a higher degree of crystallinity than Batch 3, which was considered to have a higher degree of amorphous Compound 1 - thus, Batch 2 was considered the more crystalline batch, while Batch 3 was considered the more amorphous batch. The methods used were those discussed in this document. PBS was prepared as usual (NaCl (8 g / L), KCl (0.2 g / L), disodium hydrogen phosphate anhydrous (1.42 g / L), and potassium dihydrogen phosphate anhydrous (0.24 g / L) were added to 250 mL of deionized water, and the mixture was stirred until all solids were dissolved. HCl (1 M) or NaOH (1 M) was used as needed to adjust the pH of the solution to pH 7.4).

[0202] Samples of the more amorphous Compound 1 Batch 3 were added to PBS or water to a final concentration of 258 mg / mL, sonicated for 10 minutes, and then shaken for 30 minutes. After centrifugation to remove solid material, analysis revealed that the concentration had reached 258 mg / mL.

[0203] Samples of the more crystalline Compound 1 Batch 2 of Compound 1 were added to HPLC grade water (Fisher) to a final concentration of 30 mg / mL, sonicated for 10 minutes, and then shaken for 30 minutes. After centrifugation to remove solid material, analysis revealed that the concentration had reached 17 mg / mL.

[0204] A sample of Compound 1 Batch 2, which is more crystalline, was added to HPLC-grade water (Fisher) to a final concentration of 52 mg / mL, sonicated for 20 minutes, and then shaken for 1 hour. After centrifugation to remove the solid material, analysis revealed that the concentration had reached 52 mg / mL.

[0205] As can be seen from the data presented - the more amorphous material has a much higher dynamic solubility than the more crystalline material.

[0206] A sample of Compound 1 Batch 2, which is more crystalline, was added to HPLC-grade water (Fisher) to a final concentration of 2000 mg / mL, sonicated for 20 minutes, and then shaken for 1.5 hours. After centrifugation to remove the solid material, analysis revealed that the concentration had reached 850 mg / mL. This result indicates that the water solubility of the more crystalline compound is at least 850 mg / ml, i.e., it has a high water solubility, but the dynamic solubility is higher in the case of the more amorphous Compound 1.

[0207] (Example 11 - Comparison of the Stability of Compound 1 in Purified or Unpurified Water) A comparative experiment was planned regarding the stability of Compound 1 in "purified" HPLC-grade water (Fisher Scientific) and "unpurified" tap water. Briefly, 1 mg / mL solutions of Compound 1 were prepared in "purified" HPLC-grade water (Fisher Scientific) and "unpurified" tap water. These were incubated at room temperature for up to 10 days. The concentration and purity of Compound 1 were evaluated over time by HPLC, compared to a standard (for the calculated concentration, the AUC of the 8.21 RT peak at 230 nm and for the purity analysis, the AUC of the Compound 1 peak versus the impurities). The data presented are the averages of two samples. Table 23: Compound 1 Batch 3 dissolved at 1 mg / ml in tap water and stored at room temperature for several days is shown in the table

Table 20

Table 21

[0208] As can be seen from the loss of purity and assay, significant decomposition of Compound 1 was observed in samples dissolved in (unpurified) tap water after storage at room temperature for 6 days. This was not observed in samples dissolved in (purified) HPLC-grade water (Fisher Scientific). Similar data were also seen for two independent samples of various batches of Compound 1.

[0209] (Example 12 - HP-Cyclodextrin Formulation of Compound 1) (Preparation of Excipient) Kleptose hydroxypropyl β-cyclodextrin (25% w / v), sodium dihydrogen phosphate anhydrous (0.048% w / v), disodium hydrogen phosphate anhydrous (0.295% w / v), and calcium disodium EDTA (0.5% w / v) were added to 100 mL of deionized water. The mixture was sonicated for 20 minutes and then stirred until all solids were dissolved, and then adjusted to pH 7.4 with HCl (1 M).

[0210] (Formulation Protocol) 1. Weigh out the required amount of Compound 1, and then add the required amount of the prepared excipient to the solid Compound 1 to yield the required mg / ml concentration of Compound 1 (maximum amount tested 25 mg / ml). For example, for a 20 mg / ml formulation of Compound 1, weigh out 20 mg of Compound 1 and add 1 ml of the prepared excipient. 2. Sonicate the solution for 10 minutes and then shake for 20 minutes to ensure complete dissolution of Compound 1. 3. If necessary, centrifuge the solution (13000 rpm, 10 minutes) to remove fine particles. 4. If necessary, the solution can be sterile filtered.

[0211] (Example 13 - Injection of Compound 1 supplies succinic acid, increases succinate metabolism in pigs, and decreases blood lactate concentration) Injection of Compound 1 increases plasma succinate levels, increases the metabolism of succinate to fumarate in tissues, and decreases blood lactate concentration. See Figure 7.

[0212] Yorkshire - Landrace crossbred pigs were anesthetized and implanted with an intravenous catheter for injection of Compound 1 or vehicle (PBS) and collection of blood samples. Two animals were administered increasing doses of Compound 1 (2 - 6 mg / kg / min) over 2.5 hours, in which case the dose was increased by 1 mg / kg / min every 30 minutes. One animal was infused at a constant rate of 2 mg / kg / min. Control animals were injected with PBS. Blood samples were collected at 30 - minute intervals and plasma was separated by centrifugation. Crystals and tissue samples were stored frozen and then analyzed for succinic acid by LC / MS method using a Thermo Vanquish UPLC + Thermo Quantis triple - quadrupole MS instrument, an Acquity UPLC HSS C18 (100×2.1 mm, 1.8 μm) column equipped with a guard filter, and gradient elution; A = 0.1% formic acid, B = acetonitrile. [13C] - labeled succinic acid was used as an internal standard.

[0213] Plasma succinate concentration increased proportionally to the time of Compound 1 injection (Figure 7A), indicating the release of succinic acid from Compound 1. At the end of the test, fumaric acid, a primary metabolite of succinic acid in the TCA cycle, was higher in the tissues of animals administered Compound 1 than in vehicle animals, particularly in tissues with high metabolic activity such as the retina, brain, and heart. Therefore, this data suggests that Compound 1 supplies metabolizable succinic acid to these tissues and has the ability to cross the blood - brain barrier.

[0214] Blood lactate data pooled from three animals was expressed as a percentage of the initial value and plotted as a function of the cumulative dose at the time of sample collection (Figure 7C).

[0215] After intravenous injection of Compound 1, lactic acid decreased compared to the initial value, suggesting that Compound 1 supplies succinic acid to Complex 2, increases the supply of electrons to the mitochondrial electron transport system, increases ATP production, and reduces the need for glycolytic conversion from pyruvate to lactic acid.

[0216] (Example 14 - Porcine Model of Rotenone - Induced Mitochondrial Complex 1 Dysfunction) Injection of Compound 1 restores the succinic acid level in organs depleted by rotenone and reduces rotenone - induced lactic acid in the brain. See Figure 8.

[0217] To examine the effect of rotenone - mediated inhibition of Complex 1, Yorkshire - Landrace crossbred pigs were anesthetized and implanted with an intravenous catheter for co - injection of rotenone and Compound 1 or vehicle (PBS). Rotenone (7.1 mg / hour) was injected for 1.5 hours. Compound 1 was injected at a constant rate of 2 mg / kg / min over 2.5 hours. PBS was injected into control animals. Blood samples were collected at 30 - minute intervals and plasma was separated by centrifugation. Microdialysate was collected by inserting a microdialysis probe into the striatum of the brain and analyzed for lactic acid using an ISCUS device (MDialysis). At the end of the injection, the animals were euthanized and terminal blood and organ samples were collected. Plasma and tissue samples were stored frozen and then analyzed for succinic acid by LC / MS using a Thermo Vanquish UPLC + Thermo Quantis triple quadrupole MS device, an Acquity UPLC HSS C18 (100×2.1 mm, 1.8 μm) column equipped with a guard filter, and gradient elution; A = 0.1% formic acid, B = acetonitrile. [13C] - labeled succinic acid was used as an internal standard. Lactic acid data was expressed as a percentage of the baseline value obtained after the start of rotenone injection.

[0218] Rotenone injection decreased the tissue concentration of succinic acid to a sub-detectable level (<2 μM), indicating an increased utilization of succinic acid that counteracted the decrease in electron transfer from Complex 1. Administration of Compound 1 restored the tissue succinic acid concentration to a detectable level, suggesting that the supply of succinic acid by Compound 1 exceeded the increased utilization of succinic acid caused by Complex 1 inhibition. Furthermore, administration of Compound 1 neutralized the increase in brain lactate induced by rotenone, supporting the decreased need for glycolytic conversion from pyruvate to lactate by the supply of succinic acid to the brain.

[0219] (Example 15 - Mouse Hereditary Ndufs4 Knockdown Model of Complex 1 Dysfunction) Administration of Compound 1 in drinking water (1 mg / mL) from weaning (21 days) transiently resulted in a tendency for increased body weight and extended survival in the high-dose group. See Figure 9.

[0220] C57BL / 6 mice with genetic ablation of the Complex I gene Ndufs4 (Quintana et al., Proceedings of the National Academy of Sciences. 107, 24 (2010), 10996 - 11001) were administered Compound 1 (1 mg / mL) or normal drinking water in drinking water from weaning. Body weight development was monitored every 10 days (Figure 9A), and the health of the animals was monitored daily (Figure 9B). Compound 1 increased weight gain (p < 0.05) and increased survival rate (p = 0.0697) during the first 10 days. These results suggest that the therapeutic effect of succinic acid supplementation in the form of Compound 1 can be achieved in a genetic model of mitochondrial Complex I dysfunction with features of Leigh syndrome.

[0221] (Example 16 - Rat Model of Rotenone-Induced Motor Dysfunction and Lactic Acidosis) Compound 1 administered in drinking water prevented motor dysfunction and decreased blood lactate concentration. See Figure 10.

[0222] Using the rotenone-induced rat Parkinson's disease model (Cannon et al., Neurobiol Dis. 2009 May;34(2):279-90), the effect of oral administration of compound 1 on the motor and metabolic dysfunctions caused by complex 1 inhibition was investigated.

[0223] Twelve-week-old Lewis rats (6 animals per group) were given daily intraperitoneal injections of rotenone (0.25 - 0.75 mg / kg) for 4 days. Compound 1 was dissolved in drinking water at concentrations of 0.25 and 0.75 mg / mL. Functional tests and lactate measurements were performed on day 4. The rise was measured by placing the animal in a clear glass outer cylinder (height = 30 cm; diameter = 18 cm) for 5 minutes. To be classified as a rise, the forelimbs should be lifted above shoulder height and one or both forelimbs should touch the wall of the cylinder.

[0224] Postural instability was measured on a table surface covered with P-120 sandpaper marked with lines and numbers every 1 centimeter (see below). The animal was held in a position perpendicular to the surface at an angle of approximately 90 °C (a position like a "wheelbarrow"), and one forelimb was gently restrained against the animal's body. Then, the center of gravity of the animal was moved forward onto the single forelimb touching the ground to induce a two-step "catching up" gait and to restore its balance. The change in the position of the nose was recorded as the distance to induce the catching up gait of the unrestrained forelimb. This experiment was repeated 3 times for each forelimb, and the average of both forelimbs was calculated. Blood lactate (Figure 10C) was measured with a VetScan iSTAT-1 Analyser.

[0225] Rotenone treatment resulted in a decrease in rising activity. Administration of compound 1 (0.75 mg / mL) in drinking water resulted in a significant increase in rising (Figure 10A) and postural instability (Figure 10B) compared to the treated animals given water. Therefore, this data suggests that compound 1 is orally bioavailable and can improve the motor dysfunction caused by mitochondrial complex 1 dysfunction.

[0226] The blood lactate concentration increased significantly in animals treated with rotenone. There was a tendency for the lactate concentration in the blood to decrease from a low concentration (0.25 mg / mL) to a high concentration (0.75 mg / mL) of Compound 1 in the drinking water. This data suggests that succinic acid supplied from Compound 1 can achieve metabolic compensation at the level of glycolysis and the conversion from pyruvate to lactate when supplied via the oral route by intermittent administration in drinking water, implying its suitability as an oral treatment.

[0227] (Example 17 - Succinic Acid Release Data) Briefly, a stock of Compound 1 batch 12 was prepared in 50 / 50 DMSO / MeCN (200 mM, ×200). This mixture was then diluted 10 - fold into microsomal buffer (20 mM, ×20) consisting of K2HPO4 (Sigma Aldrich, 13.9 g / L, anhydrous), KH2PO4 (Sigma Aldrich, 2.72 g / L, anhydrous), MgCl2.6H2O (Fisher, 1.02 g / L), and EDTA (Sigma Aldrich, 0.375 g / L) dissolved in HPLC grade water. A 200 mM malonic acid (Sigma Aldrich) stock was prepared in microsomal buffer (200 mM, ×20). A 20 mM NADPH stock was also prepared in microsomal buffer (20 mM, ×10). A stock of microsomes (Sekisui XenoTech, 0.625 mg / ml) was prepared in 7 ml vials. Samples were prepared for each time point (T = 0, 5, 15, 60 minutes) as follows: 80 μL of microsome stock (final concentration 0.5 mg / mL), 5 μL of compound stock (final concentration 2 mM), 5 μL of malonic acid stock (final concentration 10 mM). The T = 0 sample was quenched by adding 100 μL MeOH. The reaction for all time points was then initiated by adding 10 μL NADPH stock (final concentration 2 mM). At each time point, the reaction was stopped by adding 100 μL MeOH. Samples were shaken for 1 minute, placed on ice for 10 minutes, and then centrifuged at 3000 rpm for 10 minutes. The supernatant was then analyzed by LCMS. Table 25

Table 22

[0228] As can be seen from the data, Compound 1 gradually releases succinic acid when incubated with microsomes.

[0229] (Example 18 - Mineral Instability) Compound 1 batch 14 (2 - 3 mg) was dissolved in room temperature Water for Injection (WFI) (1 mg / ml) containing various sources of minerals commonly found in tap water (containing various accompanying counterions that can distinguish the difference in the effect of cations or anions), and samples were taken at various time intervals for HPLC analysis.

[0230] Mineral sources used in the form of inorganic salts: CuCl2, CaCl2, NiSO4, CoCl2, NH4Cl, MnCl2, NaF, NaNO3, CuSO4, Ca(NO3)2, AlSO4, CaCO3, (NH4)2CO3. Table 26

Table 23

[0231] As can be seen from the data, Compound 1 decomposes more rapidly when in an aqueous solution containing carbonate ions.

[0232] (Example 19 - Carbonic Acid Instability) Compound 1 batch 14 (2 - 3 mg) was dissolved in room temperature HPLC - grade water (1 mg / ml) containing calcium and carbonate ions from various sources (recording two concentrations and pH for each source), and samples were taken at various time intervals for HPLC analysis.

[0233] Sources of calcium and carbonate used in the form of inorganic salts: CaCl2, Ca(NO3)2, CaCO3, (NH4)2CO3 Table 27

Table 24

[0234] As can be seen from the data, Compound 1 decomposes more rapidly when it is in an aqueous solution containing carbonate ions.

[0235] (Example 19 - Carbonate Concentration Dependence) Compound 1 batch 14 (2 - 3 mg) was dissolved in HPLC-grade water (1 mg / ml) containing calcium carbonate at different concentrations, and samples were taken at different time intervals for HPLC analysis. Table 28:

Table 25

[0236] (Example 20 - Dynamic Solubility) Solid Compound 1 batches s3, s12 - 17 (~80 mg) were added to the wells of a transparent flat-bottom 96-well plate. When PBS at 5 °C was added, a final concentration of 460 mg / ml of 01 - 354 for all batches was obtained. After adding PBS, the plate was stirred for 10 seconds and then immediately analyzed in real-time for 3 minutes at 620 nm turbidity using an Epoch Microplate Spectrophotometer (BioTek). The rate constant of dissolution was calculated from the exponential decay fit of the recorded data.

[0237] Table 29:

Table 26

[0238] (General Method for Evaluating Crystallinity) An X-ray powder diffraction test was performed using a Bruker AXS D8 Discover HTS. Anode: Cu anode at 40 kV and 4 mA; Gobel mirror and line optics Detector: Linear detector (LYNXEYE XE) with a receiver slit of 2.95 °C Measurement: Scan range 2 - 45 °C 2θ, 1 s / step, 0.005 °C / step Data acquisition software: Diffrac.Commander v7.3.3.0.0 Data analysis software: Diffrac Eva v4.2.1

[0239] Neither background correction nor smoothing was applied. The data is reported as peak 2θ angles and intensities. To determine the degree of crystallinity, the combined area of all defined peaks was divided by the area under the total curve and expressed as a percentage.

[0240] (Peak list) The following list details the peaks returned after XRPD for several crystal batches. The underlined peaks are common to most batches. Those with an asterisk next to the angle may be peaks common or specific to polymorphic forms. Peak list NV354-s3-batch 3 [Table 27] TIFF0007699551000033.tif152170Peak list NV354-s12-batch 18 [Table 28] Peak list NV354-s13-batch 13 [Table 29] TIFF0007699551000036.tif235170Peak list NV354-s14-batch 14 [Table 30] TIFF0007699551000038.tif54170Peak list NV354-s15-batch 19

Table 31

Table 32

Table 33

Claims

1. Isolated methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (Compound 1) in solid form, having a purity of at least 80% w / w and an X-ray powder diffraction pattern having one or more signals at 9.4, 11.1, 11.4, 12.9, 14.9, 16.2, 16.9, 19.7, 20.1, 21.4, 24.8 and 41.6 (±0.2 degrees, 2-theta value).

2. The isolated Compound 1 according to Claim 1, in free form or in the form of its salt, hydrate, solvate, or complex.

3. The isolated Compound 1 according to any one of Claims 1 or 2, having a melting point or melting range in the range of 35 to 55°C.

4. The isolated Compound 1 according to any one of Claims 1 to 3, which is a crystalline product or an amorphous product, or a mixture thereof.

5. The isolated Compound 1 according to any one of Claims 1 to 4, having a water solubility at room temperature of at least 300 mg / mL.

6. The isolated Compound 1 according to Claim 5, wherein the water solubility at room temperature is in the range of 300 mg / mL to 900 mg / mL.

7. 0.005 to 0.2 s -1 The isolated compound 1 according to any one of claims 1 to 6, having a dynamic solubility corresponding to a rate constant in the range of

8. A pharmaceutical composition comprising the isolated Compound 1 according to any one of Claims 1 to 7.

9. The pharmaceutical composition according to Claim 8, for use in medicine.

10. The pharmaceutical composition according to Claim 9, wherein the pharmaceutical composition comprises the isolated Compound 1 according to any one of Claims 1 to 7 as an active pharmaceutical ingredient.

11. The pharmaceutical composition according to Claim 9 or 10, for use in the treatment or prevention of metabolic diseases, mitochondrial dysfunction diseases, diseases associated with mitochondrial dysfunction, mitochondrial disorders, mitochondrial energy deficiency, drug-induced mitochondrial side effects, cancer, diabetes, traumatic brain injury, acute liver injury, and atrial fibrillation.

12. The pharmaceutical composition according to Claim 9 or 10, for use in the treatment or prevention of metabolic dysfunction.

13. The pharmaceutical composition according to claim 9 for use in the treatment or prevention of Leigh syndrome, Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, mitochondrial deletion syndrome, mitochondrial myopathy, secondary mitochondrial diseases, such as Parkinson's disease and hereditary enzyme deficiencies that limit the supply of NADH to complex I, such as pyruvate dehydrogenase complex deficiency.

14. Use of the isolated compound 1 according to any one of claims 1 to 7 for the manufacture of a composition for non-pharmaceutical use in humans or animals.

15. A pharmaceutical cosmetic comprising the isolated compound 1 according to any one of claims 1 to 7.

16. A nutricosmetic comprising the isolated compound 1 according to any one of claims 1 to 7.

17. An energy drink comprising the isolated compound 1 according to any one of claims 1 to 7.

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