EA045539B1 - SUCCINATE PRODRUG, COMPOSITIONS CONTAINING SUCCINATE PRODRUG, AND THEIR APPLICATION - Google Patents

Description

Field of invention

The present invention relates to the field of chemistry, pharmacologically active compounds, pharmaceutical compositions containing such compounds, and nutrition. In particular, the invention relates to cell-permeable succinate precursors used as medicines and food additives.

BACKGROUND OF THE INVENTION

Mitochondria are organelles in eukaryotic cells that produce most of the adenosine triphosphate (ATP) reserves used as an energy source. Thus, mitochondria are essential for energy production, for the survival of eukaryotic cells, and for proper cellular function. In addition to providing energy, mitochondria are involved in a number of other processes such as redox and ion balance, cell signaling, cell differentiation, cell death, and control of metabolic processes, the cell cycle, and cell growth. In particular, mitochondria are critical regulators of cell apoptosis and also play an important role in several forms of non-apoptotic cell death, such as necrosis.

Mitochondrial dysfunction contributes to a wide range of diseases and can be caused by mutations or deletions in the mitochondrial or nuclear genome, a primary or secondary disorder of the mitochondrial respiratory system, or other mechanisms associated with abnormal mitochondrial function. There is currently no treatment for mitochondrial diseases.

The oxidation of nutrients to produce useful chemical energy in the form of ATP occurs largely in the mitochondria through a series of chemical reactions in the tricarboxylic acid cycle and the electron transport chain. NADH, produced in the tricarboxylic acid cycle, is part of complex I in the electron transport chain. Succinate is a metabolic intermediate of the tricarboxylic acid cycle in mitochondria and is unique in that it is directly metabolized by the enzyme succinate dehydrogenase complex II in the electron transport chain. Succinate may also act as a signaling molecule reflecting the state of cellular metabolism.

Because of the recognized importance of maintaining or restoring normal mitochondrial function or increasing cellular energy production (ATP) in treating diseases and conditions associated with mitochondrial dysfunction or improving mitochondrial function, there is a need for compounds having cell permeability, the ability to release intracellular succinate or a succinate precursor, low toxicity of the compound and intracellular release by-products, as well as physicochemical properties appropriate for administration to a subject or patient.

Succinate compounds have been prepared as prodrugs of other active agents, for example, WO 2002/28345 describes bis(2,2-dimethylpropionyloxymethyl)succinic acid ester, dibutyryloxymethyl succinic acid ester and bis(1-butyryloxyethyl)succinic acid ester. These compounds deliver formaldehyde in finished form and are intended for different medical applications compared to existing compounds.

Various succinate ester derivatives are known in the art.

WO 97/47584 describes succinate polyols containing several succinate moieties linked together.

WO 2015/155231 describes succinates and succinate precursors that are cell permeable.

Murli et al. (Appl. Environ. Microbiol., 71:2005:4503-4509) described an attempt to chemobiosynthesize 6-deoxyerythronolide B analogs by feeding acyl thioesters to Escherichia coli and Streptomyces coelicolor bacteria. The structure table shows various acyl thioesters, including the formal structure of methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate, but the synthesis was unsuccessful and did not produce the desired product.

There is a need for effective and safe new treatment options for diseases resulting from mitochondrial dysfunction or for improving metabolism by delivering a metabolic substrate. There is also a need for new dietary supplements, nutricosmetics, cosmeceuticals and cosmetics to stimulate energy in a subject and as an antioxidant. Such new therapies, dietary supplements, nutricosmetics, cosmeceuticals and cosmetics must have attractive combinations of properties, including high potency to enhance mitochondrial energy production and/or as an antioxidant, good bioavailability, long plasma half-life, stability in product formulation and low toxicity. In particular, there is a need for such new therapeutic agents, dietary supplements, nutricosmetics, cosmeceuticals and cosmetics that are based on an active ingredient having high water solubility, good cell permeability and, if desired, high penetration of the blood-brain barrier , and/or lead to a decrease in lactate production.

The essence of the invention

In a first aspect, the present invention relates to isolated methyl 3-[(2acetylaminoethylthio)carbonyl]propionate (compound 1) in solid form. It can be free

- 1 045539 form or in the form of a salt, hydrate, solvate or complex.

Compound 1 has the following structure (formula 1)

Oh oh

It has now been unexpectedly discovered that cell permeable compound 1 has a remarkable combination of beneficial properties. It has potent in vivo activity to stimulate mitochondrial energy production and has good oral bioavailability, blood-brain barrier permeability, good plasma stability, and reduces lactate production and restores succinate levels. At the same time, compound 1 has extremely high solubility in water and aqueous systems. Solubility assessment showed a solubility greater than 500 mg/mL, corresponding to ~2.1 M. This extremely high solubility in water is likely due to the low melting point of compound 1 (less than 55 °C), and when an aqueous solvent is added it miscible with aqueous solvent. This allows for very high concentrations of aqueous formulations, allowing high doses of a compound to be orally administered, which is essentially a prodrug of the substrate for metabolism.

In one embodiment, isolated compound 1 is a solid with a melting point or melting range in the range of about 35°C to about 55°C. In preferred embodiments of the invention, 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, but it may also 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% wt./wt. or at least 75% wt./wt. Depending on the production method and storage conditions, Compound 1 may contain crystals and/or it may contain non-crystals, such as amorphous forms of Compound 1 and mixtures thereof. As can be seen from the examples provided herein, all methods used lead to compound 1 with a degree of crystallinity.

It is believed that more than one crystalline form of compound 1 may exist, and compound 1 may also exist as an amorphous solid. As used herein, all forms of compound 1 are included within the scope of this application, including mixtures of two or more forms of compound 1. Thus, the term compound 1 means a compound of formula 1 in solid form, but whether the compound is in crystalline form, in amorphous form, polymorphic form, powder form or mixtures thereof.

In particular, compound 1 was found to have a number of solid forms with different properties. For example, as an amorphous solid or as a mostly amorphous solid, it shows higher kinetic solubility.

It also has crystalline forms (or mostly crystalline forms) that have been formed and show other improved properties when handled in solid form. Purity has also been found to affect the properties of the drug. In particular, the melting point is low and close to body temperature and varies due to the presence of impurities. The stability of Compound 1 preparations is also affected by the presence of impurities, such as in untreated water, which reduce the stability of Compound 1.

In a second aspect, the present invention relates to a composition containing isolated compound 1.

In a third aspect, the present invention relates to a cosmeceutical product containing the isolated compound 1.

In a fourth aspect, the present invention relates to nutricosmetics containing isolated compound 1.

In a fifth aspect, the present invention relates to a method for producing isolated compound 1, wherein said method includes the following steps:

a) reacting N-acetylcysteamine and monomethylsuccinate in the presence of a coupling reagent in an organic solvent at a temperature in the range between 0 and 100°C; And

b) isolating compound 1, thereby obtaining isolated compound 1.

The method typically includes a purification step to improve the purity of the compound.

The compounds represented by Compound 1 of the invention can be used to increase or restore energy production in mitochondria. In particular, the compounds can be used in medicine, nutricosmetics, food supplements, cosmeceuticals and cosmetics. The compounds represented by compound 1 can be used for the prevention or treatment of disorders or diseases

- 2 045539 vaniyah, having a component related to mitochondrial dysfunction, and/or an energy deficiency (ATP) component, as well as to exploit the cell signaling properties of succinate and its anaplerotic effects on metabolic intermediates.

In addition, compared with known succinate prodrugs (such as those mentioned in WO 97/47584), the isolated compound 1 of the present invention shows improved properties for treatment and use as a dietary supplement and cosmetic product, including better cell permeability, more long plasma half-life, good oral bioavailability, reduced toxicity, increased mitochondrial energy release and improved drug properties, for example due to improved water solubility.

In another aspect, the invention relates to a pharmaceutical composition containing the compounds represented by Compound 1.

The pharmaceutical composition may be a solid composition or a solid composition for reconstitution before use.

Alternatively, it may be in the form of a liquid, such as an aqueous solution, including, for example, an aqueous phosphate-buffered saline (PBS) formulation. Typically, the pharmaceutical composition of the invention contains 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 one embodiment, the pharmaceutical composition is a solution of compound 1 in purified water, optionally isotonic with blood.

In another aspect, the present invention relates to the use of Compound 1 in a composition for the treatment or prevention of a metabolic disease, mitochondrial dysfunction, a disease associated with mitochondrial dysfunction, a mitochondrial disorder, mitochondrial energy deficiency, drug-induced mitochondrial side effects, cancer, diabetes, cranial -brain injury, acute liver injury and atrial fibrillation.

In one aspect, the present invention relates to a method for preparing a pharmaceutical composition containing Compound 1.

Brief description of the figures

In fig. Figure 1 shows LCMS analysis of compound 1, lot 3, plotting absorbance units (AU) versus time using HPLC method 2.

In fig. Figure 2 shows LCMS analysis of compound 1, lot 3, plotting total ions on a normalized scale versus time using HPLC method 2.

In fig. Figure 3 shows LCMS analysis of compound 1, lot 3, plotting intensity (%) versus m/z using HPLC method 2.

In fig. Figure 4 shows LCMS analysis of compound 1, batch 12, plotting absorbance units (AU) versus time using HPLC method 2.

In fig. Figure 5 shows LCMS analysis of compound 1, lot 12, plotting total ions on a normalized scale versus time using HPLC method 2.

In fig. Figure 6 shows LCMS analysis of compound 1, lot 12, plotting intensity (%) versus m/z using HPLC method 2.

In fig. 7A and 7B show intravenous infusion of PBS or Compound 1 into an anesthetized pig, showing plasma succinate concentration as a function of infusion time (A), as well as tissue fumarate concentration (B).

In fig. 7C shows intravenous infusion of PBS or Compound 1 into an anesthetized pig, demonstrating the effect on blood lactate concentration.

In fig. 8 shows an intravenous infusion of PBS or Compound 1 into an anesthetized pig co-administered with the complex 1 inhibitor rotenone, showing tissue succinate concentrations at the end of infusion (A), as well as lactate concentrations in brain microdialysates expressed as a percentage of the pre-infusion baseline value as a function of from time (B).

In fig. 9 shows treatment of Ndufs4 KO mice with Compound 1 in drinking water, showing the change in body weight over time (A) and percent survival over time (B).

In fig. 10 shows treatment of rotenone-injected rats with Compound 1 in drinking water, showing the number of upright stances compared with treatment (A), displacement distance in the postural instability test compared with treatment (B), and blood lactate concentration compared with treatment ( WITH).

In fig. Figure 11 shows X-ray powder diffraction analysis of compound 1, batch 12.

In fig. Figure 12 shows X-ray powder diffraction analysis of compound 1, batch 15.

In fig. 13 shows LCMS analysis of compound 1, lot 3, plotting absorbance units (AU) versus time using HPLC method 1.

In fig. 14 shows LCMS analysis of Compound 1, Lot 3, plotting total ions on a normalized scale versus time using HPLC Method 1.

In fig. Figure 15 shows LCMS analysis of Compound 1, Lot 3, showing intensity (%) per

- 3 045539 depending on m/z using HPLC method 1.

In fig. 16 shows LCMS analysis of compound 1, lot 12, plotting absorbance units (AU) versus time using HPLC method 1.

In fig. 17 shows LCMS analysis of Compound 1, Lot 12, plotting total ions on a normalized scale versus time using HPLC Method 1.

In fig. 18 shows LCMS analysis of compound 1, lot 12, plotting intensity (%) versus m/z using HPLC method 1.

In fig. Figure 19 shows X-ray powder diffraction analysis of compound 1, batch 3.

In fig. Figure 20 shows X-ray powder diffraction analysis of compound 1, batch 18.

In fig. Figure 21 shows X-ray powder diffraction analysis of compound 1, lot 13.

In fig. Figure 22 shows X-ray powder diffraction analysis of compound 1, batch 14.

In fig. Figure 23 shows X-ray powder diffraction analysis of compound 1, batch 19.

In fig. Figure 24 shows X-ray powder diffraction analysis of compound 1, batch 16.

In fig. Figure 25 shows X-ray powder diffraction analysis of compound 1, batch 17.

Description of the invention

In a first aspect, the present invention relates to isolated methyl 3-[(2acetylaminoethylthio)carbonyl]propionate (compound 1) in solid form. It may be in free form or as a salt, hydrate, solvate or complex.

Compound 1 has the following structure (formula 1):

Oh Oh

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

Compound 1 may also be a solvate. Suitable solvates may include hydrates, ethanolates.

Compound 1 can also be in the form of a complex. Examples of suitable complexes include compound 1 complexed with cyclodextrin, lipids, triglycerides, carbohydrates, PVA,

In one embodiment, isolated compound 1 is a solid with 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 has different melting points, most likely depending on the content of different forms of Compound 1, such as crystalline forms, amorphous forms, etc. In particular, melting points in the range of 39 to 51°C were found, such as a melting point of 39°C and melting temperatures in the range of 46 to 51°C, such as about 46-47, 48-49 and 50-51° WITH.

In preferred embodiments of the invention, isolated compound 1 has a purity of at least 80% w/w, such as at least 85% w/w, at least 90% w/w, such as at least 95% wt./wt. or at least 97% w/w, but it may also be of lower purity, for example at least 30% w/w, at least 40% w/w, at least 45% w/w ./wt., at least 50% wt./wt., at least 55% wt./wt., at least 60% wt./wt., at least 65% wt./wt., according to at least 70% w/w or at least 75% wt./wt. Depending on the production method and storage conditions, Compound 1 may contain crystals and/or it may contain non-crystals, such as amorphous forms of Compound 1 and mixtures thereof. As can be seen from the examples provided herein, all methods used lead to compound 1 with a degree of crystallinity. Compound 1 may also be in the form of a powder.

As can be seen from the examples herein, Compound 1 has excellent solubility in water at room temperature (20-25°C). At pH 7.4 and in the aqueous environment tested in the Examples, Compound 1 has a water solubility of at least 300 mg/ml. The solubility of compound 1 in water depends on the crystallinity of the compound; thus, the lower the degree of crystallinity, the higher the solubility in water. As seen in Example 10 herein, the substantially amorphous material may have a water solubility of 850 mg/mL. Therefore, the solubility of compound 1 in water is expected to range from 300 mg/ml to about 900 mg/ml.

Kinetic solubility was also determined and kinetic solubility rate constants were found to be in the range of 0.005 to 0.2 s ^-1 , for example in the range of 0.01 to 0.15 s ^-1 . Kinetic solubility may depend on various factors such as particle size, crystallinity, amorphous material content, etc.

Regarding the crystallinity of Compound 1, it may have a degree of crystallinity ranging from 0 to 100%, such as 10 to 100%, 20 to 100%, 30 to 100%, 40 to 100%, 50 to 100% , from 60 to 100%. As can be seen from the examples provided herein, many batches received

- 4 045539 by the method described herein, have a crystallinity of at least 50%, for example in the range of from about 50% to about 80%.

As can be seen from the powder X-ray diffraction data given in the examples, the crystals of compound 1 are characterized by a powder X-ray diffraction pattern with signals at 21.4, 22.2, 22.8, 23.1 and 23.3 (±0.2°, values 2 -theta).

Compound 1 crystals may also have one or more signals at 10.9, 13.1, 14.9, 16.2, 20.1, 24.0, 24.8, 26.1, such as two or more, three or more, four or more, six or more, seven or more or eight. As can be seen from the examples, almost all studied compounds have signals corresponding to these degrees (±0.2°, 2-theta values).

From the data presented in the Examples, it is assumed that the signals at 11.1 and 16.9 (±0.2°, 2-theta values) are due to the polymorphic form of compound 1 (form 1). Thus, crystals of compound 1 may have an X-ray powder diffraction pattern with signals at 11.1 and 16.9 (±0.2°, 2-theta values) either in addition to one or more of the signals mentioned above or as an alternative .

As mentioned above, compound 1 is in solid form, particularly including crystals of the compound. The melting point is quite low, but it is preferable that Compound 1 is not in oil form. First, Compound 1 will be easier to handle in the manufacture of a pharmaceutical/cosmeceutical composition (eg, grinding, powder flowability, and compressibility). Second, the crystalline form is usually the most stable form, and non-crystalline (less ordered) material tends to change to a crystalline (more ordered, lower energy) form over time.

Definitions.

The term compound 1 means a compound of formula 1 in solid form, and the term includes all crystalline forms, all amorphous forms, all polymorphic forms, and mixtures thereof, including mixtures in the same form or in different forms. Compound 1 may also be in powder form.

The term purity as used herein in relation 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 and related impurities, being by-products, aberrant forms of compound 1 (closely related structure) and synthesis precursors for compound 1. Therefore, in a composition containing 10% wt./wt. compound 1, the purity of said compound 1 may be, for example, 95 or 50 wt.%, which means that the compound 1 used to obtain said composition has a purity of 95 or 50 wt.%, respectively. Purity can be assessed by one of a number of methods including qNMP (quantitative NMR spectroscopy), HPLC, etc. In qNMP, a known amount of analyte is dissolved in an NMR solvent with a known amount of internal standard. Obtain a 1H NMR spectrum with a sufficient number of scans to reduce the signal-to-noise ratio. Approximate resonance in internal standard and analyte combined. The ratio of these integrals, combined with knowledge of how many protons the signal contains and the molecular weights of both the analyte and the internal standard, is then used to determine the purity in % w/w. In HPLC, purity is assessed as the area under the curve (AUC) for an analyte compared to other signals with different retention times.

The term isolated, as used herein in relation to compound 1, means the product of compound 1, methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate, obtained by a synthetic reaction and isolated, for example, by purification from various by-products, synthetic precursors and aberrant forms connections 1.

The term nutricosmetic as used herein refers to dietary supplements or cosmetics specifically formulated to support healthy skin, hair and nails with active ingredients that support physiological functions to achieve a healthier and more youthful appearance over time. Unlike topical creams or treatments, nutricosmetics are taken orally and work from the inside out to promote healthy skin, hair, or nails from the inside out.

The term cosmeceutical, as used herein, is intended to refer to a cosmetic product with bioactive ingredients that are believed to have medicinal benefits. Cosmeceutical products are marketed as cosmetics but generally contain at least one biologically active ingredient. Examples of cosmeceuticals include anti-wrinkle skin creams with ingredients such as alpha lipoic acid and dimethylaminoethanol, and creams containing cell repair serum that are claimed to have anti-aging properties.

The term treatment, as used herein, is intended to refer to the provision of therapy to reduce the severity or frequency of symptoms. As used herein, the term treatment refers to both therapeutic treatment and prophylactic or preventive measures.

- 5 045539

The term prevention, as used herein, is intended to mean prevention in whole or in part, or improvement, reduction or control.

Compound 1 is generally covered by formula (I) specified in WO 2015/155231 describing succinates and succinate precursors that are cell permeable. However, upon qualitative analysis of compound I, the inventors made a number of new and unexpected discoveries showing that it has an unexpectedly good combination of properties that make it suitable for a number of therapeutic and non-therapeutic applications. In addition, surprising discoveries have been made regarding the benefits of certain forms and compositions of compound I.

General Use of the Compounds of the Invention.

Methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (compound 1) in free form or its salt, hydrate, solvate or complex as described herein can be used in medicine, particularly in the drug treatment or prevention of conditions , diseases or disorders related to mitochondria, in nutricosmetics or cosmetics. Compound I can also be used in the preparation of a composition for such medicinal treatment or prevention in nutricosmetics or cosmetics. Compound 1 or a salt, hydrate, solvate or complex thereof can be used in any situation where increased or restored energy (ATP) production is desired, such as in the drug treatment of a disease. Drug treatment may relate to metabolic diseases or in the treatment of diseases or conditions of mitochondrial dysfunction or diseases associated with decreased levels of succinate or functional activity of succinate, or a disease that benefits from the anaplerotic effect of succinate or its signaling properties, treatment or suppression of mitochondrial disorders. The compounds represented by Compound 1 can be used to stimulate mitochondrial energy production and to restore drug- or chemical-induced mitochondrial dysfunction such as, for example, sensorineural hearing loss or tinnitus (a side effect of some antibiotics due to mitochondrial toxicity), chemical poisoning substances or gases that affect mitochondrial metabolism, or lactic acidosis. The compounds can be used in the treatment of cancer, diabetes, acute starvation, endotoxemia, sepsis, systemic inflammatory response syndrome, multiple organ failure syndrome and after hypoxia, ischemia, stroke, myocardial infarction, acute angina, acute kidney injury, coronary occlusion and atrial fibrillation or for the prevention reperfusion injury or counteraction to it. Moreover, it is believed that the compounds of the invention may be useful in the treatment of male infertility and menopausal symptoms in women.

The compounds represented by Compound 1 of the invention are expected to provide cell-permeable precursors to components of the Krebs cycle and, optionally, glycolytic pathways. It is assumed that after penetration into the cell, succinate will be released as a result of enzymatic or chemical hydrolysis. This hydrolysis of compound 1 is also considered to be particularly beneficial because the released thiol group has reducing properties. Many diseases have a component of unwanted oxidative stress that can damage cellular structure and cell function. Oxidative stress is also thought to be involved in the aging process. Accordingly, the release of a component that can act as an antioxidant and scavenge free radicals or reduce oxygen-reactive species is expected to provide additional benefit for both medical, nutriocosmetic and cosmetic applications.

Compound 1 can be used to increase or restore mitochondrial energy production. Compound 1 can also be used as an antioxidant and to scavenge free radicals or reduce oxygen-reactive species. Compound 1 can be used for the prevention or treatment of disorders or diseases having a component related to mitochondrial dysfunction and/or an energy deficiency (ATP) component, as well as diseases associated with reduced levels of succinate or functional activity of succinate, or diseases in which it is useful anaplerotic effect of succinate or its signaling properties.

Increasing energy production, for example, is relevant for subjects suffering from a mitochondrial defect, disorder or disease. Mitochondrial diseases result from dysfunction of mitochondria, specialized compartments found in every cell of the body except red blood cells. When mitochondrial function declines, the energy produced inside the cell decreases, leading to cell damage or death.

Mitochondrial diseases most often occur in organs that are very energy-demanding, such as the retina, cochlea, brain, heart, liver, skeletal muscle, kidney, and endocrine and respiratory systems. Symptoms of mitochondrial disease may include loss of motor control, muscle weakness and pain, seizures, vision/hearing problems, heart disease, liver disease, gastrointestinal problems, difficulty swallowing, fatigue and more. Mitochondrial disease may be inherited or may be caused by

- 6 045539 spontaneous mutations that lead to changes in the functions of proteins or RNA molecules usually found in mitochondria. Many diseases have been found to involve mitochondrial deficiency, such as complex I, II, III, or IV deficiency, or an enzyme deficiency, such as pyruvate dehydrogenase deficiency. However, the picture is complex and many factors may be involved in the diseases.

There are still no available treatment procedures. The only treatments available are those that can relieve symptoms and slow the progression of the disease.

Accordingly, the discoveries of the present inventors described herein are very important because they demonstrate the beneficial effect of the cell-permeable succinic acid thioester prodrug Compound 1 on mitochondrial energy production.

In addition, compared with known succinate prodrugs (such as those mentioned in WO 97/47584, for example), the isolated compounds represented by Compound 1 of the present invention show improved properties for treatment and use as nutricosmetic, dietary supplement, cosmeceutical and cosmetic product, including better cell permeability, longer plasma half-life, reduced toxicity, increased mitochondrial energy release, and improved formulation (due to improved properties including increased solubility). In some cases, the isolated compounds represented by compound 1 are also orally bioavailable, making their administration easier.

Thus, the beneficial properties of an isolated compound of the invention may include one or more of the following:

increased cell permeability;

increased bioavailability when taken orally;

longer plasma half-life;

reduced toxicity;

increased energy release into mitochondria;

increased antioxidant activity;

improved composition;

increased solubility.

The present invention relates to compound 1 for use in medicine as a pharmaceutically active substance, in particular for the treatment of cellular energy (ATP) deficiency.

The compound of the present invention can be used to treat a disorder of complex I, or dysfunction of the complex itself, or any condition or disease that limits the supply of NADH to complex I, for example, dysfunction of the Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even glucose transport or substrates associated with complex I.

The present invention also relates to a method of treating disorders associated with mitochondrial complex I, such as, but not limited to, Leigh syndrome, Leber hereditary optic neuropathy (LHON), MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), mitochondrial deletion syndromes, mitochondrial myopathies, and MERRF (myoclonic epilepsy with ragged red fibers), which involves administering to a subject in need thereof an effective amount of a compound of the present invention.

The present invention also relates to the use of the compounds of the invention for the manufacture of a medicament for the treatment of toxin- or drug-induced lactic acidosis/mitochondrial dysfunction.

Isolated Compound 1 can also be used in any conditions that could potentially benefit from additional energy production, such as, but not limited to, long-term surgery and intensive care.

Mitochondria.

Mitochondria are organelles in eukaryotic cells that are commonly referred to as the powerhouse of the cell. One of their main functions is oxidative phosphorylation. The adenosine triphosphate (ATP) molecule functions as the currency or energy carrier of the cell, and eukaryotic cells obtain most of their ATP from biochemical processes carried out by mitochondria. These biochemical processes include the citric acid cycle (tricarboxylic acid cycle or Krebs cycle), which 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 again to NAD+ and FAD.

The electrons released by the oxidation of NADH travel through a series of protein complexes (complex I, complex II, complex III, and complex IV) known as the electron transport chain or respiratory chain. Oxidation of succinate occurs in complex II (succinate dehydrogenase complex), and FAD is a prosthetic group in the succinate dehydrogenase enzyme complex (complex II). Respiratory complexes are embedded in the inner membrane of the mitochondria. Complex

- 7 045539

IV at the end of the chain transfers electrons to oxygen, which is reduced to water. The energy released as these electrons pass through the complexes is used to create a proton gradient across the inner membrane of the mitochondrion, 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 in the electrochemical gradient to convert ADP to ATP.

The citric/tricarboxylic acid cycle and oxidative phosphorylation are preceded by glycolysis, in which a molecule of glucose is split into two molecules of pyruvate, netting two molecules of ATP per molecule of glucose. Pyruvate molecules then enter the mitochondria, where they are fully oxidized to CO2 and H2O through oxidative phosphorylation (a general process known as aerobic respiration). Complete oxidation of two molecules of pyruvate to carbon dioxide and water produces about 28-29 molecules of ATP in addition to the 2 molecules of ATP obtained by converting glucose into two molecules of pyruvate. If oxygen is not available, the pyruvate molecule does not enter the mitochondria, but is converted to lactate through the process of anaerobic respiration.

Thus, the total net yield per glucose molecule is approximately 30-31 ATP molecules. ATP is used to power, directly or indirectly, almost every other biochemical reaction in the cell. Thus, the additional (approximately) at least 28 or 29 ATP molecules contributed by oxidative phosphorylation during aerobic respiration are critical for proper cell function. Lack of oxygen interferes with aerobic respiration and can lead to the death of almost all aerobic organisms; some organisms, such as yeast, can survive using either aerobic or anaerobic respiration.

When body cells are temporarily deprived of oxygen, anaerobic respiration is used until oxygen becomes available again or the cell dies. Pyruvate produced during glycolysis is converted to lactate during anaerobic respiration. Lactic acid buildup is thought to cause muscle fatigue during periods of intense activity when oxygen cannot reach muscle cells. When oxygen becomes available again, lactate is converted back to pyruvate for use in oxidative phosphorylation.

Mitochondrial dysfunction contributes to various disease conditions. Some mitochondrial diseases arise from mutations or deletions in the mitochondrial genome or nucleus. If a threshold proportion of mitochondria in a cell is defective, and if a threshold proportion of such cells in a tissue have defective mitochondria, symptoms of tissue or organ dysfunction may occur. Almost any tissue can be affected, and a wide variety of symptoms may be present, depending on the extent of the different tissues affected.

Use of the compound according to the invention.

The compound of the present invention can be used in any situation where increased or restored energy (ATP) production is desired. Examples, for example, in all clinical conditions where there is potential benefit from increasing mitochondrial ATP production or restoring mitochondrial function, such as in restoring drug or chemically induced mitochondrial dysfunction or lactic acidosis conditions associated with reduced succinate levels or functional succinate activity, conditions, in which the anaplerotic effect of succinate or its signaling properties is useful, as well as the treatment of inborn errors of metabolism, cancer, diabetes, acute starvation, endotoxemia, sepsis, decreased visual acuity, hearing, systemic inflammatory response syndrome and multiple organ dysfunction syndrome.

In particular, compound 1 can be used in medicine, in particular for the treatment or prevention of a condition, disease or disorder associated with mitochondria, in nutricosmetics or cosmetics.

Mitochondrial dysfunction has also been described in association with renal tubular acidosis; motor neuron diseases; other neurological diseases; epilepsy; genetic diseases; Huntington's disease; mood disorders; schizophrenia; bipolar disorder; age-related diseases; cerebrovascular accidents, macular degeneration; diabetes; symptoms of menopause and cancer.

Compound 1 for use in mitochondria-related disorders or diseases.

The compound of the invention can be used to prevent or treat a mitochondrial related disease selected from the following:

aging;

Alpers disease (progressive childhood polyodystrophy);

Alzheimer's disease;

amyotrophic lateral sclerosis (ALS);

autism;

Barth syndrome (lethal childhood cardiomyopathy);

disorders of beta oxidation, deficiency of bioenergy metabolism;

carnitine-acyl-carnitine deficiency;

- 8 045539 carnitine deficiency;

creatine deficiency syndromes (cerebral creatine deficiency syndromes (CCDS) include guanidinoacetate methyltransferase deficiency (GAMT deficiency), L-arginine:glycinamidinotransferase deficiency (AGAT deficiency), and creatine transporter deficiency associated with SLC6A8 (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 aciduria type II;

KSS (Kearns-Sayre syndrome);

lactic acidosis;

LCAD (long-chain acyl-CoA dehydrogenase deficiency);

LC-FAOD (long chain fatty acid oxidation disorders);

LCHAD, Leigh's disease or syndrome (subacute necrotizing encephalomyelopathy);

LHON (Leber's hereditary optic neuropathy);

Luft's disease;

MCAD (medium chain acyl-CoA dehydrogenase deficiency);

MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes);

MERRF (myoclonic epilepsy and ragged red fiber disease);

methylmalonyl-KoA epimerase deficiency;

methylmalonyl-CoA mutase deficiency;

Mitochondrial DNA depletion syndrome 5;

Mitochondrial DNA depletion syndrome 9;

Mitochondrial DNA depletion syndrome 15 (hepatocerebral type) (family 1);

maternally inherited diabetes and deafness;

MIRAS (mitochondrial recessive ataxia syndrome);

mitochondrial cytopathy;

Mitochondrial DNA depletion;

mitochondrial encephalopathy, including encephalomyopathy and encephalomyelopathy, mitochondrial myopathy;

MNGIE (myoneurogastointestinal disorder and encephalopathy);

NARP (neuropathy, ataxia and retinitis pigmentosa);

neurodegenerative disorders associated with Parkinson's, Alzheimer's or Huntington's disease;

Parkinson's disease;

Pearson syndrome;

progressive external ophthalmoplegia;

propionic acidemia;

pyruvate dehydrogenase deficiency;

POLG mutations;

respiratory chain failure;

SCAD (Short Chain Acyl-CoA Dehydrogenase Deficiency)

SCHAD and

VLCAD (very long chain acyl-CoA dehydrogenase deficiency).

Of particular interest is the use of Compound 1 in the treatment of Leigh's syndrome, LHON, MELAS, MERRF (myoclonic epilepsy with ragged muscle fibers) and other diseases/conditions associated with complex I defects.

Use of the compounds according to the invention in cosmetics.

The compounds of the invention can be used in cosmetics for the following purposes:

improvement of metabolic function in dermal cells (aging skin);

astringent (acne);

use of the compounds of the invention as food additives.

- 9 045539

The compounds of the invention can be used as food additives for the following purposes:

increased need for energy due to heavy physical activity;

increased energy demand due to metabolic decompensation during infections and surgical interventions;

Improved muscle recovery through rapid tissue distribution and bypass of glycolysis.

Pharmaceutical compositions containing a compound of the invention.

The present invention also relates to a pharmaceutical composition containing the isolated compounds represented by Compound 1 of the present invention, together with one or more pharmaceutically acceptable diluents or carriers.

A compound of the invention or a composition thereof may be administered by any conventional route, for example, but without limitation, it may be administered parenterally, orally, topically (including mucosally, buccally, sublingually, transdermally, or cutaneously), by means of a medical device (eg, a stent), by inhalation, injection or infusion (intravenous, subcutaneous, intramuscular, etc.). Treatment may consist of a single dose or multiple doses over a period of time.

Treatment may consist of a single dose or multiple doses over a period of time.

Treatment can be carried out by intravenous drip once a day, twice a day, three times a day, four times a day, etc.

Although the compound of the invention can be administered alone, it is preferable to present it in the form of a pharmaceutical preparation together with one or more suitable carriers. The carrier(s) must be acceptable in the sense that they are compatible with the compound of the invention and not harmful to recipients thereof. Examples of suitable media are described in more detail below.

The compositions may be conveniently presented in dosage form, such as unit dosage form, and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of combining the active ingredient (a compound of the invention) with a carrier, which is one or more additional ingredients. Typically, formulations are prepared by uniformly and thoroughly combining the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compound of the invention is typically administered intravenously, orally or by any parenteral route as a pharmaceutical preparation containing the active ingredient, optionally as a non-toxic organic or inorganic acid or addition salt, in a pharmaceutically acceptable dosage form. The compositions can be administered in varying doses depending on the disease and patient being treated, as well as the route of administration.

Pharmaceutical compositions must be stable under manufacturing and storage conditions; thus, it is preferable that they be protected from the contaminating action of microorganisms such as bacteria and fungi. Depending on the type of formulation and route of administration chosen, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

For example, a compound of the invention may also be administered orally, buccally or sublingually in the form of tablets, capsules, ovoid suppositories, elixirs, gels, solutions, emulsions or suspensions, which may contain flavorings or colors, for immediate, delayed or controlled release applications .

The compositions of the present invention suitable for oral administration may be presented in the form of discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; in the form of powder or granules; in the form of a solution or suspension in an aqueous liquid or non-aqueous liquid; or in the form of a liquid emulsion of oil in water or a liquid emulsion of water in oil. The active ingredient may also be presented as a bolus, electuary or paste.

Solutions or suspensions of a compound of the invention suitable for oral administration may also contain excipients, for example solvents such as water, ethanol, etc., N,N-dimethylαcetαmide, dispersants such as polysorbate 80, surfactants and solubilizers eg polyethylene glycol, Phosal 50 PG (which consists of phosphatidylcholine, soy fatty acids, ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate). The compositions of the present invention may also be in the form of emulsions, where Compound 1 may be present in a water-in-oil or oil-in-water emulsion. The oil may be any oil-like substance such as, for example, soybean oil, safflower oil, etc., triglycerides, such as medium chain triglycerides (MCT oil), such as, for example, coconut oil, palm oil, etc. .d. or combinations thereof.

Tablets may contain pharmaceutically acceptable excipients such as fillers, binders, dispersing agents, disintegrants, glidants, pH adjusting agents, stabilizers,

- 10 045539 taste masking agents, etc. Specific examples include microcrystalline cellulose, lactose (such as lactose monohydrate or lactose anhydrous), 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, sodium croscarmellose and some complex silicates, as well as granulating binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), macrogol 8000, sucrose, gelatin and acacia. Additionally, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

The tablet may be made by compression or molding, optionally with one or more pharmaceutically acceptable excipients. Compressed tablets can be prepared by compressing the active ingredient in a suitable device into a free-flowing form such as powder or granules, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surfactant or dispersant. Molded tablets can be prepared by molding in a suitable apparatus a mixture of a powdered compound moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated to provide slow or controlled release of the active ingredient, using, for example, hydroxypropyl methylcellulose in varying proportions to provide the desired release profile.

Solid compositions of this type can also be used as fillers in gelatin capsules. Preferred fillers in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the compounds of the invention may be combined with various sweeteners or flavoring agents, coloring agents or dyes, emulsifying and/or suspending agents, and diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

Formulations suitable for topical administration to the oral cavity include film compositions or lozenges containing the active ingredient in a flavored base, typically sucrose and gum arabic or tragacanth; lozenges containing the active ingredient on an inert base such as gelatin and glycerin or sucrose and gum arabic; and mouthwashes containing the active ingredient in a suitable liquid carrier.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, emulsions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, powders, and the like. These compositions can be prepared by conventional methods containing the active agent. Thus, they may also include compatible conventional carriers and additives such as preservatives, solvents to aid drug penetration, emollient in creams or ointments, and ethanol or oleyl alcohol for lotions. Such carriers may comprise from about 1% to about 98% of the composition. Most often they make up about 80% of the composition. By way of illustration only, a cream or ointment is prepared by mixing sufficient quantities of the hydrophilic material and water containing about 5-10% by weight of the compound in quantities sufficient to obtain the cream or ointment of the desired consistency.

Pharmaceutical compositions adapted for transdermal administration may be presented in the form of individual patches designed to maintain close contact with the recipient's epidermis over an extended period of time. For example, the active agent can be delivered from the patch using iontophoresis.

For application to external tissues, such as the mouth and skin, the compositions are preferably applied as a topical ointment or cream. In an ointment formulation, the active agent can be used with either a paraffin or water-miscible ointment base.

Alternatively, the active agent may be formulated as a cream in an oil-in-water or water-in-oil cream base.

For parenteral administration, liquid unit dosage forms or infusions are prepared using the active ingredient and a sterile carrier, such as, but not limited to, water, alcohols, polyols, glycerin and vegetable oils, preferably water. The active ingredient, depending on the vehicle and concentration used, may be colloidal, suspended or dissolved in the vehicle. When preparing solutions, the active ingredient may be dissolved in water for injection and sterilized, for example by filtration sterilization, before filling into a suitable vial or ampoule and sealing.

Agents such as local anesthetics, preservatives and buffering agents may advantageously be dissolved in the vehicle. To improve stability, the composition can be frozen, for example by freeze drying, after filling the vial and removing the water under vacuum. Then su

- 11 045539 Hoy lyophilized powder is sealed in a vial and an accompanying vial of water for injection may be provided to reconstitute the fluid before use.

Pharmaceutical compositions of the present invention suitable for injection include sterile aqueous solutions or dispersions. In addition, the compositions may be in the form of sterile powders for the immediate preparation of such sterile injectable solutions or dispersions. In all cases, the final injection form must be sterile and must be liquid so that it can be drawn up with a syringe.

The pharmaceutical compositions of the present invention include those suitable for intraocular administration. They consist of a therapeutically effective amount of Compound 1, one or more pharmaceutically acceptable excipients or a pharmaceutically acceptable carrier. Such pharmaceutical compositions may be a conventional eye drop dosage form or another composition having better bioavailability. Such compositions that overcome ocular drug delivery barriers and have improved ocular bioavailability are, for example, emulsions, ointments, suspensions, aqueous gels, nanomicelles, nanoparticles, liposomes, dendrimers, nanosuspensions, microneedles, and in situ temperature-sensitive gels.

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

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

It should be appreciated that, in addition to the ingredients specifically mentioned above, the compositions of the present invention may include other agents commonly used in the art, taking into account the type of composition in question, for example, agents suitable for oral administration may include flavorings. One skilled in the art will know how to select a suitable formulation and how to prepare it (see, for example, Remington's Pharmaceutical Sciences 18 ed. or later). One skilled in the art will also know how to select the appropriate route of administration and dosage.

The present invention relates to a method for producing a liquid pharmaceutical composition according to any of the preceding claims, wherein said method includes the following steps:

a) obtaining methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (compound 1) in free form or its salt, hydrate, solvate or complex;

b) optionally heating to a temperature below 90°C, for example 60°C, or maintaining at room temperature;

c) adding an aqueous liquid (eg phosphate-buffered saline pH 7.4), saline or pure water;

d) optionally promoting dissolution by sonication;

e) stirring at room temperature to obtain said pharmaceutical composition.

One skilled in the art will appreciate that the optimal amounts and individual dosage ranges of a compound of the invention will be determined by the nature and extent of the condition being treated, the type, route and site of administration, as well as the age and condition of the particular subject being treated, and that ultimately the appropriate doses to be used are determined by the doctor. The indicated dose may be repeated as often as necessary. If side effects develop, the dosage amount and/or frequency may be adjusted or reduced in accordance with normal clinical practice.

All % values mentioned herein are expressed in % w/w unless the context otherwise requires.

Nutricosmetic compositions containing a compound of the invention.

Nutricosmetics are products for oral use. The present invention also relates to a nutricosmetic composition containing compounds represented by Compound 1. Nutricosmetic compositions contain Compound 1 in free form or a salt, hydrate, solvate or complex thereof together with one or more orally acceptable diluents or carriers. Nutricosmetic compositions are very similar to pharmaceutical compositions for oral administration.

Therefore, typical compositions are tablets, capsules, ovules, elixirs, gels, solutions or suspensions.

Cosmeceutical compositions containing a compound of the invention.

Cosmeceutical compositions are usually applied to the skin or mucous membrane. Sometimes they can also be given by injection. The present invention also relates to a cosmeceutical composition containing the compounds represented by Compound 1. Cosmeceutical compositions contain Compound 1 in free form or a salt, hydrate, solvate or complex thereof together with one or more orally acceptable diluents or carriers.

Exemplary cosmeceutical compositions include those mentioned herein above, suitable for application to the skin, mucous membrane or by injection.

Other aspects of the invention.

The present invention also relates to the combination (for example, for the treatment of mitochondrial dysfunction) of a compound of formula (I) or a pharmaceutically acceptable form thereof, as defined above, and one or more agents independently selected from the following:

quinone derivatives, eg ubiquinone, idebenone, MitoQ;

vitamins such as tocopherols, tocotrienols and trolox (vitamin E), ascorbate (C), thiamine (B1), riboflavin (B2), nicotinamide (B3), menadione (K3);

antioxidants in addition to vitamins, for example TPP compounds (MitoQ), Sk compounds, epicatechin, catechin, lipoic acid, uric acid, melatonin;

dichloroacetate;

methylene blue;

L-arginine;

Szeto-Schiller peptides, elamipretide and elamipretide analogues;

creatine;

benzodiazepines;

PGC-1a modulators;

AMPK modulators;

modulators of mitochondrial fission and fusion;

PPAR alpha/beta/gamma agonists;

Trolox analogues, carboxamide derivatives;

NRF-2 activators;

NAD+ modulators;

NAD+ precursors;

ketogenic diet.

Another aspect of the invention is that any of the compounds represented by Compound 1 described herein can be administered together with any other compounds, such as, for example, sodium bicarbonate (as a bolus (for example, 1 mEq/kg ) followed by continuous infusion) as a concomitant drug with the compounds described herein.

Lactic acidosis or drug-induced side effects due to complex I-related impairment of mitochondrial oxidative phosphorylation.

The present invention also relates to the prevention or treatment of lactic acidosis and mitochondrial related side effects. In particular, the compounds represented by Compound 1 of the invention are used for the prevention or treatment of side effects of a drug related to mitochondria, or toxin-induced at or above the level of complex I, or otherwise expressed in the invention proposed in accordance with the invention for preventing or treating direct drug-induced inhibition of complex I, or any drug effect that limits the supply of NADH to Complex I (such as, but not limited to, effects on the Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism, and even drugs that affect transport or levels of glucose or other substrates associated with complex I).

Drug-induced mitochondrial toxicity may be part of a desired therapeutic effect (eg, mitochondrial toxicity caused by anticancer drugs), but in most cases, drug-induced mitochondrial toxicity is an undesirable effect. Mitochondrial toxicity can markedly increase glycolysis to compensate for the cellular loss of mitochondrial ATP production through oxidative phosphorylation. This can lead to elevated plasma lactate levels, which, if excessive, results in lactic acidosis, which can be fatal. Type A lactic acidosis is primarily associated with tissue hypoxia, whereas type B aerobic lactic acidosis is associated with drugs, toxins, or systemic disorders such as liver disease, diabetes, cancer, and inborn errors of metabolism (eg, mitochondrial genetic defects).

Many known drugs adversely affect mitochondrial respiration (eg, antipsychotics, local anesthetics, and antidiabetic agents), and accordingly, there is a need to identify or develop agents that can be used to circumvent or mitigate the negative mitochondrial effects caused by the use of such a drug. In addition, some chemicals and gases negatively affect metabolism and mitochondrial function.

- 13 045539

The present invention relates to the compounds represented by Compound 1 for use in the prevention or treatment of lactic acidosis and side effects caused by mitochondrial drugs or toxins. In particular, succinate prodrugs are used to prevent or treat mitochondrial-related side effects caused by drugs at or above the level of complex I, or otherwise expressed, the invention provides succinate prodrugs to prevent or treat drug-induced direct inhibition of complex I, other respiratory complexes or any drug-induced effect that limits the supply of NADH to complex I (such as, but not limited to, effects on the Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism, and even drugs that affect transport or levels of glucose or other substrates associated with complex I).

As stated above, elevated plasma lactate levels are often observed in patients taking medications that may have mitochondrial-related side effects. The present invention is based on experimental results showing that metformin (a first-line therapy for the treatment of type 2 diabetes and which has been associated with lactic acidosis as a rare side effect) suppresses the mitochondrial function of human peripheral blood cells in complex I in a time-dependent manner: concentrations corresponding to metformin intoxication. Metformin also causes a significant increase in lactate production by intact platelets over time.

Accordingly, the invention relates to compounds corresponding to formula (I) for use in the prevention or treatment of lactic acidosis. However, since the results presented herein are based on lactic acidosis associated with direct inhibition of complex I or associated with a defect at or above the level of complex I, it is believed that the compounds of the invention are suitable for use in the prevention or treatment of mitochondria-related side effects. drug-induced effects at or above the level of complex I. The compounds of the present invention may also antagonize drug effects that interfere with pre-complex I metabolism (indirect inhibition of complex I, which may include any drug effect that limits the supply of NADH to complex I, e.g., effects on the Krebs cycle, glycolysis, beta-oxidation, pyruvate metabolism and even drugs that affect levels of glucose or other substrates associated with complex I). The compounds can also counteract defects downstream of complex I (complex III, IV and V by increasing proton driving force.

It is envisaged that Compound 1 can be used in industrial applications, for example, in vitro to reduce or inhibit lactate formation or to increase ATP availability in commercial or industrial cell lines. Examples include use in cell culture, organ preservation, etc.

The compounds of the present invention are used to treat or prevent drug-induced side effects associated with mitochondria, or to increase or restore cellular energy (ATP) or succinate levels during treatment. In particular, they are used to treat or prevent direct or indirect side effects associated with complex I mitochondria. In particular, they are used to treat or prevent lactic acidosis, such as drug-induced lactic acidosis.

The invention also relates to a combination of compound 1 and a drug that can cause a side effect related to mitochondria, in particular a side effect that is caused by direct or indirect disruption of complex I by the drug. Such a combination can be used as a prophylactic warning against a mitochondrial side effect or, if a side effect occurs, to alleviate and/or treat a mitochondrial side effect.

Compound 1 is expected to be effective in the treatment or prevention of drug-induced side effects, in particular side effects associated with direct or indirect inhibition of complex I.

Medicinal substances that are known to cause defects, malfunctions or disturbances in complex I and/or are known to cause lactic acidosis as a side effect, namely analgesics, including paracetamol, capsaicin;

antianginal agents, including amiodarone, perhexiline;

antibiotics, including linezolid, trovafloxacin, gentamicin;

anticancer drugs, including quinones, including mitomycin C, adriamycin;

anticonvulsants, including valproic acid;

antidiabetic agents including metformin, phenformin, butyl biguanide, troglitazone and rosiglitazone, pioglitazone;

anti-hepatitis B, including fialuridine;

antihistamines;

antiparkinsonism, including tolcapone;

- 14 045539 antipsychotics risperidone;

antischizoprenic zotepine, clozapine;

antiseptics, quaternary ammonium compounds (QAC);

anti-tuberculosis drugs, including isoniazid;

fibrates, including clofibrate, ciprofibrate, simvastatin;

sleeping pills, including propofol;

immunosuppressive disease-modifying antirheumatic drug (DMARD) leflunomide;

local anesthetics including bupivacaine, diclofenac, indomethacin and lidocaine;

muscle relaxant, including dantrolene;

antipsychotics, including antipsychotics such as chlorpromazine, fluphenazine and haloperidol;

NRTIs (nucleotide reverse transcriptase inhibitors), including efavirenz, tenofovir, emtricitabine, zidovudine, lamivudine, rilpivirine, abacavir, didanosine;

NSAIDs, including nimesulfide, mefenamic acid, sulindac;

barbituric acids.

Other drugs that are known to cause lactic acidosis as a side effect include beta2-agonists, epinephrine, theophylline, or other herbicides. Alcohols and cocaine can also cause lactic acidosis.

Moreover, it is contemplated that the compounds of the invention may also be effective in the treatment or prevention of lactic acidosis, even if it is not associated with a complex I defect.

Combination of drugs and compounds according to the invention.

The present invention also provides a combination of a drug and a compound of the present invention for use in the treatment and/or prevention of a drug-induced side effect selected from lactic acidosis and side effects associated with complex I defect, inhibition or disruption, wherein

i) the medicinal substance is used to treat the disease for which the medicinal substance is indicated; and ii) the compound of the invention is used to prevent or alleviate side effects induced or induced by a drug, wherein the side effects are selected from lactic acidosis and side effects associated with defect, inhibition or disruption of complex I.

Any combination of such a drug substance with any compound of the invention is within the scope of the present invention. Accordingly, based on the description provided herein, one skilled in the art will understand that the essence of the invention is to discover beneficial properties of the compounds of the invention to avoid or reduce the side effects described herein. Thus, the potential use of compounds of the invention capable of penetrating cells and delivering succinate and possibly other active moieties in combination with any drug that has or potentially has the side effects described herein is apparent from the present disclosure.

The invention also relates to

i) a composition containing a drug substance and a compound of the invention, wherein the drug substance has a potential drug-induced side effect selected from lactic acidosis and side effects associated with defect, inhibition or disruption of complex I;

ii) the composition described in paragraph i) above, wherein the compound of the present invention is used to prevent or alleviate side effects induced or induced by a drug, wherein the side effects are selected from lactic acidosis and side effects associated with defect, inhibition or disruption of the complex I.

The composition can be in the form of two separate packages.

A first package containing a drug substance or a composition containing a drug substance, and a second package containing Compound 1 of the invention or a composition containing a compound of the invention. The composition may also be a single composition containing both the drug substance and Compound 1 of the invention.

In case the composition includes two separate packages, the drug substance and Compound 1 of the invention can be administered by different routes of administration (for example, the drug substance by oral administration and the compound of the invention by parenteral or mucosal administration) and/or they can be administered essentially simultaneously, or the drug substance can be administered before the compound of the invention, or vice versa.

Sets.

The invention also relates to a kit containing:

i) a first container containing a drug substance that has a potential drug-induced side effect selected from lactic acidosis and side effects associated

- 15 045539 those with a defect, inhibition or disruption of complex I; and ii) a second container containing Compound 1 of the invention, which has the potential to prevent or alleviate drug-induced or drug-induced side effects, wherein the side effects are selected from lactic acidosis and side effects associated with defect, inhibition or disruption of complex I .

Method of treatment/prevention of side effects.

The invention also relates to a method of treating a subject suffering from a drug-induced side effect selected from lactic acidosis and a side effect associated with defect, inhibition or disruption of complex I, wherein the method comprises administering to the subject an effective amount of a compound represented by Compound 1 of the invention, and to a method of preventing or alleviating a drug-induced side effect selected from lactic acidosis and a side effect associated with defect, inhibition or disruption of complex I in a subject who suffers from a disease being treated with a drug that potentially causes a side effect selected from lactic acid acidosis and a side effect associated with a defect, inhibition or disruption of complex I, where the method includes administering to a subject an effective amount of a compound represented by Compound 1 of the invention before, during or after treatment with said drug.

Metformin.

Metformin is an antidiabetic drug belonging to the biguanide class. It is the first-line treatment for type 2 diabetes, which accounts for about 90% of diabetes cases in the United States. The antidiabetic effect is attributed to a decrease in hepatic glucose production, an increase in the biological effect of insulin by increasing glucose uptake into peripheral tissues, and a decrease in intestinal glucose uptake, but the exact mechanisms of action are not fully understood. Despite its advantages over other antidiabetic agents, it is associated with rare cases of lactic acidosis (LA) as a side effect. LA is defined as an enlarged anion gap, arterial lactate levels greater than 5 mM, and pH <7.35.

The following list of non-limiting embodiments further illustrates the invention.

1. Isolated methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (compound 1) in free form or its salt, hydrate, solvate or complex.

2. Isolated compound 1 according to embodiment 1, which is a solid product.

3. Isolated compound 1 in accordance with any of the preceding embodiments, which is or contains a crystalline product, such as a polymorph having an x-ray powder diffraction pattern of compound 1, lot 12 (Fig. 7), or having an x-ray powder diffraction pattern of compound 1, lot 15 ( Fig. 8), or having a position (°2theta) equal to 11.2 (±0.2) and 16.9 (±0.2).

4. Isolated compound 1 according to any of embodiments 1-2, which is or contains an amorphous product.

5. Isolated compound 1 according to any of the preceding 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% wt./wt.

6. Isolated compound 1 according to any of the preceding 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. Isolated compound 1 according to any of the preceding embodiments, having a synthesis 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. Isolated compound 1 according to any of the preceding embodiments, having a purity sufficient for pharmaceutical use.

9. Isolated compound 1 according to any of the preceding embodiments, which is in free form.

10. Isolated compound 1 according to any one of embodiments 1-8, which

- 16 045539 is a salt.

11. Isolated compound 1 according to embodiment 10, which is a hydrochloride salt, hydrobromide salt, acetate salt, citrate salt, lactate salt, maleate salt or malonate salt.

12. Isolated compound 1 according to any of embodiments 1 to 8, which is a hydrate such as a monohydrate.

13. Isolated compound 1 according to any of the preceding embodiments for use in humans or animals.

14. Isolated compound 1 according to any of the preceding embodiments for use in humans.

15. Isolated compound 1 according to any of the preceding embodiments for use in medicine.

16. Isolated Compound 1 according to any of the preceding embodiments for use as an active pharmaceutical ingredient in a pharmaceutical product.

17. Isolated compound 1 according to any of the preceding embodiments for use in the treatment or prevention of metabolic disease, mitochondrial dysfunction, mitochondrial dysfunction-related disease, mitochondrial disorder, mitochondrial energy deficiency, mitochondrial disorder, mitochondrial energy deficiency, drug-induced mitochondrial side effects effects, cancer, diabetes, traumatic brain injury, hypoxia during cardiac arrest, ischemia, stroke, myocardial infarction, acute angina, acute liver injury, coronary occlusion, atrial fibrillation, male infertility and menopausal symptoms in women.

18. Isolated compound 1 according to embodiment 17, wherein said mitochondrial dysfunction disease or mitochondrial dysfunction-related disease is selected from the following:

aging;

Alpers disease (progressive childhood polyodystrophy);

Alzheimer's disease;

amyotrophic lateral sclerosis (ALS);

autism;

Barth syndrome (lethal childhood cardiomyopathy);

disorders of beta oxidation, deficiency of bioenergy metabolism;

carnitine-acyl-carnitine deficiency;

carnitine deficiency;

creatine deficiency syndromes (cerebral creatine deficiency syndromes (CCDS), including guanidinoacetate methyltransferase deficiency (GAMT deficiency), L-arginine:glycinamidinotransferase deficiency (AGAT deficiency), and creatine transporter deficiency associated with SLC6A8 (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 aciduria type II;

KSS (Kearns-Sayre syndrome);

lactic acidosis;

LCAD (long-chain acyl-CoA dehydrogenase deficiency);

LC-FAOD (long chain fatty acid oxidation disease);

LCHAD, Leigh's disease or syndrome (subacute necrotizing encephalomyelopathy);

LHON (Leber's hereditary optic neuropathy);

Luft's disease;

MCAD (medium chain acyl-CoA dehydrogenase deficiency);

MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes);

MERRF (myoclonic epilepsy and ragged red fiber disease);

- 17 045539 methylmalonyl-CoA epimerase deficiency;

methylmalonyl-KoA mutase deficiency;

Mitochondrial DNA depletion syndrome 5;

Mitochondrial DNA depletion syndrome 9;

Mitochondrial DNA depletion syndrome 15 (hepatocerebral type) (family 1);

maternally inherited diabetes and deafness;

MIRAS (mitochondrial recessive ataxia syndrome);

mitochondrial cytopathy;

Mitochondrial DNA depletion;

mitochondrial encephalopathy, including encephalomyopathy and encephalomyelopathy, mitochondrial myopathy;

MNGIE (myoneurogastointestinal disorder and encephalopathy);

NARP (neuropathy, ataxia and retinitis pigmentosa);

neurodegenerative disorders associated with Parkinson's, Alzheimer's or Huntington's disease;

Pearson syndrome;

Parkinson's disease;

progressive external ophthalmoplegia;

propionic acidemia;

pyruvate dehydrogenase deficiency;

POLG mutations;

respiratory chain failure;

SCAD (short-chain acyl-CoA dehydrogenase deficiency);

SCHAD;

VLCAD (very long chain acyl-CoA dehydrogenase deficiency).

19. Isolated compound 1 according to embodiment 18, wherein said mitochondrial dysfunction disease or mitochondrial dysfunction related disease is attributed to 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 muscle fibers).

20. Isolated compound 1 according to any of embodiments 1-17 for use in treating or preventing metabolic dysfunction.

21. Isolated compound 1 according to embodiment 20, wherein said metabolic dysfunction is diabetes, such as impaired insulin secretion (type 2 diabetes).

22. Isolated compound 1 according to embodiment 20, wherein said metabolic dysfunction is side effects of the drug on mitochondria.

23. Isolated compound 1 according to embodiment 22, wherein said drug-induced mitochondrial side effects are selected from inhibition of complex I induced by metformin (lactic acidosis), paracetamol/acetaminophen induced (liver failure) or drug-induced mitochondrial depletion .

24. Isolated compound 1 according to embodiment 20, wherein said metabolic dysfunction is chemically induced side effects on mitochondria.

25. Isolated compound 1 according to embodiment 24, wherein said chemically induced side effects on mitochondria are selected from rotenone inhibition of complex I (Parkinson's disease-like symptoms), inhibition of pesticide-induced respiratory complexes and mitochondrial enzymes, inhibition of chemically induced respiratory complexes, and mitochondrial enzymes and gas poisoning of respiratory complexes and mitochondrial enzymes, such as carbon monoxide poisoning.

26. Isolated compound 1 according to embodiment 20, wherein said metabolic dysfunction is a genetic mitochondrial dysfunction.

27. Isolated compound 1 according to embodiment 26, wherein said genetic mitochondrial dysfunction is selected from dysfunctional energy production due to decreased number of mitochondria, dysfunctional mitochondrial transcription factors, dysfunctional transcription factors for mitochondrial mitochondrial membrane proteins that contribute to the stabilization of large mitochondrial DNA complexes (mtDNA)-protein called nucleoids, dysfunctional energy production, pyruvate dehydrogenase deficiency, complex I, II, III or IV deficiency or enzyme deficiency, e.g. pyruvate dehydrogenase deficiency, dysfunction of enzymes involved in succinate synthesis, e.g. propionyl-CoA carboxylase, methylmalonylCoA mutase and succinylCoA synthetase.

28. Selected compound 1 according to embodiment 27, wherein said effect

- 18 045539 the active amount is in the range from 1 mg to 5.0 g per day, from 10 mg to 2.0 g per day, from 25 mg to 1 g per day, from 50 to 500 mg per day, from 100 to 1000 mg per day, 250 to 1000 mg per day, or 50 to 500 mg per day of Compound 1 or its salt, hydrate, solvate, or complex.

29. Isolated compound 1 according to any one of embodiments 15-28, wherein said compound 1 or a salt, hydrate, solvate or complex thereof is administered to the subject from once a day to 10 times a day or from once a day to 4 times a day once a day.

30. Isolated Compound 1 according to any one of Embodiments 15-29, wherein said treatment or prevention is pre-treatment, e.g., use before surgery, use before elective medical intervention with high metabolic demand, and before the subject goes into hospital. combat zone or other hazardous environment.

31. Isolated compound 1 according to any one of embodiments 15-30, wherein said treatment or prevention is a chronic treatment.

32. Isolated compound 1 according to any of embodiments 1-14, for non-pharmaceutical use in humans or animals.

33. Isolated compound 1 according to embodiment 19, for use as a cosmeceutical or nutricosmetic.

34. Isolated compound 1 according to any of embodiments 28, 29, for use as an energy drink or cream.

35. A composition comprising isolated compound 1 according to any of the preceding embodiments.

36. A cosmeceutical containing isolated compound 1 according to any one of embodiments 1-14.

37. Nutricosmetics containing isolated compound 1 in accordance with any of embodiments 1-14.

38. An energy drink comprising isolated compound 1 according to any one of embodiments 1-14.

39. A pharmaceutical composition containing isolated compound 1 in accordance with any of embodiments 1-33.

40. A method for producing isolated compound 1 according to any one of embodiments 1-33, wherein said method includes the following steps:

a) reacting N-acetylcysteamine and monomethylsuccinate in the presence of a coupling reagent in an organic solvent at a temperature in the range between 0 and 100°C;

b) isolating compound 1, thereby obtaining isolated compound 1.

41. The method according to embodiment 40, wherein step a) is carried out when, independently, the solvent is dichloromethane, the coupling reagent is carbonyldiimidazole, and the temperature is 15-30°C.

42. The method according to any one of embodiments 40-41, wherein step b) comprises extraction with an aqueous acidic solution (optionally 20% ammonium chloride) and then extraction of the organic layer with another aqueous medium (suitable brine or water).

43. The method according to embodiment 42, wherein the organic layer is removed in vacuo and the residue is dissolved in an organic solvent with suitable dissolution properties for crystallization, such as methyl tert-butyl ether (MTBE).

44. The method according to embodiment 42, wherein the solution is cooled, suitably to about 5°C, and an antisolvent such as n-heptane is added and after stirring for a period of time, preferably about 24 hours, compound 1 is collected and filtered and washed with antisolvent.

45. Selected compound 1 according to any one of embodiments 1 to 3, wherein the position (°2θ) is 11.2 (±0.2) and 16.9 (±0.2).

46. The pharmaceutical composition according to an embodiment is a solid composition.

47. The pharmaceutical composition according to an embodiment is a solid composition for reconstitution before use.

48. The pharmaceutical composition according to an embodiment is an aqueous composition.

49. The pharmaceutical composition according to an embodiment is an aqueous phosphate buffered saline (PBS) formulation.

39,

46,

46,

48, which which which which predepred-

50. The pharmaceutical composition according to any of embodiments 46-49, which has a concentration of Compound 1 of at least 10% w/w, at least 30% w/w, at least 50% w/w wt., at least 60% or at least 70% wt./wt.

51. The pharmaceutical composition according to any of embodiments 46-50, which is intended for oral administration, for subcutaneous administration, for intravenous administration,

- 19 045539 for parenteral administration, for ocular administration or for local administration.

52. The pharmaceutical composition according to embodiment 51, which is a drink or gel.

53. The pharmaceutical composition in accordance with any of embodiments 46-52, which contains from 1 mg to 5.0 g, from 10 mg to 2.0 g, from 25 mg to 1 g, from 50 to 500 mg, from 100 up to 1000 mg, 250 to 1000 mg or 50 to 500 mg of compound 1 or its salt, hydrate, solvate or complex.

54. The pharmaceutical composition according to any of embodiments 46-53, which is an immediate release formulation.

Examples

General methods, substances and analyses.

HPLC method for purity analysis.

HPLC method 1.

Solvent A is water + 0.1% NH ₄ OH.

Solvent B is 2.5 l of acetonitrile + 130 ml of H ₂ O + 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 a Waters XSelect CSH C18 3.5 µm, 2.1 x 50 mm.

Absorption is monitored using a diode array detector at 234 nm.

Sample concentration 1 mg/ml, injection volume 1 µl.

HPLC method 21.

Solvent A is water + 1.57 g NH4HCO2 + 5 ml formic acid.

Solvent B is 2.5 l of acetonitrile + 130 ml of H ₂ O + 4.5 ml of formic acid.

Gradient: T=0 min, V%=0, flow rate=1 ml/min; T=1 min, V%=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 a Waters XSelect CSH C18 3.5 µm, 2.1 x 50 mm.

Absorption is monitored using a diode array detector at 230 nm.

Sample concentration 1 mg/ml, injection volume 1 µl.

Example 1. Synthesis of methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (compound 1).

Detailed description of the synthesis and isolation of compound 1.

OOO

intermediate connection 1 connection 1

Compound 1 was prepared by three separate methods (A, B and C, below).

Method A.

Acetic anhydride (204 g, 2 mol) was added dropwise to a solution of 2-aminoethanethiol hydrochloride (226 g, 2 mol), KON (114 g, 2 mol) and NaHCO ₃ (168 g, 2 mol) in water (4 L). . The mixture was stirred at room temperature for 45 minutes. The reaction mixture was extracted with EtOAc (8x2 L), dried over MgSO ₄ and the solvent was removed under reduced pressure to give intermediate 1 (190 g, 80% yield) as a light yellow oil.

N-(3-dimethylaminoppropyl)-N'-ethylcarbodiimide hydrochloride (304 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 NaHCO ₃ solution (2x2 L), dried over Na2SO4 and concentrated under reduced pressure to give crude 1 (350 g) as a yellow oil. Crude compound 1 was purified by silica gel column chromatography (2000 g silica gel, eluting with CH ₂ Cl ₂ /MeOH=100/1 to 80/1) to give compound 1 (201 g, 94.9% by LCMS) as white solid. The crude side fractions after purification (110 g) were purified by silica gel column chromatography (1200 g silica gel, eluting with CH ₂ Cl ₂ /MeOH=100/1 to 80/1) to give compound 1 (40 g, 96.7% according to LCMS data) in the form of a white solid.

Method B.

To a solution of 2-aminoethanethiol hydrochloride (11.3 g, 0.1 mol), KON (5.6 g, 0.1 mol) and NaHCO ₃ (5.88 g, 0.07 mol) in water (200 ml) At room temperature, acetic anhydride (7.14 g, 0.07 mol) was added dropwise. The mixture was stirred at room temperature for 45 minutes. Reactionary

- 20 045539 the mixture was extracted with EtOAc (8x200 ml), dried over MgSO ₄ (1 h) and then the solvent was removed in vacuo at 50°C to give crude intermediate 1 (7 g, 84% yield) as a slightly liquid yellow color.

To a solution of 4-methoxy-4-oxobutyric acid (9.24 g, 0.07 mol) in dichloromethane (200 ml) was added portionwise 1,1'-carbonyldiimidazole (11.34 g, 0.07 mol). The mixture was stirred at room temperature for 1 hour. Intermediate 1 (7 g, 0.059 mol) was added dropwise and the mixture was then stirred at room temperature for 3 hours. The resulting mixture was washed with HCl (1 N, 3x150 ml) and saturated solution NaHCO ₃ (3x150 ml), dried over Na ₂ SO ₄ (1 hour) and then the solvent was removed in vacuo at 50°C to obtain 9.5 g of compound 1 as a yellow solid.

Method C.

To a solution of KON (0.71 kg, 13.2 mol) and Na ₂ CO ₃ (1.00 kg, 9.43 mol) in water (15 L) was added 2-aminoethanethiol hydrochloride (1.5 kg, 13.2 mole). To the resulting clear, dark purple solution, acetic anhydride (0.96 kg, 9.43 mol) was added dropwise at +22°C, maintaining the internal temperature below +30°C during the addition (addition time was 24 min). 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 (2x15 l), bringing the internal temperature to +28±2°C during the extraction process. The organic phases were combined and the solvent was removed in vacuo. Intermediate 1 was then 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.

Distillation. 933 g of Intermediate 1 was distilled using a thin layer distillation unit under the following conditions: T=+110°C, P=1 mbar, speed 202 g/h. Intermediate 1 was obtained as a clear, colorless oil.

To a solution of 4-methoxy-4-oxobutyric acid (1.33 kg, 10.07 mol) in DCM (10 L), 1,1'-carbonyldiimidazole (CDI) (1.63 kg, 10.07 mol) was added portionwise. . During the addition, intense foaming and gas evolution were observed. After addition was complete, the mixture was stirred at +20 to +25°C for 1 hour. A dichloromethane (5 L) solution of intermediate 1 (1.00 kg, 8.39 mol) was added, maintaining the internal temperature below +30° WITH. The reaction mixture was stirred at a temperature of +20 to +25°C for 2 hours. A 20% aqueous solution of NH ₄ Cl (10 l) was added and the mixture was stirred for 20 minutes. The layers were separated. The organic phase was extracted with a 13% aqueous NaCl solution and water, sequentially (10 and 5 L separately). After this, the solvent (DCM) was replaced with MTBE during distillation. A solution of compound 1 in MTBE (approximately 6 L) was gradually cooled to +5°C. Crystallization began when the internal temperature reached +12°C. N-heptane (15 L) was added to the slurry and the mixture was stirred at a temperature of 0 to +5°C for 20 hours (overnight). The suspension was filtered and the filter cake was washed with N-heptane (2x3l). The product was dried by passing air through it for 42 hours. Yield was 1.26 kg (64%), purity 98.5% by area (HPLC).

The synthesis described above prepared several batches of Compound 1. The batches were purified by various purification methods as described in Methods A, B and C.

Batch 3 (or compound 1-s3) was prepared by Method A.

Batch 12 (or compound 1-s12), 13 (or compound 1-s13) and 14 (or compound 1-s14) were prepared by Method B.

Batch 15 (or compound 1-s15), 16 and 17 were prepared by Method C.

Example 2. Characteristics of connection 1 of different batches._________________________

Batch number Method of obtaining XRPD data Crystallinity according to XRPD 2 Method A 3 Method A Figure 19 74.4% 4 Method A 5 Method B 6 Method B

- 21 045539

eleven Method A was then purified using Method B 12 Method B Figure 11 13 Method B Figure 21 70.2% 14 Method B Figure 22 56.2% 15 Method C Figure 12 16 Method C Figure 24 65.2% 17 Method C was then purified by pre-HPLC and lyophilized Figure 25 63.7% 18 (stored lot 12) Batch 12, stored as a solid at -20°C for 20 months Figure 20 70.4% 19 (stored lot 15) Batch 15, stored as a solid at -20°C for 14 months Figure 23 69.7%

It should be noted that during powder X-ray diffraction analysis the temperature increases. Since compound 1 has a low melting point (and the amorphous form is assumed to have a lower melting point than the crystalline form), the degrees of crystallinity listed in the table above can be considered as minimum values.

Batch 3.

In TGA analysis, batch 3 loss was 0.04% by weight at temperatures of 20-150°C. In fig. 1-3 show the LCMS analysis spectra of Batch 3. The melting point of Batch 3 was 50.4°C as determined by differential scanning calorimetry (DSC).

Batch 12.

Batch 12 was analyzed using the same methods used to analyze Batch 3 above.

Losses in TGA analysis were 0.12% by weight at temperatures of 20-150°C. The LCMS results are shown in FIG. 4-6, the purity by qYMP was 96.1%, and the melting point was 48.6°C.

Batch 13.

Batch 13 was analyzed using the same methods used to analyze the above batches.

Losses in TGA analysis were 0.18% by weight at temperatures of 20-150°C. LCMS spectra are not shown, but the results are summarized in Table. 1. The purity according to qNMR was 96.3% and the melting point was 49.0°C.

Batch 14.

Lot 14 was analyzed using the same methods used to analyze the above lots.

Losses in TGA analysis were 0.45% by weight at temperatures of 20-150°C. LCMS spectra are not shown, but the results are summarized in Table. 1. Purity by qNMR was 91.6% and melting point was 46.9°C.

Batch 15.

Lot 15 was analyzed by some of the same methods used to analyze the above lots.

LCMS spectra are not shown, but the results are summarized in Table. 1. The purity according to qNMR was 98.9% and the melting point was 39°C.

Comparison of batch properties: batches 3, 12, 13, 14, 15 and 16.

In table 1 also summarizes the purity, melting point, and visual description of Solid Compound 1. Batches of Solid Compound 1 appeared as a white, free-flowing powder; see table 1.

In addition, in table. 1 shows that the solubility of all prepared batches of Compound 1 was at least 366-398 mg/ml, and the appearance of such composition in water visually appeared as a clear and clear or translucent solution.

- 22 045539

Summary of analytical results from various batches of Compound 1

Table 1

Batch 3 Batch 12 Batch 13 Batch 14 Batch 15 Batch 16 Final cleaning method Purified by silica gel column chromatography Wash, dry and remove solvent Wash, dry and remove solvent Wash, dry and remove solvent Washed, precipitated and dried Washed, precipitated and dried High pH HPLC Purity (%) 99.1 97.7 96.7 99.3 98.5 98.1 Purity analysis by cNMR data (%) 98.1 96.1 96.3 91.7 98.9 95.6 Melting point (°C) 50.4 48.6 49.0 46.9 39 Visual description of forms White free-flowing powder; slightly lumpy substance Large white waxy lumps; some white, free-flowing powder Large white waxy lumps; some white, free-flowing powder Large white waxy lumps; some white, free-flowing powder White free flowing powder White free flowing powder 400 mg of compound in 0.7 ml of water, measured concentration (mg/ml) 381 378 395 366 414 435 Visual description of the composition in water Colorless transparent solution Cloudy translucent solution Colorless transparent solution Colorless transparent solution Colorless transparent solution Colorless transparent solution

Summary of purity and impurity analysis of batches

Compound 1 Table 2 (High pH LCMS2 Impurity Profiling)

RT (min) 0.69 1.57 1.86 2.18 2.22 6.24 RRT (min) 0.439 1,000 1.185 1,389 1.414 3.975 Apparent molecular weight 161.0 233.0 319.9 247.0 305.0 278.0 Structure (inferred from m/z data) O D. ⁿ 2 Oh Oh TVS TVS TVS TVS % at 234 nm, batch 3 0.52 99.14 0.35 % at 234 nm, batch 12 97.65 1.41 0.24 0.38 0.32 % at 234 nm, batch 13 0.47 96.71 2.44 0.39 % at 234 nm, batch 14 0.34 99.34 0.32

Example 3. Preparation of aqueous compositions of compound 1.

Protocol for preparing the composition.

1. Weigh the required amount of Compound 1, then add the required amount of excipient (0.9% w/v saline, 100 mM PBS pH 7.4, water) to solid Compound 1 to obtain the desired concentration in mg/ml of Compound 1 For example, to obtain a composition with a concentration of 400 mg/ml of compound 1 in water, weigh out 400 mg of compound 1 and add 0.7 ml of water.

2. The solution is sonicated for 10 minutes and then shaken for 20 minutes to ensure that Compound 1 is completely dissolved.

- 23 045539

3. The solution can be centrifuged (13000 rpm, 10 min) to remove any solid particles if necessary.

4. If necessary, the solution can be sterilized by filtration.

Table 3

Data for formulations in water of various batches of Compound 1 prepared according to the protocol above

Sample Target concentration (mg/ml) Injection volume (µl) Dilution factor Calculated conc. (mg/ml) Purity (%) Party 3 in tap water 400 2 1000 381 98.8 Batch 12 tap water 400 2 1000 378 99.2 Batch 13 tap water 400 2 1000 395 99.1 Batch 14 in tap water 400 2 1000 366 99.4

Preparation of a composition with a concentration of 50% w/v.

Four batches of compound 1 were prepared in PBS at 50% w/v.

~500 mg Compounds were weighed into a vial, ~500 μl of 100 mM PBS pH 7.4 was added and the vial was sonicated for 10 min, then shaken for 20 min. A sample was then taken, diluted 1/2000 and the concentration calculated by HPLC analysis.

Table 4

Preparation of compound 1 formulations in PBS at pH pH 7.4

Sample Amount of Compound 1 Added (mg) Amount of added lOOMMPBSpH 7.4 (µl) Calculated concentration from HPLC data (mg/ml) Batch 3 525 525 535 Batch 4 514 514 526 Batch 5 494 494 531 Batch 6 481 481 543

The concentration of compound 1 was determined in the soluble formulation using HPLC.

Preparation of the liquid composition of compound 1.

Compound 1 from Batch 3 was heated in an oven at 60°C for 20 minutes, after which it became a translucent pale yellow liquid. 100 mM PBS pH 7.4 (20% v/v) was added and the solution was stirred for 20 min on a shaker. After this time, the solution was cooled to room temperature and it remained a translucent liquid. The solution was kept at 4°C for 72 hours. Observation after this time confirmed that it remained a translucent liquid.

Brief conclusions on the composition.

The amount of compound 1 that can be prepared in aqueous solutions such as 0.9 w/v% saline, 100 mM PBS, pH 7.4 or water does not appear to have an achievable limit. This can perhaps be explained by the melting point of compound 1, which was measured to be ca. 47-50°C in some batches. When an aqueous solution is added to a solid, it disrupts the intramolecular interactions of the molecules of compound 1, it becomes miscible with water.

Example 4. Preparation of the gel composition.

Preparation of the composition of compound 1 in the form of gel packets with a concentration of 2.25 mg/ml.

Experiment details.

First, blue food coloring was used to see how easily the water solution could mix with the gel. Two gel samples were taken, one was kept at room temperature, the other was melted in a microwave oven (1 min). Blue food coloring (1% v/v) was added and the solutions were mixed. As the gel melted, mixing became much more effective. The dye can be fully incorporated after stirring for <10 s.

Then the dye solution was replaced with a solution of compound 1.

Water (225 mg/ml, 100 times the required concentration) was added to compound 1 and sonicated for 20 min, then shaken for 30 min. A sample was taken for HPLC analysis to check the concentration.

- 24 045539

Table 5

Measured concentration of compound 1 in aqueous stock solution

Sample Estimated conc. (mg/ml) Volume injection (µl) Coefficient dilute Comput. mg/ml Purity (%) Water source solution for adding to the gel 225 1 100 222,749 98.8

The analysis showed that compound 1 was completely dissolved.

Another 2 parts of the gel were melted and an aqueous solution of compound 1 (1% v/v) was added. The gels were shaken for the same time as when adding the dye (10 s). The gels were left to harden.

After the gels had solidified, samples were taken for HPLC analysis to check whether compound 1 was evenly distributed.

Sampling procedure.

1. A gel sample (100 mg) was added to Eppendorf tubes and MeOH (0.9 ml, 1/10 dilution) was added.

2. The sample was shaken on a Vibrax for 30 minutes.

3. The sample was centrifuged (13000 rpm, 10 min).

4. The supernatant was collected for HPLC analysis.

concentration of compound 1 in gel

Table 6 ah

Sample Estimated conc. (mg/ml) Volume injection (µl) Dilution factor Comput. mg/ml Purity (%) 4°С 2.25 5 10 2.07 99.4 37°C 2.25 5 10 2.03 99.5

The concentration of Compound 1 was slightly lower than expected, 2.25 mg/mL, but the two samples agreed well, suggesting that Compound 1 was evenly distributed and that the dilution factor was slightly higher.

One of the gel samples was kept at room temperature and the other at 4°C to test the stability of compound 1 in the gel.

Stability of the gel composition.

Gel samples were taken as above at regular intervals to check stability at both 4°C and room temperature. The data from these experiments are presented in table. 7, 8.

Table 7

Concentrations of compound 1 in gel samples taken during 20 days of storage at 4°C

Time (days) 0

Estimated conc. (mg/ml) 77

Injection Volume (µL)

Dilution factor 16

Comput. mg/ml 2.071 2.024 1.937 1.979 2.007 2.167 2.218

AUC (% at T=0)

100.0

97.8

93.5

95.6

96.9

104.7

107.1

Purity at 230 nm (%) 77

663

- 25 045539

Table 8

Concentrations of compound 1 in gel samples taken during 20 days of storage at room temperature (approx. 20°C)

Time (days) Estimated conc. (mg/ml) Injection Volume (µL) Dilution factor Comput. mg/ml AUC (% at T=0) Purity at 230 nm (%) 0 2.25 5 10 2,034 100.0 99.5 1 2.25 5 10 1,746 85.8 99.5 5 2.25 5 10 2,053 100.9 99.4 7 2.25 5 10 1.721 84.6 99.2 AND 2.25 5 10 1,889 92.9 99.1 14 2.25 5 10 2,739 134.6 99.4 20 2.25 5 10 2,273 111.7 99.4

The overall trend of the data, especially the purity data at 230 nm, suggests that Compound 1 is stable in the gel formulation for at least 20 days. The AUC data contains more errors due to inaccuracies in weighing the extraction gel samples and possibly differences in the local concentration of compound 1 in the gel.

Protocol for obtaining the gel composition.

A 100-fold concentrated solution of compound 1 in water was prepared so that it could be added to the gel in an amount of 1/100 of the volume of the gel. The example given is for a final concentration of 2.25 mg/mL in a 200 mL gel pack, so 2 mL of 100x Compound 1 in water (225 mg/mL) is required. It is also suggested to add food coloring to the stock solution and inject the combined solution into the gel pack (provided this will not adversely affect the study). This provides a visual check that the compound 1 solution is evenly distributed throughout the gel. If food coloring is included, it is recommended that you also add food coloring to the control gel.

1. Weigh 500 mg of compound 1 into a 3 ml vial (or equivalent).

2. Add 2 ml water (adding 2 ml water to 500 mg of compound 1 is the volume of solid compound 1 and was confirmed by the HPLC calibration curve to be 225 mg/ml), sonicate for 20 min, then shake for 30 min (the solution may remain slightly cloudy). 1 ml of natural food coloring was added (the food coloring will indicate that the distribution was even).

3. If necessary, the solution can be sterile filtered.

4. Heat the unopened gel pack by immersing it in water at 70°C for 10 minutes, and the gel becomes a mobile liquid.

5. Draw up a solution of compound 1 into a needle/syringe (with or without food coloring).

6. Add the compound 1 solution (and food coloring) into the gel pack by poking a small hole in the gel pack with a needle.

7. Re-seal the gel pack at the injection site with tape.

8. Shake the gel pack vigorously for 5 min to evenly distribute Compound 1 (if food coloring is included, this will become apparent when the injected solution is evenly distributed as the color of the gel changes).

9. Let the gel harden, this took about 45 minutes at room temperature.

10. Use the gel or store it unopened at a temperature of 4°C (after opening, it is recommended to store the gel at a temperature of 4°C for no more than 14 days).

11. Compound 1 is stable in gel for at least 14 days at 4°C and room temperature.

Test report.

The gel formulation was prepared according to the protocol, and HPLC analysis indicated that the correct concentration of Compound 1 was achieved in the aqueous solution and in the gel formulation.

- 26 045539

Table 9

Concentrations of compound 1 in aqueous stock solution and gel formulation

Sample Estimated conc. (mg/ml) Volume injection (µl) Dilution factor Comput. mg/ml Purity at 230 nm (%) Water source solution for adding to the gel 225 2 200 229,496 99.0 Gel composition 2.25 5 10 2.355 99.7

ml of the original aqueous solution was added to 1 ml of food coloring. It was diluted and analyzed by HPLC. The AUC for the aqueous solution diluted with food coloring was 0.63 times higher than for the aqueous solution before dilution, indicating that Compound 1 remains soluble when diluted with food coloring.

Summary/conclusion.

Compound 1 can be formulated in aqueous gel packs at a concentration of 2.25 mg/ml and is stable at both 4°C and room temperature for at least 20 days.

Example 5. Preparation of a saline solution of compound 1.

1. Weigh out 400 mg of compound 1 and add 0.7 ml of saline (0.9% w/v).

2. Sonicate for 20 min, then shake for 30 min or until all compound is visually dissolved.

Resistance of compound 1 to freezing/thawing.

Way.

1. Saline (0.9 w/v%) was added to compound 1 (1 mg/ml), then sonicated for 10 min until complete dissolution.

2. The sample was taken for HPLC analysis (F/T 0).

3. The solution was frozen at -80°C overnight.

4. The solution was thawed and a sample was taken for HPLC analysis (F/T 1).

5. Stages 3&4 were repeated in 3 cycles.

Table 10

Compound 1 cleanliness after freeze/thaw (FT) cycles

Sample F.T. cycle We assume that conc. (mg/ml) Volume injection (µl) Coeff, diluted nya Comput. mg/ml Purity (%) Compound 1 in saline solution 1 2 1 1.157 98.9 1 2 1 1.148 98.8 1 2 1 1.153 98.9 1 2 1 1.146 98.8

The results (Table 10) show that there is no significant change in the assay data or purity of Compound 1 in saline, so Compound 1 is stable for at least 3 freeze/thaw cycles.

Stability of compound 1 at room temperature.

Way.

1. Saline (0.9% w/v) was added to compound 1 (1 mg/ml), then sonicated for 10 min until complete dissolution.

2. A sample was taken for HPLC analysis (T=0).

3. The solution was stored at room temperature.

4. Samples were taken periodically for stability analysis by HPLC.

Results.

The results indicated that there was no significant change in the assay data or purity of Compound 1 in saline (data not shown), so Compound 1 was stable for at least 14 days at room temperature.

Preparation of the composition of compound 1 at a concentration of 200-500 mg/ml.

-27045539

Way.

1. Saline (0.9% w/v) was added to Compound 1, Batch I, in varying amounts, then sonicated for 10 min - the samples remained slightly cloudy.

2. The samples were shaken for 30 minutes, after which the solutions became translucent.

3. Solutions were diluted for HPLC analysis.

Results.

Table 11

Purity of Compound 1 at Increasing Concentrations in Saline

Sample RT (min) Volume injection (µl) Coefficient diluted Research Institute Comput. mg/ml Purity (%) 400 mg of compound 1 in 1 ml of saline solution 8.21 5 1000 261 98.7 400 mg compound 1 in 0.7 ml saline 8.21 5 1000 403 98.7 400 mg compound 1 in 0.4 ml saline 8.21 5 1000 545 98.6

The results show (Table 11) that concentrations of more than 500 mg/ml of compound 1 in saline can be achieved.

Preparation of the transfer composition (composition 400 mg/ml).

3. Weigh out 400 mg of compound 1, lot 11, and add 0.7 ml of saline (0.9% w/v).

4. Sonicate for 20 min, then shake for 30 min or until all compound is visually dissolved.

Summary/conclusion.

Resistance of compound 1 to freezing/thawing.

Compound 1 is stable for at least 3 freeze/thaw cycles.

Stability of compound 1 at room temperature.

Compound 1 is stable for at least 14 days when kept at room temperature (data collection will continue).

Preparation of the composition of compound 1 with a concentration of 400 mg/ml.

Concentrations greater than 500 mg/ml of Compound 1 in saline can be achieved.

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 maintained at room temperature, 37 and 65°C for stability studies over a 40-day period.

Table 12

Compound 1 when kept in water at room temperature in the dark (concentration 1 mg/ml) with determination of purity

Time (days) AUC at 230 nm AUC (% at T=0) Purity at 230 nm (%) Purity by data LCMS (%) 0 9041 100.0 100.0 100.0 1 8847 97.9 100.0 99.2 2 8807 97.4 100.0 98.3 4 8045 89.0 100.0 99.0 7 8588 95.0 100.0 99.2 AND 8554 94.6 100.0 98.7 14 8662 95.8 100.0 95.6 17 8873 98.1 100.0 98.1 23 8561 94.7 100.0 98.9 38 8555 94.6 100.0 98.7

There was a slight deterioration in purity and analytical data for Compound 1 upon aging

-28045539 in water at room temperature for 38 days.

Table 13

Compound 1 when kept in water at a temperature of 37°C in the dark (concentration 1 mg/ml) with determination of purity

Time (days) AUC at 230 nm AUC (% at T=0) Purity at 230 nm (%) Purity by data LCMS (%) 0 9044 100.0 100.0 100.0 1 9798 108.3 100.0 99.6 2 8681 96.0 100.0 97.5 4 8156 90.2 100.0 99.0 7 9106 100.7 100.0 98.9 AND 8577 94.8 100.0 98.4 14 7777 86.0 100.0 96.9 17 7928 87.7 100.0 93.2 23 7567 83.7 100.0 98.4 38 7579 83.8 100.0 98.3

There was a deterioration in purity and analytical data for compound 1 when kept in water at 37°C for 38 days. The degradation of the assay data was greater than that observed with immersion in water at room temperature.

Table 14

Compound 1 when kept in water at a temperature of 65°C in the dark (concentration 1 mg/ml) with determination of purity

Time (days) AUC at 230 nm AUC (%atT=0) Purity at 230 nm (%) Purity by data LCMS (%) 0 8741 100.0 100.0 100.0 1 8614 98.5 100.0 98.3 2 8410 96.2 100.0 97.3 5 7512 85.9 100.0 95.9 8 8750 100.1 100.0 99.1 14 9095 104.0 100.0 99.0 29 4521 50.0 100.0 89.1

Deterioration in purity and analytical data was noted for compound 1 when kept in water for 29 days. The deterioration of the analytical data was more significant than that observed when kept in water at a temperature of 37°C.

Table 15

Compound 1 when kept in DMSO at 37°C in the dark (concentration 1 mg/ml) with determination of purity

Time (days) AUC at 230 nm AUC (% at T=0) Purity at 230 nm (%) Purity by data LCMS (%) 0 16363 100.0 100.0 98.4 1 17465 106.7 100.0 98.4 4 17041 104.1 100.0 98.2 8 17644 107.8 100.0 98.2 AND 17434 106.5 100.0 99.0 14 17557 107.3 100.0 98.2 22 17353 106.1 100.0 98.0 37 17904 109.4 100.0 97.6

For Compound 1, no significant deterioration in purity or analytical data was observed when maintained in DMSO at 37°C for 37 days.

-29045539

Table 16

Compound 1 when kept in DMSO at 65°C in the dark (concentration 1 mg/ml) with determination of purity.

Time (days) AUC at 230 nm AUC (% at T=0) Purity at 230 nm (%) Purity according to data LCMS (%) 0 15834 100.0 100.0 98.1 1 16974 107.2 100.0 98.3 2 16185 102.2 100.0 96.8 5 17175 108.5 100.0 99.2 8 14415 91.0 100.0 96.7 14 13846 87.4 100.0 93.9 29 16472 100.7 100.0 97.7

For Compound 1, no significant deterioration in purity or analytical data was observed when held in DMSO at 65°C for 29 days.

Example 7 Analysis of Compound 1

Method C (batch 15) from Example 1 was analyzed by powder X-ray diffraction. The data is presented in Fig. 12 and show crystalline material.

Method A material (lot 12) was also analyzed by X-ray powder diffraction and was found to show crystalline material with the same polymorph (FIG. 11).

Example 8: Comparison of the solubility of compound 1 with other succinate prodrugs.

The solubility of compound 1 in aqueous formulations relative to other succinate prodrugs was assessed by dissolving the solid in aqueous formulations and measuring its amount in solution using HPLC(-MS). Compound 1 was observed to be soluble in water at a concentration greater than 350 mg/ml, PBS (pH 7.4) at a concentration of 190 mg/ml, and greater than 500 mg/ml in 0.9% saline, whereas the other succinate prodrugs evaluated had much lower solubility. In many cases, the maximum solubility of other prodrugs was below 100 μM. See table. 17, which provides examples of solubility data in PBS pH 7.4 for other succinate prodrugs.

Table 17

Solubility of examples of succinate prodrugs

Test sample

Solubility (µM)

100 (see text)

Example 9 Comparison of the bioavailability of compound 1 with other succinate prodrugs.

Cell penetrance and oral bioavailability of Compound 1 were examined using a standard in vitro Caco-2 bioavailability assay (in short, Caco-2 confluent cells (L1, A.P., 1992; Grass, G.M. et al., 1992, Volpe, D.A. et al., 1992) al., 2001) in a 24-well Corning Costar Transwell format provided by In Vitro Technologies Inc. (IVT Inc., Baltimore, Md., USA).The apical chamber contained 0.15 ml Hank's Balanced Buffer Solution (HBBS) pH 7, 4.1% DMSO,

0.1 mM Lucifer yellow. The basal chamber contained 0.6 ml HBBS pH 7.4, 1% DMSO. Control and test products were incubated at 37°C in a humidified incubator, shaking at 130 rpm for 1 hour. Lucifer yellow penetrates only through the paracellular pathway (between tight junctions), high apparent permeability (Papp) for lucifer yellow indicates cellular damage during analysis and all such wells were rejected. Propranolol (good passive permeability without known transporter effects) and acebutalol (poor passive permeation attenuated by active P-gp efflux) were used as control compounds. Compounds were tested in unidirectional and bidirectional formats by applying the compound to the apical or basal chamber (0.01 mM). Compounds in the apical or basal chambers were analyzed by HPLC-MS. Results were expressed as apparent permeability, Papp, (nm/s) compared to other succinate prodrugs, including several from WO 2015/155231. The data is presented in table. 18 below and show that apical to basolateral transmission is highest for compound 1, showing improved permeability and bio

- 30 045539 cell availability. This was confirmed by in vivo pharmacokinetic studies, which showed that compound 1 also has high oral bioavailability and penetration into the brain, unlike other prodrugs tested.

Table 18

Bioavailability of Caco-2 compound 1 compared to other succinate prodrugs

Connection ID Rarr(A-B) nm/s o □ L.s. A. Ό γ M - 1° connection 4.12 □ L. 1 O ' NH g Ad ⁰ A A A OO 3.13 O d About NH Y In OO 1.92 o o .- ₀ A^ _v s.^ s ° ⁿ 1.28 1 o o 1 d ,-, ,s,d O T Τ'Ν' o T ⁿ D ° 1.42 / oA ΖΙ < ) with Vo / \ ! Oh k ^- sh <0.21 O G \ And ___/ } / IZ A / 1.45 O O AS .I O V γ N G ° ' ^N 0.87 JAo G U o o J 1 .S L A ⁰ y A * ί Υ O HN^ J G HI ⁰ 2.62 1 o o Aa^ _v ..^ _n a. , ° ⁿ 1.35 O o Α _Ν η - o - - X - k oo 1.68 o o ^V-^yV'n'A _l a 3 b 0.16

-31 045539

Example 10 Comparison of thermodynamic solubility of different batches of compound 1.

Two batches of compound 1 with different crystallinities were obtained. Batch 2 had higher crystallinity than Batch 3, which was considered to have a higher degree of amorphousness of Compound 1 - Batch 2 was therefore considered to be a more crystalline batch, while Batch 3 was considered to be a more amorphous batch. The methods used are discussed in this document. PBS was prepared as usual (NaCl (8 g/L), KC1 (0.2 g/L), anhydrous sodium hydrogen phosphate (1.42 g/L) and anhydrous potassium dihydrogen phosphate (0.24 g/L) added to 250 ml deionized water and the mixture was stirred until all the solid was dissolved.The pH of the solution was adjusted to pH 7.4 using HC1 (1 M) or NaOH (1 M) if necessary).

A sample of the more amorphous compound 1, lot 3, was added to PBS or water to a final concentration of 258 mg/mL and sonicated for 10 min, then vortexed for 30 min. After centrifugation to remove solid material, the analysis indicated that a concentration of 258 mg/ml had been reached.

A sample of the more crystalline compound 1, batch 2, was added to HPLC-grade water (Fisher) to a final concentration of 30 mg/mL and sonicated for 10 min, then vortexed for 30 min. After centrifugation to remove solid material, analysis indicated that a concentration of 17 mg/ml had been achieved.

A sample of the more crystalline compound 1, lot 2, was added to HPLC water (Fisher) to a final concentration of 52 mg/ml and sonicated for 20 min, then vortexed for 1 h. After centrifugation to remove solid material, analysis indicated that there was a concentration of 52 mg/ml was achieved.

As can be seen from the data presented, a more amorphous substance has a much higher kinetic solubility than a more crystalline material.

A sample of the more crystalline compound 1, lot 2, was added to HPLC water (Fisher) to a final concentration of 2000 mg/mL and sonicated for 20 min, then vortexed for 1.5 h. After centrifugation to remove solid material, analysis indicated that a concentration of 850 mg/ml was achieved. This result shows that the water solubility of the more crystalline compound is at least 850 mg/ml, that is, it has high water solubility, but the kinetic solubility is higher for the more amorphous compound 1.

Example 11 Comparison of the stability of compound 1 in purified or untreated water.

A parallel stability experiment was conducted for compound 1 in HPLC-grade purified water (Fisher Scientific) and unpurified tap water. Briefly, 1 mg/mL solutions of compound 1 were prepared in HPLC-grade purified water (Fisher Scientific) and unpurified tap water. They were incubated at room temperature for up to 10 days. The concentration and purity of compound 1 were assessed over time by HPLC compared to a standard (AUC peak 8.21, RT at 230 nm for calculated concentration, and AUC peak of compound 1 compared to impurities for purity analysis). Data presented are the average of two samples.

Table 19

Time (days) Estimated conc. (mg/ml) Calculated conc. (mg/ml) Purity (%) 0 1 1.06 98.5 3 1 0.98 95.7 5 1 0.93 94.4 6 1 0.89 93.3 10 1 0.83 93.0

-32045539

Compound 1, batch 3, dissolved in tap water at a concentration of 1 mg/ml, was kept at room temperature for the number of days indicated in the table.

Table 20

Time (days) Estimated conc. (mg/ml) Calculated conc. (mg/ml) Purity (%) 0 1 1.02 99.0 3 1 1.04 98.8 5 1 1.03 99.0 6 1 1.01 99.0

Compound 1, Lot 3, dissolved in HPLC grade water (Fisher Scientific) at a concentration of 1 mg/mL, was kept at room temperature for the number of days indicated in the table.

Significant degradation of compound 1 is observed in samples dissolved in (untreated) tap water after incubation at room temperature for 6 days, as evidenced by loss of purity and analysis. This is not observed for samples dissolved in (purified) HPLC grade water (Fisher Scientific). Similar data were observed for two independent samples from different batches of compound 1.

Example 12. Composition of compound 1 with HP-cyclodextrin.

Obtaining the excipient.

Kleptozohydroxypropyl-b-cyclodextrin (25% w/v), sodium dihydrogen phosphate anhydrous (0.048 w/v), sodium hydrogen phosphate dihydrate (0.295% w/v) and calcium disodium EDTA (0.5% w/v) was added to 100 ml of deionized water. The mixture was sonicated for 20 min, then stirred until all solid dissolved, then adjusted to pH 7.4 with HCl (1 M).

Protocol for preparing the composition.

1. Weigh out the required amount of compound, 1 then add the required amount of the resulting excipient to the solid compound 1 to obtain the required mg/ml concentration of compound 1 (maximum concentration 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 resulting excipient.

2. The solution is sonicated for 10 minutes and then shaken for 20 minutes to ensure that compound 1 is completely dissolved.

3. The solution can be centrifuged (13000 rpm, 10 min) to remove any solid particles if necessary.

4. If necessary, the solution can be sterilized by filtration.

Example 13 Infusion of Compound 1 delivers succinate, increases succinate metabolism in pigs, and reduces blood lactate concentrations.

Infusion of compound 1 increases plasma succinate levels, increases tissue metabolism of succinate to fumarate, and decreases blood lactate concentrations; see fig. 7.

Yorkshire Landrace hybrid pigs were anesthetized and implanted with venous catheters for infusion of Compound 1 or vehicle (PBS) and collection of blood samples. Two animals received an escalating dose of Compound 1 (2-6 mg/kg/min) over 2.5 hours, with the dose increasing by 1 mg/kg/min every 30 minutes. One animal was infused at a constant rate of 2 mg/kg/min. The control animal was infused with PBS. Blood samples were collected at 30-minute intervals, and plasma was separated by centrifugation. Plasma and tissue samples were kept frozen and then analyzed for succinate by LC/MS using a Thermo Vanquish UPLC+Thermo Quantis triple quadrupole MS instrument, an Acquity UPLC HSS C18 column (100 x 2.1 mm, 1.8 μm) with a guard filter and elution gradient; A=0.1% formic acid, B=acetonitrile. Labeled [13C] succinate was used as an internal standard.

Plasma succinate concentrations increased proportionally to the time of infusion of Compound 1 (Fig. 7A), demonstrating the release of succinate from Compound 1. At the end of the study, fumarate, the primary metabolite of succinate in the TCA cycle, was higher in tissues of the Compound 1-treated animal than in the Compound 1-treated animal. carrier, especially in tissues with high metabolic activity such as the retina, brain and heart. Thus, the data suggest that compound 1 delivers metabolizable succinate to these tissues and has the ability to cross the blood-brain barrier.

Blood lactate data obtained from three animals were expressed as a percentage of the initial value and plotted as a function of the cumulative dose at the time of sampling (Fig. 7C).

Lactate content decreased compared to baseline values after intravenous infusion

- 33 045539 of compound 1, suggesting that compound 1 delivers succinate to complex 2 and increases the supply of electrons to the mitochondrial electron transport chain to increase ATP production and reduce the need for the glycolytic conversion of pyruvate to lactate.

Example 14 Porcine model of rotenone-induced mitochondrial complex 1 dysfunction.

Infusion of compound 1 restores rotenone-depleted succinate levels in organs and reduces rotenone-induced lactate in the brain; see fig. 8.

To study the effect of rotenone-mediated inhibition of complex 1, Yorkshire Landrace hybrid pigs were anesthetized and implanted with venous catheters for simultaneous infusion of rotenone and compound 1 or vehicle (PBS). Rotenone (7.1 mg/h) was administered over 1.5 h. Compound 1 was infused at a constant rate of 2 mg/kg/min over 2.5 h. The control animal was infused with PBS. Blood samples were collected at 30-minute intervals, and plasma was separated by centrifugation. Microdialysates were collected by inserting a microdialysis probe into the striatum and analyzed for lactate content using the ISCUS instrument (MDialysis). At the end of the infusion, the animal was sacrificed and blood and end-stage organ samples were collected. Plasma and tissue samples were kept frozen and then analyzed for succinate by LC/MS using a Thermo Vanquish UPLC+Thermo Quantis triple quadrupole MS instrument, an Acquity UPLC HSS C18 column (100 x 2.1 mm, 1.8 μm) with a guard filter and gradient elution; A=0.1% formic acid, B=acetonitrile. Labeled [13C] succinate was used as an internal standard. Lactate data were expressed as a percentage of the baseline value obtained before the start of the rotenone infusion.

Infusion of rotenone reduces tissue succinate concentrations to subquantitative levels (<2 μM), indicating increased utilization of succinate to compensate for decreased electron transfer from complex 1. Administration of compound 1 restores tissue succinate concentrations to detectable levels, suggesting that delivery succinate by compound 1 exceeded the increased succinate utilization caused by inhibition of complex 1. In addition, administration of compound 1 counteracted the rotenone-induced increase in brain lactate, confirming the reduced requirement for the glycolytic conversion of pyruvate to lactate by delivery of succinate to the brain.

Example 15. Genetic model of Ndufs4 knockout mouse with complex 1 dysfunction.

Administration of Compound 1 in drinking water (1 mg/mL) after weaning (21 days) resulted in a transient increase in body weight and a trend towards increased survival in the high concentration group; see fig. 9.

C57BL/6 mice with a genetic ablation of the Ndufs4 complex I gene (Quintana et al., Proceedings of the National Academy of Sciences. 107, 24 (2010), 10996-11001) were administered compound 1 (1 mg/ml) in drinking water or normal water. drinking water after weaning from a nursing female. Body weight development was monitored every 10 days (Fig. 9A) and animal health was monitored daily (Fig. 9B). Compound 1 increased weight gain (p<0.05) during the first 10 days and increased survival (p=0.0697). The results showed that in a genetic model of mitochondrial complex 11 dysfunction with signs of Leigh syndrome, a therapeutic effect could be achieved by adding succinate in the form of compound 1.

Example 16: Rotenone-induced motor dysfunction and lactic acidosis model in rats.

Compound 1, administered with drinking water, prevents motor dysfunction and reduces blood lactate concentrations; see fig. 10.

A rat model of rotenone-induced Parkinson's disease was used to study the effect of oral administration of Compound 1 on motor and metabolic dysfunctions caused by inhibition of complex 1 (Cannon et al., Neurobiol Dis. 2009 May;34(2):279-90).

Twelve-week-old Lewis rats (6 animals per group) were intraperitoneally administered rotenone (0.25-0.75 mg/kg) daily for 4 days. Compound 1 was dissolved in drinking water at a concentration of 0.25 and 0.75 mg/ml. Functional tests and lactate measurements were performed on day 4. Vertical standing was determined by placing the animals in a clear glass cylinder (height = 30 cm; diameter = 18 cm) for 5 min. To be classified as upright, the forelimbs must be raised above shoulder level and in contact with the cylinder wall with one or both forelimbs.

Postural instability was measured on a tabletop covered with P-120 sandpaper marked with lines and numbers every centimeter (see below). The animal was held in a vertical position (wheelbarrow position) at an angle of almost 90° to the surface, with one forelimb gently pressed against the body of the animal. The animal's center of gravity was then shifted forward over the single mounted forelimb to induce two catch-up steps to restore balance. The change in nose position was recorded as the distance at which the catch-up step of the free forelimb began. The experiment was repeated three times for each anterior

- 34 045539 limbs and calculated the average value for both forelimbs. Blood lactate (Fig. 10C) was determined using a VetScan iSTAT-1 analyzer.

Treatment with rotenone resulted in decreased standing activity. Administration of Compound 1 (0.75 mg/ml) to drinking water resulted in a significant increase in upright posture (Figure 10A) and ostural instability (Figure 10B) compared to water-treated animals. Thus, the data suggest that compound 1 is orally bioavailable and has the potential to improve motor dysfunction caused by mitochondrial complex 1 dysfunction.

Blood lactate concentrations increased significantly in animals treated with rotenone. There was a trend for blood lactate concentrations to decrease from a low concentration (0.25 mg/mL) of Compound 1 in drinking water to a high concentration (0.75 mg/mL). Data suggest that succinate delivered from Compound 1 can achieve metabolic compensation at the level of glycolysis and conversion of pyruvate to lactate when delivered orally via intermittent administration in drinking water, and imply its utility as an oral treatment.

Example 17 Succinate Release Data.

Briefly, a stock of compound 1 of batch 12 was prepared in a 50/50 DMSO/MeCN mixture (200 mM, x200). This mixture was then diluted 1:10 in microsomal buffer (20 mM, x20) consisting of K ₂ HPO ₄ (Sigma Aldrich, 13.9 g/L, anhydrous), KH2PO4 (Sigma Aldrich, 2.72 g/L, anhydrous ), MgCl ₂ 6H ₂ O (Fisher, 1.02 g/L) and EDTA (Sigma Aldrich, 0.375 g/L) dissolved in HPLC grade water. Then, a stock solution of 200 mM malonic acid (Sigma Aldrich) was prepared in microsomal buffer (200 mM, x20). A stock of 20 mM NADPH was also prepared in microsomal buffer (20 mM, x10). A stock of microsomes (Sekisui XenoTech, 0.625 mg/mL) was prepared in a 7 mL vial. Samples were prepared for each time temperature (T=0, 5, 15, 60 min) as follows: 80 μl of microsomal stock solution (final concentration 0.5 mg/ml), 5 μl of compound stock solution (final concentration 2 mM), 5 µl of malonate stock solution (final concentration 10 mM). The T=0 sample was quenched by adding 100 μL MeOH. The reaction was then initiated at all time points by adding 10 μL of NADPH stock solution (final concentration 2 mM). At each time point, the reaction was stopped by adding 100 μL MeOH. Samples were vortexed for 1 minute, placed on ice for 10 minutes, then centrifuged at 3000 rpm for 10 minutes. The supernatant was then analyzed by LCMS.

Table 21

Time (min) Samples detected by LCMS Compound 1 (mM) succinic acid Mono-methyl (mm) succinate(mM) 0 0.934 0.058 0.088 5 0.688 0.136 0.161 15 0.388 0.285 0.205 60 0.080 0.599 0.308

As can be seen from the data, compound 1 releases succinic acid over time when incubated with microsomes.

Example 18: Mineral instability.

Lot 14 Compound 1 (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 (with different accompanying counterions to be able to distinguish differences between cation or anion effects), and samples were taken at different time intervals for HPLC analysis.

Mineral sources used in the form of inorganic salts: CuCl ₂ , CaCl ₂ , NiSO ₄ , CoCl ₂ , N^Cl, MnCl2, NaF, NNO3, CUSO4, Ca(NO3^, MSO4, CaCO3, NbCO3.

- 35 045539

Table 22

Time (days) Percentage (%) of compound 1 remaining compared to time T=0 for each different salt added to HPLC water WFI CuC12 CaC12 NiSO4 CoC12 NH4C1 MnC12 0 100 100 100 100 100 100 100 7 98.7 99.5 100 98.4 101 101 98.9 14 97.7 98.3 97.9 99.1 101 99.8 99.2 Time (days) Percentage (%) of connection 1 remaining compared to time point T=0 for each different salt added to HPLC water NaF NaNO3 CuSO4 Ca(NO3)2 A1SO4 CaPOPs (NH4)2CO3 0 100 100 100 100 100 100 100 7 101 99.2 100 99.4 99.8 79.5 91.3 14 100 99.4 99.4 98.9 100 66.0 87.4

As can be seen from the data, compound 1 decomposes faster in an aqueous solution with carbonate ions.

Example 19a. Carbonate instability.

Compound 1, Lot 14, (2-3 mg) was dissolved in HPLC grade water at room temperature (1 mg/mL) containing various sources of calcium and carbonate ions (two concentrations for each source, with pH recorded) and at various time intervals samples were collected for HPLC analysis.

Sources of calcium and carbonate used in the form of inorganic salts: CaC1 ₂ , Ca(NO ₃ ) ₂ ,

CaCO3, (NH ₄ ) ₂ CO ₃ .

Table 23

Time (days) Percentage (%) of connection 1 remaining compared to time point T=0 for each individual mineral added to HPLC water HPLC water (control, pH 6.5) CaPOP (3.4 mM, pH 7.6) CaPOP (0.34 mM, pH 7.6) CaC12 (3.4 mM, pH 7.5) CaC12 (0.34 mM, pH 7.4) 0 100 100 100 100 100 3 94.3 93.6 93.8 101 98.4 17 91.4 49.5 50.4 96.7 95.1 Time (days) Percentage (%) of connection 1 remaining compared to time point T=0 for each individual mineral added to HPLC water Ca(NO3)2 (3.4 mM, pH 7.2) Ca(NO3)2 (0.34 mM, pH 7.3) (NH4)2CO3 (3.4 mM, pH 9.3) (NH4)2CO3 (3.4 mM, pH 8.9) 0 100 100 100 100 3 100 101 98.9 99.6 17 97.1 97.5 57.5 85.5

As can be seen from the data, compound 1 decomposes faster in an aqueous solution with carbonate ions.

Example 19b. Dependence on carbonate concentration.

Compound 1, lot 14, (2-3 mg) was dissolved in HPLC grade water (1 mg/mL) containing various concentrations of calcium carbonate and samples were taken at various time intervals for HPLC analysis.

Table 24

Time (days) Percentage (%) of connection 1 remaining compared to time point T=0 for each individual mineral added to HPLC water CaPOP (0 mM) CaPOP (0.425 mM) CaPOP (0.85 mM) CaPOP (1.7 mM) CaPOP (3.4 mM) 0 100 100 100 100 100 7 98.7 98.1 97.8 93.8 83.4 14 98.6 98.4 97.1 90.8 71.4

Example 20: Kinetic solubility.

Batches of solid compound s3, si2-17, (~80 mg) were added to the wells of a 96-well plate

-36045539 with a transparent bottom. PBS was added at 5°C to obtain a final concentration of 460 mg/ml 01-354 for all batches. After addition of PBS, the plate was shaken for 10 s and then immediately analyzed for turbidity at 620 nm in real time for 3 min using an Epoch Microplate Spectrophotometer (BioTek). The dissolution rate constant was calculated from the exponential decay of the recorded data.

Table 25

Compound 1, batch no. Rate constants, k (s-1) 3 0.014 12 0.022 13 0.013 14 0.011 15 0.074 16 0.055 17 0.111

General method for assessing crystallinity.

X-ray powder diffraction studies were carried out using a Bruker AXS D8 discover HTS.

Anode: Si anode at 40 kV and 4 mV; Goebel mirror and linear optics.

Detector: linear detector (LYNXEYE XE) with a receiver slit of 2.95°.

Measurement: scan range 2-45° 2θ, is/stage, 0.005°/stage.

Data collection program: Diffrac.Commander v7.3.3.0.0.

Data analysis software: Diffrac Eva v4.2.i.

No background correction or smoothing was applied. Data are presented as 2θ peak angle and intensity. To determine the degree of crystallinity, the combined area for all identified peaks was divided by the total area under the curve and expressed as a percentage.

List of peaks.

The following list details the peaks obtained from X-ray powder diffraction analysis for a number of crystal batches. The underlined peaks are common to most batches. Those with an asterisk near the corner may be peaks, more common, or unique to polymorphic forms.

NV354-s3 peak list - batch 3

Corner d value Intensity Rel. intensity 9.4 9,389 54 1.4% 10.9 8,136 453 12.1% 11.1* 7,997 1270 33.7% 11.4 7,777 110 2.9% 12.9 6,870 208 5.5% 13.1 6,776 1240 32.9%

- 37 045539

13.1 6,728 120 3.2% 13.8 6,420 36 1.0% 14.5 6.104 60 1.6% 14.9 5,939 406 10.8% 15.6 5.659 63 1.7% 16.2 5,476 179 4.8% 16.9* 5,246 89 2.4% 17.9 4,941 89 2.4% 18.5 4.785 49 1.3% 19.2 4.624 176 4.7% 19.6 4.528 226 6.0% 19.7 4,499 214 5.7% 20.1 4,418 111 3.0% 21.4 4,143 831 22.1% 21.7 4,100 95 2.5% 22.2 3,994 163 4.3% 22.7 3,919 698 18.6% 22.8 3,893 1410 37.4% 23.1 3.847 2760 73.4% 23.3 3.813 307 8.2% 24.0 3,711 3760 100.0% 24.4 3,640 58 1.5% 24.8 3,591 602 16.0% 25.2 3.538 78 2.1% 25.5 3,487 283 7.5% 26.1 3,409 1340 35.5% 27.2 3,282 53 1.4% 27.7 3.221 76 2.0% 28.0 3.185 58 1.5% 28.5 3.128 48 1.3% 29.9 2,989 69 1.8% 30.2 2.956 115 3.1% 30.8 2,901 89 2.4% 30.9 2,890 73 1.9%

- 38 045539

31.3 2.857 64 1.7% 31.6 2,829 29 0.8% 32.2 2,776 42 1.1% 32.8 2,730 27 0.7% 33.0 2,711 42 1.1% 33.2 2,694 59 1.6% 35.0 2,560 73 1.9% 35.4 2,536 37 1.0% 36.6 2,456 86 2.3% 38.6 2.333 49 1.3% 40.1 2,248 52 1.4% 41.6 2,169 58 1.5% 44.7 2,026 86 2.3% 44.8 2,022 57 1.5%

List of NV354-s12 peaks - batch 18

Corner d value Intensity Rel. intensity 5.6 15,805 68 3.3% 9.4 9,390 99 4.9% 10.9 8,141 233 11.5% I,1 7,949 365 18.0% AND 2 7,883 635 31.3% I,4 7,744 252 12.4% 12.9 6,848 183 9.0% 13.2 6,710 103 5.1% 13.8 6,432 115 5.7% 14.9 5,941 446 22.0% 15.7 5.657 91 4.5% 16.2 5,484 312 15.4% 16.9 5,243 288 14.2% 17.3 5.119 107 5.3% 18.1 4,902 87 4.3% 18.6 4,779 60 3.0% 19.3 4,607 274 13.5% 19.3 4,594 154 7.6%

- 39 045539

19.7 4,506 164 8.1% 20.1 4,411 505 25.0% 21.4 4,148 1030 51.1% 21.6 4.110 160 7.9% 22.2 4,001 395 19.5% 22.7 3,919 2030 100.0% 22.8 3,892 1960 96.6% 23.0 3.865 1990 98.3% 23.2 3.824 204 10.1% 24.0 3,709 90 4.5% 24.5 3.637 122 6.0% 24.8 3,593 226 11.1% 25.1 3.539 227 11.2% 25.5 3,487 126 6.2% 26.1 3,407 151 7.4% 27.8 3,210 196 9.7% 28.5 3.124 53 2.6% 32.8 2.728 41 2.0% 35.0 2,560 64 3.2% 37.7 2,386 75 3.7% 41.6 2,169 62 3.0% 43.0 2.104 26 1.3%

NV354-S13 Peak List - Batch 13

Corner d value Intensity Rel. intensity 7.9 11,190 39 2.3% 9.4 9,405 118 6.9% 10.9 8,136 668 39.3% I,1 7,963 408 24.0% AND 2 7,884 294 17.3% I,4 7,743 179 10.5% 12.9 6,858 209 12.3% 13.2 6,709 103 6.1% 13.8 6,432 26 1.6% 14.5 6,099 71 4.2%

-40045539

14.9 5,954 474 27.9% 15.6 5.659 155 9.1% 16.2 5,472 326 19.2% 16.9 5,250 134 7.9% 17.4 5.102 44 2.6% 18.1 4,906 89 5.2% 19.0 4.677 57 3.3% 19.2 4.619 269 15.8% 19.4 4,577 180 10.6% 19.7 4,505 162 9.5% 20.1 4,414 392 23.0% 21.4 4.145 536 31.5% 21.6 4.106 79 4.7% 22.3 3,990 396 23.3% 22.7 3,913 898 52.7% 22.9 3.887 1700 100.0% 23.1 3.855 1380 80.9% 23.3 3,812 336 19.8% 24.0 3,711 84 4.9% 24.6 3,620 131 7.7% 24.8 3,590 371 21.8% 25.2 3.536 152 8.9% 25.5 3,487 94 5.5% 26.1 3,407 195 11.4% 27.2 3,280 71 4.2% 27.8 3,210 100 5.9% 28.0 3.185 90 5.3% 29.6 3.015 47 2.8% 30.2 2,959 34 2.0% 30.8 2,902 72 4.2% 31.3 2.858 37 2.2% 31.6 2.827 27 1.6% 32.0 2,795 68 4.0% 32.8 2,730 79 4.6% 34.2 2,620 65 3.8% 34.4 2,602 52 3.0% 35.1 2.555 63 3.7% 35.4 2.535 66 3.9% 37.7 2,384 44 2.6% 40.1 2,246 35 2.1% 41.6 2,170 68 4.0% 44.2 2,049 27 1.6%

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List of NV354-s14 peaks - batch 14

Corner d value Intensity Rel. intensity 7.9 11,145 111 5.5% 9.4 9,370 132 6.5% 10.8 8.175 130 6.4% 10.9 8,132 74 3.7% I,1 7,992 343 16.9% I,4 7,750 184 9.0% 12.9 6,841 73 3.6% 13.2 6,713 169 8.3% 14.9 5.935 FROM 5.6% 15.6 5,663 80 3.9% 16.2 5,484 161 7.9% 16.9 5,248 47 2.3% 18.0 4.937 33 1.6% 18.6 4,777 52 2.6% 19.4 4,564 220 10.9% 19.7 4,502 195 9.6% 20.1 4.421 46 2.3% 21.4 4,143 1150 56.5% 22.2 4,000 355 17.5% 22.7 3.921 2030 100.0% 22.9 3.888 509 25.1% 23.0 3.857 812 40.0% 24.5 3.624 173 8.5% 24.8 3,592 40 2.0%

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25.2 3.537 89 4.4% 25.5 3,486 346 17.0% 26.0 3.427 122 6.0% 26.2 3,397 169 8.3% 27.1 3,282 63 3.1% 27.8 3,208 262 12.9% 28.0 3.182 57 2.8% 28.6 3.123 43 2.1% 28.7 3.113 52 2.6% 29.6 3,012 thirty 1.5% 30.3 2,951 82 4.0% 30.8 2,901 55 2.7% 31.2 2,864 31 1.5% 32.2 2.775 22 1.1% 33.0 2,711 55 2.7% 34.2 2.618 83 4.1% 34.8 2,577 44 2.2% 35.1 2,556 57 2.8% 36.2 2,483 167 8.2% 37.3 2,408 69 3.4% 37.7 2,386 44 2.2% 39.8 2,266 26 1.3% 41.6 2,169 83 4.1% 41.7 2,164 45 2.2%

NV354-s15 Pick List - Batch 19

Corner d value Intensity Rel. intensity 5.6 15,862 72 4.6% 7.9 11,170 52 3.3% 9.4 9,389 134 8.5% 10.8 8,151 388 24.6% I,1 7,976 412 26.2% AND 2 7,907 400 25.4% I,4 7,765 121 7.7% 12.9 6,846 108 6.8%

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13.2 6,714 211 13.4% 13.7 6,440 41 2.6% 14.9 5.936 456 29.0% 15.6 5,660 134 8.5% 16.2 5,478 312 19.8% 16.9 5.252 122 7.7% 18.1 4,906 49 3.1% 19.0 4,673 58 3.7% 19.2 4,614 96 6.1% 19.7 4,506 170 10.8% 20.1 4,413 162 10.3% 21.4 4,143 943 59.9% 21.6 4.104 306 19.5% 22.2 3,997 318 20.2% 22.8 3,894 1400 88.7% 23.1 3.854 1570 100.0% 23.3 3.813 488 31.0% 23.9 3,716 81 5.1% 24.5 3.628 74 4.7% 24.5 3.627 109 6.9% 24.8 3,592 139 8.8% 25.2 3.535 147 9.3% 25.6 3,482 146 9.3% 26.1 3,406 139 8.8% 27.2 3.281 52 3.3% 27.8 3.211 122 7.8% 29.6 3,012 34 2.2% 30.8 2,900 62 3.9% 33.0 2.715 25 1.6% 41.6 2,170 38 2.4%

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NV354-S16 Peak List - Batch 16

Corner d value Intensity Rel. intensity 5.6 15,858 91 4.2% 7.9 11,148 63 2.9% 9.4 9.395 138 6.3% 10.9 8,134 401 18.4% I,1 7,945 354 16.3% AND 2 7,891 514 23.6% I,4 7,748 167 7.7% 12.9 6,852 100 4.6% 13.2 6,709 175 8.0% 13.8 6,424 72 3.3% 14.5 6,091 54 2.5% 14.9 5,943 370 17.0% 15.7 5.656 151 7.0% 16.2 5,479 233 10.7% 16.9 5,244 248 11.4% 17.4 5.106 31 1.4% 18.1 4.895 106 4.9% 18.6 4,777 43 2.0% 19.2 4,613 284 13.1% 19.4 4,583 214 9.8% 19.7 4,507 179 8.2% 20.1 4,413 274 12.6% 21.4 4,147 622 28.6% 21.6 4.102 210 9.7% 22.2 3.995 293 13.5% 22.7 3.921 834 38.3% 22.8 3,891 1490 68.2% 23.1 3.855 2180 100.0% 23.3 3.813 279 12.8% 24.0 3,711 87 4.0% 24.5 3.627 100 4.6% 24.8 3,592 266 12.2% 25.2 3.533 180 8.3% 25.5 3,489 122 5.6% 26.2 3.403 162 7.4% 27.1 3,283 36 1.6%

-45 045539

27.8 3,207 173 7.9% 28.0 3,184 49 2.2% 28.5 3.125 34 1.6% 29.6 3.015 33 1.5% 30.8 2,905 82 3.8% 31.6 2,829 44 2.0% 32.0 2,791 24 1.1% 32.8 2.725 42 1.9% 34.2 2.618 71 3.3% 35.0 2,559 61 2.8% 35.4 2.537 28 1.3% 36.1 2,484 35 1.6% 37.7 2,387 48 2.2% 40.2 2,244 47 2.2% 41.6 2,170 98 4.5%

List of NV354-s17 peaks - batch 17

Corner d value Intensity Rel. intensity 7.9 11,171 108 2.6% 9.4 9.422 77 1.8% 10.8 8,158 297 7.1% ID 7,984 379 9.0% AND 2 7,873 790 18.9% I,4 7,753 134 3.2% 12.8 6,892 114 2.7% 12.9 6,845 185 4.4% 13.2 6,715 179 4.3% 13.8 6,420 77 1.8% 14.9 5,931 349 8.3% 15.7 5,649 225 5.4% 16.2 5,462 184 4.4% 16.9 5,248 303 7.2% 17.9 4,948 52 1.3% 18.5 4.785 40 0.9% 19.2 4.625 183 4.4%

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Claims (10)

19.3 4,586 263 6.3% 19.7 4,500 307 7.3% 20.1 4,416 220 5.3% 21.4 4,148 501 12.0% 21.6 4.105 145 3.5% 22.2 3,998 1100 26.3% 22.8 3,890 1700 40.6% 23.1 3.853 2000 47.7% 23.2 3.832 4190 100.0% 24.6 3.616 264 6.3% 24.8 3,590 771 18.4% 25.2 3.535 230 5.5% 25.5 3,489 127 3.0% 26.1 3,410 242 5.8% 27.8 3,207 285 6.8% 29.6 3.015 24 0.6% 30.8 2,901 151 3.6% 30.9 2,896 155 3.7% 31.2 2,861 314 7.5% 31.6 2,830 160 3.8% 33.1 2,707 38 0.9% 33.7 2.658 164 3.9% 34.2 2.621 62 1.5% 35.0 2,561 103 2.5% 38.6 2.331 95 2.3% 38.9 2,311 44 1.0% 40.7 2.216 77 1.8% 41.6 2,171 48 1.1% 42.4 2.129 34 0.8% CLAIM 1. Isolated methyl 3-[(2-acetylaminoethylthio)carbonyl]propionate (compound 1) in solid form having a purity of at least 80% w/w. 2. The isolated compound according to claim 1, having a purity of at least 90% w/w, at least 95% w/w, at least 97% w/w, at least 98% w/w ./wt. or at least 99% wt./wt. 3. Isolated compound 1 according to claim 1 or 2 in free form or its salt, hydrate, solvate or complex. 4. Isolated compound 1 according to claim 1 or 2, having a melting point or melting range from 35 to 55°C. 5. Isolated compound 1 according to any of the preceding paragraphs, which is a crystalline product, or an amorphous product, or a mixture thereof. 6. Isolated compound 1 according to any of the preceding paragraphs, having a solubility in water at room temperature of at least 300 mg/ml. 7. Isolated compound 1 according to claim 5, where the solubility in water at room temperature is in the range from 300 to 900 mg/ml. 8. Isolated compound 1 according to any one of the preceding claims, having a crystallinity in the range from 0 to 100%, for example from 50 to 100%. 9. Isolated compound 1 according to any of the preceding paragraphs, having a kinetic solubility in water corresponding to a rate constant in the range from 0.005 to 0.2 s ^-1 . 10. Isolated compound 1 according to any of the previous paragraphs, having a powder di -