EA044945B1 - NEW FERROPORTIN INHIBITOR SALTS - Google Patents

Description

Field of technology to which the invention relates

The invention relates to new salts of compounds having the general formula (I), pharmaceutical compositions containing them, and their use as medicines, in particular use as ferroportin inhibitors, more specifically for use in the prevention and/or treatment of diseases caused by hepcidin deficiency or disorders of iron metabolism, such as, in particular, iron overload conditions, such as, in particular, thalassemia, sickle cell anemia and hemochromatosis.

Prior Art

Iron is an essential trace element for almost all organisms and is necessary, in particular, for growth and the formation of blood elements. The balance of iron metabolism in this case is regulated mainly at the level of the return of iron from the hemoglobin of aging red blood cells and the absorption of dietary iron in the duodenum. The released iron is absorbed in the intestine, in particular with the help of specific transport systems (DMT-1, ferroportin), enters the circulatory system and is delivered to the corresponding tissues and organs (transferrin, transferrin receptors).

In the human body, iron plays a huge role in, among other things, oxygen transport, oxygen absorption, cellular functions such as electron transport in mitochondria, cognitive functions, etc., and energy metabolism in general.

On average, the human body contains 4-5 g of iron, which is present in enzymes, in hemoglobin and myoglobin, as well as stored or reserve iron in the form of ferritin and hemosiderin. About half of this iron, about 2 g, is heme iron bound in the hemoglobin of red blood cells. Since red blood cells have a limited lifespan (75-150 days), continuous formation of new and removal of old red blood cells is necessary (over 2 million red blood cells are formed every second). High regenerative capacity is provided by macrophages that engulf old red blood cells through phagocytosis, lyse them and regenerate the thus obtained iron for further iron metabolism. This covers most of the daily iron requirement for erythropoiesis, equal to 25 mg.

The daily iron requirement for an adult is 0.5 to 1.5 mg per day; children and pregnant women need 2-5 mg of iron per day. Daily loss of iron, for example due to the detachment of skin cells and epithelium, is small. Increased iron loss occurs, for example, during menstrual bleeding in women. In general, blood loss can significantly reduce iron levels, since approximately 1 mg of iron is lost per two milliliters of blood. In a healthy adult, daily iron loss, normally about 1 mg, is usually replaced by daily dietary intake, which brings the daily iron requirement back to normal levels.

Iron levels are regulated by absorption, and the percentage of iron absorption from food ranges from 6 to 12%; in case of iron deficiency, the percentage of absorption increases to 25%. The level of absorption is regulated by the body depending on the need for iron and the size of iron stores. During this process, the human body absorbs both ferrous and ferric iron ions. Typically, iron(III) compounds dissolve in the stomach at sufficiently acidic pH values and thus become available for absorption. Iron absorption occurs in the upper part of the small intestine by mucosal cells. During this process, non-heme ferric iron is first reduced in the intestinal cell membrane to Fe(II) for absorption, for example by iron reductase (membrane-bound duodenal cytochrome b), so that it can then be transported into intestinal cells via the transport protein DMT1 (divalent metal transporter 1). In contrast, heme iron enters enterocytes through the cell membrane without any changes. In enterocytes, iron is either stored in ferritin as stored iron or released into the blood by the transport protein ferroportin. Hepcidin plays a central role in this process, since it is the most important regulatory factor in iron absorption. Ferrous iron, transported into the blood by ferroportin, is converted into ferric iron by oxidase (ceruloplasmin, hephaestin), and then ferric iron is transported to the appropriate parts of the body by transferrin (see, for example, Balancing acts: molecular control of mammalian iron metabolism. M.W. Hentze, Cell 117 , 2004, 285-297).

Mammalian bodies are unable to actively remove iron. Iron metabolism is primarily controlled by hepcidin through cellular iron release from macrophages, hepatocytes and enterocytes.

Hepcidin is a peptide hormone produced in the liver. The predominant active form contains 25 amino acid residues (see, for example, the article Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation. T. Ganz Blood 702,2003,783-8), although two forms, shortened by amino end: hepcidin-22 and hepcidin-20. Hepcidin affects the absorption of iron through the intestines, across the placenta, and the release of iron from the reticuloendothelial system. In the body, hepcidin is synthesized from the so-called pro-hepcidin in the liver, and pro-hepcidin is encoded by the so-called HAMP gene. The formation of hepcidin is regulated in direct correlation with the level of iron in the body, i.e. If the body receives enough iron and oxygen, increased amounts of hepcidin are formed, and if the supply of iron and oxygen is low, or in the case of increased erythropoiesis, less hepcidin is formed. In the mucosal cells of the small intestine and in macrophages, hepcidin binds to the transport protein ferroportin, through which phagocytically recycled iron is normally transported from cells to the blood.

The transport protein ferroportin is a transmembrane protein containing 57 amino acid residues that is produced in the liver, spleen, kidneys, heart, intestines and placenta. In particular, ferroportin is localized in the basolateral membrane of intestinal epithelial cells. Ferroportin bound in this way affects the export of iron into the blood. In this case, it is most likely that ferroportin transports iron in the form of Fe ²⁺ . If hepcidin is associated with ferroportin, ferroportin is transported into the cell, where it is destroyed, which then completely blocks the exit of phagocytically recycled iron from the cells. If ferroportin is inactivated, for example by hepcidin, so that it is unable to export the iron contained in the mucous cells, the iron contained in them is lost due to the natural excretion of the cells in the stool. Thus, intestinal iron absorption is reduced when ferroportin is inactivated or suppressed, for example by hepcidin. In addition, ferroportin is prominently localized in the reticuloendothelial system (RES), to which macrophages also belong. Hepcidin plays an important role in the deterioration of iron metabolism in chronic inflammation. In the case of such inflammation, in particular, the level of interleukin-6 increases, which leads to an increase in the content of hepcidin. As a result, increased amounts of hepcidin bind to macrophage ferroportin, thereby blocking the release of iron, ultimately leading to the development of anemia caused by inflammation (ACD or AI).

On the other hand, if the iron content in the blood plasma decreases, then the production of hepcidin in the liver hepatocytes decreases, and therefore less hepcidin is released, accordingly less ferroportin is inactivated, which allows more accumulated iron to be transported into the blood.

From the above, it becomes clear that the hepcidin-ferroportin system directly regulates iron metabolism and that disruption of the hepcidin regulation mechanism directly affects iron metabolism in the body. In principle, the mechanism of hepcidin-ferroportin regulation works on two opposing principles described below.

On the one hand, an increase in hepcidin content leads to inactivation of ferroportin, which blocks the release of accumulated iron from cells into the blood, thereby reducing the iron content in the blood. In pathological cases, low iron levels in the blood lead to low hemoglobin levels, decreased red blood cell production and, as a result, iron deficiency anemia.

On the other hand, a decrease in hepcidin content leads to an increase in the content of active ferroportin, which allows for an increase in the release of accumulated iron and an increase in the supply of iron, for example, from food, thereby increasing the level of iron in the blood. In pathological cases, elevated levels of iron in the blood lead to iron oversaturation.

Iron overload conditions and diseases are characterized by excess iron levels. Problems arise due to excess iron in the blood, which leads to the appearance of so-called free iron (NTBI). Free iron is quickly nonspecifically absorbed by organs, which leads to the accumulation of iron in organs and tissues. Iron overload causes many diseases and adverse medical conditions, including damage to the heart, liver and endocrine system. In addition, iron accumulation in the brain has been observed in patients suffering from neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. As a particularly harmful aspect of excess free iron, the undesirable formation of radicals should be mentioned. In particular, iron(III) ions catalyze the formation (among other things, via the Fenton reaction) of reactive oxygen species (ROS). ROS cause damage to DNA, fats, proteins and carbohydrates, which has long-term effects on cells, tissues and organs. The formation of ROS is well known and described in the literature as a cause of the development of so-called oxidative stress.

A well-known current treatment for iron overload is based on the concept of reducing the amount of iron in the blood by increasing the removal of iron from the body. The oldest known method, and still routinely used, for otherwise healthy patients is regular phlebotomy (bloodletting). After initial diagnosis, phlebotomy is usually ordered quite frequently, such as once a week, until iron levels are within the normal range, after which phlebotomy is ordered once a month or every three months, depending on the rate of iron accumulation in the individual patient.

For patients unable to tolerate regular phlebotomy, chelating agents are available for use. For example, deferoxamine (also known as desferrioxamine B, N'-{5-[acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl }amino)pen

- 2 044945 til]-N-hydroxysuccinamide or Desferal®), which is a bacterial siderophore, is a known drug for use in chelation therapy. Deferoxamine binds iron in the circulatory system as a chelator and enhances its excretion in urine and feces. Typical treatment for chronic iron overload requires subcutaneous administration for 8-12 hours every day. Parenterally injectable compositions of desferrioxamine-B salts are described, for example, in WO 1998/25887.

Two new drugs licensed for use in patients receiving regular blood transfusions to treat thalassemia causing iron overload are deferasirox and deferiprone.

Deferasirox (Exjade®, 4-(3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl)benzoic acid), described for example in WO 1997/49395, and deferiprone ( Ferriprox®, 3-hydroxy-1,2-dimethylpyridin4(1H)-one) similarly work as iron chelating agents and are therefore suitable for use as medicinal compounds for iron chelating therapy.

Other compounds have also been described to act as iron chelators for the treatment of iron overload. For example, WO 2013/142258 concerns encapsulated particles of diethylenetriamine pentaacetate (DTPA) and zinc salt. WO 2003/041709 concerns 4-hydroxy-2-alkylquinolines, such as 4-hydroxy-2-nonylquinoline, as iron chelators. WO 1998/09626 concerns chelating agents based on dithiocarbamate-containing compositions for the treatment of iron overload conditions.

WO 2015/077655 concerns deferrithiocin derivatives having the general formula (A) or (J)

for use in the treatment of diseases associated with iron oversaturation. According to WO 2015/077655, these deferrithiocin derivatives have been found to work as iron chelating agents.

WO 2005/051411 concerns new antibiotics or antifungals based on oxaheline and its derivatives, having the formula

which have been described as working as iron chelators and suitable for use in the treatment of diseases associated with iron overload.

The disadvantage of treating iron overload with chelation therapy is that it removes chelated iron from the body after iron overload has already occurred, rather than preventing the onset of the disease. In addition, the drugs used for iron chelating therapy are known to have toxic potential.

It can be expected that modern approaches will gradually replace this method, particularly as knowledge of the underlying mechanisms increases and suitable therapies are developed based on this knowledge. Hepcidin agonists or compounds that have an inhibitory or supportive effect on the biochemical pathways regulating iron metabolism are generally known in the art.

Iron overload can occur, for example, if hepcidin production is impaired, for example due to a genetic defect such as the well-known iron overload disease hemochromatosis. Hemochromatosis is an iron overload disorder caused by mutations in the genes that control hepcidin synthesis or in the hepcidin gene itself. Low or no hepcidin levels in such patients lead to increased amounts of active ferroportin, which increases the absorption of iron from food, leading to severe iron overload, causing damage to the heart, liver and endocrine system. Was shown,

- 3 044945 that hepcidin mimetic proteins, i.e. proteins that similarly bind and inactivate ferroportin effectively reverse tissue iron accumulation in hepcidin knockout mice, a model of Type 2 (juvenile) hemochromatosis (Ramos et al., Blood 2012).

In the well-known iron overload disease beta thalassemia, a mutation in the beta globin gene causes decreased hemoglobin production and ineffective erythropoiesis, the inability to produce enough red blood cells due to damage and death of developing red blood cells in the bone marrow. This causes an increase in the rate of erythropoiesis and a decrease in hepcidin levels in order to make more iron available for the needs of increased erythropoiesis. This inadequate response leads to iron overload due to decreased hepcidin levels, resulting in increased amounts of active ferroportin and increased absorption of dietary iron, as described above. Red blood cells in thalassemia have a shortened half-life due to the toxicity of an unbalanced ratio of alpha and beta subunits of hemoglobin. The use of hepcidin mimetic proteins has also been described in the treatment of beta thalassemia, the therapeutic rationale for which is based on increasing the activity of hepcidin, leading to limiting iron levels and reducing iron-induced damage in red blood cells. Administration of hepcidin mimetic proteins to th3/+ mice, in a transfusion-independent model of thalassemia, resulted in attenuated symptoms of ineffective erythropoiesis, increased red blood cell lifespan, and decreased anemia. In this model, prevention of iron overload due to decreased dietary iron absorption was identified as an additional benefit of hepcidin mimetics therapy (Gardenghi et al. 2010; Casu et al. 2013).

The described methods of therapy are based on direct intervention in the impaired iron metabolic pathway by direct action through the primary regulator hepcidin through the administration of a hepcidin mimetic or a hepcidin agonist, i.e. effects by replacing or increasing the amount of hepcidin. This approach is based on the therapeutic principle of treating iron overload, i.e. increased levels of iron in the blood, by suppressing ferroportin through the mechanism of hepcidin inactivation, which leads to blocking excess iron absorption.

Other known diseases associated with iron overload are diseases associated with ineffective erythropoiesis such as myelodysplastic syndrome (also known as MDS or myelodysplasia), polycythemia vera, etc.

In addition, mutations in genes involved in the regulation of systemic iron stores, such as hepcidin (Hamp1), hemochromatosis protein (HFE), hemojuvelin (HJV), and transferrin receptor 2 (TFR2), cause iron overload in mice and humans. Accordingly, diseases associated with HFE gene mutations, diseases associated with chronic hemolysis, sickle cell anemia, red blood cell membrane disorders, as well as glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency), erythropoietic porphyria and Friedreich's ataxia can be specified. In addition, subtypes of iron overload include transfusion iron overload, iron toxicity, pulmonary hemosiderosis, osteopenia, insulin resistance, African iron overload, Hallervorden-Spatz disease, hyperferritinemia, ceruloplasmin deficiency, neonatal hemochromatosis, and red blood cell disorders including thalassemia, alpha thalassemia, intermediate thalassemia, sickle cell disease and myelodysplastic syndrome.

Other diseases and/or disorders and/or disease states associated with elevated iron levels include, but are not limited to, diseases caused by elevated iron levels including ataxia, Friedreich's ataxia, age-related macular degeneration, age-related cataracts, age-related retinal diseases and neurodegenerative diseases. diseases, wherein said neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, pantothenate kinase-associated neurodegeneration, restless legs syndrome and Huntington's disease.

Hepcidin is a host-protective protein that is a component of the innate immune system that responds to invading organisms.

It has been reported that many bacteria are highly dependent on iron supply from the host (so-called siderophilic organisms) and have evolved mechanisms to capture iron from surrounding tissues. The ability to limit the amount of iron available to such organisms through the use of ferroportin inhibitors may be an effective adjuvant therapy. One such siderophilic organism is Vibrio vulnificus, which causes rare but extremely severe infections in coastal communities, often in patients with undiagnosed iron overload. Studies in animals inoculated with lethal doses of Vibrio vulnificus have shown nearly 100% survival when treated with hepcidin mimetic proteins that inactivate ferroportin, regardless of whether treatment was initiated before or after infection (Arezes et al. 2015).

Known hepcidin mimetics include the so-called minihepcidins described, for example, in WO 2013/086143. Minihepcidins are small synthetic peptide analogues of the N-terminus of hepcidin, which is responsible for the interaction of hepcidin with ferroportin. Minihepcidins were developed based on the discovery that the first 9 amino acids

- 4 044945 hepcidin (DTHFPICIF) is sufficient to exhibit activity in vitro (measured by destruction of ferroportin-GFP). Minihepcidins contain a modified sequence of nine amino acids of hepcidin to increase resistance to proteolysis and enhance biophysical interaction with ferroportin. It has been reported that minihepcidins can be used to treat iron overload conditions in humans caused by hepcidin deficiency.

WO 2015/069660 describes methods for enhancing hepcidin production to treat iron overload conditions by reducing free iron (NTBI) by administering a modified iron binding/transferrin releaser.

All the described compounds working as hepcidin agonists, hepcidin mimetics or ferroportin inhibitors, etc., are compounds with relatively high molecular weight, especially those obtained mainly by genetic engineering methods. Various other approaches based on biomolecular interactions and biomolecules have been described. The disadvantages are the difficult preparation and high sensitivity of such biomolecular compounds. In particular, methods based on antibodies to ferroportin are not effective enough, since ferroportin suppressed by antibodies is continuously reproduced by the body, and therefore the suppression is not long enough to achieve the target therapeutic effect.

Low molecular weight compounds are also known that are involved in iron metabolism and can have an inhibitory or promoting effect.

For example, WO 2008/151288, WO 2008/118790, WO 2008/115999 and WO 2008/109840 describe compounds that act as divalent metal transporter 1 (DMT1) inhibitors and their use for the treatment of iron metabolic disorders such as thalassemia or hemochromatosis .

WO 2008/123093 describes an agent for the prevention or treatment of iron overload conditions, which is 22 beta-methoxyoleane-12-ene-3 beta,24(4 beta)-diol.

EP 1074254 and EP 1072265 concern the use of plant polyphenols with catechin and flavonoid structures for the treatment of iron overload.

WO 2011/029832 concerns thiazole and oxazole compounds which act as hepcidin antagonists and are therefore described as suitable for the treatment of diseases associated with iron deficiency. This document describes that hepcidin antagonistic activity inhibits the suppression of ferroportin by hepcidin, which is an effect opposite to that found by the present inventors for the new thiazole and oxazole compounds.

Unpublished international patent applications PCT/EP2016/075305 and PCT/EP2016/075306 describe compounds having activity as ferroportin inhibitors that overlap with some of the compounds of formula (I) of the present invention and are generally in the free base form and/or pharmaceutically acceptable salts thereof. These international patent applications provide a general list of possible acids for pharmaceutically acceptable salts. In addition, these international patent applications provide some specific examples of compounds in the form of 2HCl salts, 3HCl salts or 4HCl salts, of which only some specific examples of HCl salts overlap with some of the compounds having formula (I) of the present invention. Accordingly, the present invention describes a new set of very specific group of compounds having formula (I), being in the form of a salt (instead of a free base or mixtures of salts and free bases) and further characterized by a new set of specific counterion ratios (free base/compound (C: acid).

Chemical compounds, and their salts, based on structures having general formula (I) of the present invention have not hitherto been described in connection with their activity as ferroportin inhibitors or for use in the prevention and treatment of iron metabolic disorders associated with increased iron levels, such as iron overload.

B US 2004/0138268 A1, US 2011/0224136 A1, CN 103508957, WO 2006/062224 A1, WO 2015/051362 A1, EP 1953145 A1, WO 2009/154739 A2, GB 937878 A , WO 2011/023722 A1, WO 2010 /020556 A1, WO 2005/011685 A1, WO 00/56724 A1, WO 2010/036632 A1, WO 2005/014576 A1, WO 2013/067578 A1, WO 2005/116355 A1, EP 1889842 A1, US 2013/303508 A1, WO 98/27108 A2, WO 2006/040646 A1, WO 2010/078408 A1 or Ashish K. Pathak et al. Solution-Phase Parallel Synthesis of Acyclic Nucleoside Libraries of Purine, Pyrimidine, and Triazole Acetamides, ACS Combinatorial Science Vol. 16, No. 9, pages 485-493, 2014, Zou Yiquan et al. Discovery of pyrazole as C-terminus of selective BACE1 inhibitors; Eur. J. of Medicinal Chemistry 68 (2013) 270-283, Tussing-Humphreys et al. Rethinking Iron Regulation and Assessment in Iron Deficiency, Anemia of Chronic Disease, and Obesity: Introducing Hepcidin J. Academy of Nutrition and Dietetics (2012), Vol. 122, No. 3, 391-400, Riordan et al. Bleomycin analogs. Synthesis and proton NMR spectral assignments of thiazole amides related to bleomycin A2 (1); J. Heterocyclic Chem. 18, 1213 (1981), Hideaki Sasaki Synthesis of a novel bis(2,4'-bithiazole) derivative as a Co(II)-activated DNA cleaving agent; Chem. Pharm. Bull. 42(8) 1685-1687 (1994), and Ballell et al. Fueling open-source drug discovery. 177 small-molecule leads against tuberculosis; ChemMedChem 2013, 8, 313-321 describes compounds for various medical applications and having different mechanisms of action.

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Purpose of the invention.

The purpose of the present invention is to develop, in particular, new therapeutically effective compounds that can be used for effective therapy for the prevention and treatment of disorders of iron metabolism associated with elevated iron levels, such as, in particular, iron overload. As part of another objective of the invention, said new compounds should exhibit few side effects and have very low toxicity and good bioavailability and compatibility. In addition, these new compounds, unlike known iron-chelating compounds, should be suitable for preventing an increase in iron levels and thereby preventing related diseases, rather than removing excess iron from the body once an increase in iron levels has already occurred. As part of another objective of the invention, these new compounds must have a defined structure (stoichiometry), they must be prepared using simple synthesis techniques, they must exhibit less sensitivity and improved long-term effectiveness compared to known biomolecular compounds such as antibodies.

In another aspect of the present invention, the new compounds must have optimal stability of their physical, chemical and physicochemical characteristics. Particularly for pharmaceutical applications, good or improved long-term stability (storage stability) is an important aspect for the development of new pharmaceutically active compounds that retain their physical, chemical and physicochemical properties for a long time. Also, solubility stability (i.e., a stable solubility profile) is important for pharmaceutical applications. Therefore, another object of the present invention is to develop new compounds described herein having good or improved long-term stability, including, for example, reduced or no solvent loss, and/or reduced or no mass loss at elevated temperatures, reduced hygroscopicity or lack thereof, preservation of the solid structure even during long-term storage at different temperatures and humidity, stability of the crystalline form to vacuum drying, high reproducibility with high purity and a small amount of by-products or decomposition products in the production method, preservation of the solubility profile even under long-term storage conditions at different temperatures and humidity, as well as combinations of these characteristics.

This goal was achieved by developing new salts of compounds having formula (I) as indicated herein, which have been found to work as ferroportin inhibitors, and thereby can be used to suppress iron transport, and can be effectively used in the prevention and treatment of disorders of iron metabolism associated with elevated iron levels, such as, in particular, iron overload, and in the prevention and treatment of diseases caused by hepcidin deficiency, diseases associated with or caused by elevated iron levels or iron overload, and diseases associated with ineffective erythropoiesis .

Description of the invention.

The inventors of the present invention have unexpectedly discovered that certain salts of compounds having the general structural formula (I) act as ferroportin inhibitors, thereby effectively inhibiting iron transport, and can be used as medicines, in particular for the treatment and/or prevention of diseases caused by hepcidin deficiency, diseases associated with ineffective erythropoiesis, or disorders of iron metabolism leading to elevated iron levels, such as, but not limited to, iron overload conditions such as, but not limited to, thalassemia, sickle cell disease, and hemochromatosis. In particular, the new compounds have proven suitable for the treatment of thalassemia, sickle cell anemia and hemochromatosis. These new compounds can also be used to treat diseases caused by pathologically low levels of hepcidin and to inhibit iron transport.

Accordingly, the present invention relates to new salts of compounds having the general formula (I) wherein X ₁ represents N or O; And

X2 represents N, S or O;

provided that X ₁ and X ₂ are different;

R1 is selected from the group consisting of a hydrogen atom and an optionally substituted alkyl; n is an integer from 1 to 3;

- 6 044945

A ¹ and A ² are independently selected from the group consisting of alkanediyl;

R ² represents a hydrogen atom or optionally substituted alkyl; or

A ¹ and R ² together with the nitrogen atom to which they are attached form an optionally substituted 4-6 membered ring;

R ³ represents 1, 2 or 3 optional substituents which may be independently selected from the group consisting of halogen, cyano, optionally substituted alkyl, optionally substituted alkoxy and carboxyl;

R ⁴ is selected from the group consisting of hydrogen, halogen, C ₁ -C ₃ -alkyl and halogen-substituted alkyl, wherein said salts are selected from salts of compounds having formula (I) with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of compound (I):acidsα is from 1 :2 to 1:3; and where the following ZNS1 salts are excluded:

Note 40:

Note 94:

- 7 044945

In a preferred embodiment, the present invention relates to new salts of compounds having the general formula (I)

where X ¹ represents N or O; And

X ² represents N, S or O;

provided that X ¹ and X ² are different;

R ¹ represents a hydrogen atom;

n is an integer from 1 to 3;

A ¹ and A ² are independently methylene or ethane-1,2-diyl;

R ² represents a hydrogen atom or a linear or branched C1-C ₄ alkyl; or

A ¹ and R ² , together with the nitrogen atom to which they are attached, form an unsubstituted 4-membered ring of the formula

R ³ represents H;

R ⁴ represents H, wherein said salt is selected from salts of the compound of formula (I), with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of compound (I):acid is from 1:2 to 1:3; and its solvates; and where the following ZNS1 salts are excluded:

- 8 044945

Note 114:

and where the following 2HC1-salt is excluded:

In the context of the present invention, the above substituent groups have the following definitions.

Optionally substituted alkyl preferably includes straight or branched alkyl, preferably containing 1-8, more preferably 1-6, especially preferably 1-4, even more preferably 1, 2 or 3 carbon atoms, denoted

- 9 044945 also as C1-C ₄ -alkyl or C ₁ -C ₃ -alkyl.

Optionally substituted alkyl also includes cycloalkyl containing preferably 3-8, more preferably 5 or 6 carbon atoms.

Examples of alkyl radicals containing 1 to 8 carbon atoms include a methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group, n-pentyl group, isopentyl group group, sec-pentyl group, tert-pentyl group, 2-methylbutyl group, n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, 3-ethylbutyl group, 1,1-dimethylbutyl group, 2,2-dimethylbutyl group, 3,3-dimethylbutyl group, 1-ethyl-1-methylpropyl group, n-heptyl group, 1-methylhexyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl group, 1-ethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, 4-ethylpentyl group, 1,1-dimethylpentyl group, 2,2-dimethylpentyl group, 3 ,3-dimethylpentyl group, 4,4-dimethylpentyl group, 1-propylbutyl group, n-octyl group, 1-methylheptyl group, 2-methylheptyl group, 3-methylheptyl group, 4-methylheptyl group, 5-methylheptyl group, 6- methylheptyl group, 1-ethylhexyl group, 2-ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 5-ethylhexyl group, 1,1-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4 -dimethylhexyl group, 5,5dimethylhexyl group, 1-propylpentyl group, 2-propylpentyl group, etc. Groups containing 1-4 carbon atoms (C1- _C4 alkyl), such as, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, are preferred. C1-C3 alkyl, in particular methyl, ethyl, propyl and isopropyl, are more preferred. Most preferred are C1 and C2 alkyl, such as methyl and ethyl.

Cycloalkyl radicals containing 3 to 8 carbon atoms preferably include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group and a cyclooctyl group. A cyclopropyl group, a cyclobutyl group, a cyclopentyl group and a cyclohexyl group are preferred. The cyclopropyl group is particularly preferred.

Substituents on the optionally substituted alkyl described above preferably include 1, 2 or 3 identical or different substituents selected, for example, from the group consisting of halogen as described above, such as preferably F, cycloalkyl as described above, such as preferably cyclopropyl, optionally substituted as described below a heteroaryl group, such as preferably a benzimidazolyl group, as described below, an optionally substituted amino group, such as preferably an amino group or a benzyloxycarbonylamino group, a carboxyl group, as described below, an aminocarbonyl group, and also from an alkylene group, such as, in particular, a methylene group, forming, for example, methylene- substituted ethyl group (CH ₃ -(C=CH ₂ )- or .^=, where * denotes the point of attachment).

In the context of the present invention, halogen includes fluorine, chlorine, bromine and iodine, preferably fluorine or chlorine, most preferably fluorine.

Examples of a linear or branched alkyl moiety substituted with halogen and containing 1 to 8 carbon atoms include a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, a chloromethyl group, a dichloromethyl group, a trichloromethyl group, a bromomethyl group, a dibromomethyl group, a tribromomethyl group, a 1-fluoroethyl group, 1-chloroethyl group, 1-bromoethyl group, 2-fluoroethyl group, 2-chloroethyl group, 2-bromoethyl group, difluoroethyl group such as 1,2-difluoroethyl group, 1,2-dichloroethyl group, 1,2-dibromoethyl group, 2 ,2-difluoroethyl group, 2,2-dichloroethyl group, 2,2-dibromoethyl group, 2,2,2-trifluoroethyl group, heptafluoroethyl group, 1-fluoropropyl group, 1-chloropropyl group, 1-bromopropyl group, 2-fluoropropyl group group, 2-chloropropyl group, 2-bromopropyl group, 3-fluoropropyl group, 3-chloropropyl group, 3-bromopropyl group, 1,2-difluoropropyl group, 1,2-dichloropropyl group, 1,2-dibromopropyl group, 2,3-difluoropropyl group , 2,3-dichloropropyl group, 2,3-dibromopropyl group, 3,3,3-trifluoropropyl group, 2,2,3,3,3-pentafluoropropyl group, 2-fluorobutyl group, 2-chlorobutyl group, 2-bromobutyl group, 4-fluorobutyl group, 4-chlorobutyl group, 4-bromobutyl group, 4,4,4-trifluorobutyl group, 2,2,3,3,4,4,4heptafluorobutyl group, perfluorobutyl group, 2-fluoropentyl group, 2-chloropentyl group group, 2-bromopentyl group, 5-fluoropentyl group, 5-chloropentyl group, 5-bromopentyl group, perfluoropentyl group, 2-fluorohexyl group, 2-chlorohexyl group, 2-bromohexyl group, 6-fluorohexyl group, 6-chlorohexyl group, 6- bromohexyl group, perfluorohexyl group, 2-fluoroheptyl group, 2-chloroheptyl group, 2-bromoheptyl group, 7-fluoroheptyl group, 7-chloroheptyl group, 7-bromoheptyl group, perfluoro-heptyl group, etc. Of particular note are fluoroalkyl, difluoroalkyl and trifluoroalkyl, with trifluoromethyl and mono- and di-fluoroethyl being preferred. Trifluoromethyl is particularly preferred.

Examples of the cycloalkyl-substituted alkyl group include the above alkyl radicals containing 1 to 3, preferably 1 cycloalkyl group, such as, for example: cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, 2-cyclohexylethyl, 2- or 3-cyclopropylpropyl, 2- or 3-cyclobutylpropyl, 2- or 3-cyclopentylpropyl, 2- or 3-cyclohexylpropyl, etc. Preferred is cyclopropylmethyl.

Examples of the heteroaryl-substituted alkyl group include the above alkyl moieties containing 1 to 3, preferably 1 (optionally substituted) heteroaryl group, such as, for example, a pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, benzimidazolyl, thiophenyl or oxazolyl group, such as pyridin2-yl-methyl, pyridin-3-yl-methyl, pyridin-4-yl-methyl, 2-pyridin-2-yl-ethyl, 2-pyridin-1-yl-ethyl, 2-pyridin-3-yl-ethyl , pyridazin-3-yl-methyl, pyrimidin-2-yl-methyl, pyrimidin-4-yl-methyl, pyrazin-2yl-methyl, pyrazol-3-yl-methyl, pyrazol-4-yl-methyl, pyrazol-5 -yl-methyl, imidazol-2-yl-methyl, imidazol-5-yl-methyl, benzimidazol-2-yl-methyl, thiophen-2-yl-methyl, thiophen-3-yl-methyl, 1,3-oxazole -2-ylmethyl.

Preferred is an alkyl group that is substituted with a benzimidazolyl group, such as benzimidazol-2-yl-methyl and benzimidazol-2-yl-ethyl.

Examples of the amino-substituted alkyl moiety include the above alkyl moieties containing 1-3, preferably 1 (optionally substituted) amino group described below, such as, for example, aminoalkyl (NH ₂ -alkyl) or mono- or dialkylaminoalkyl, such as aminomethyl, 2-aminoethyl , 2- or 3-aminopropyl, methylaminomethyl, methylaminoethyl, methylaminopropyl, 2-ethylaminomethyl, 3-ethylaminomethyl, 2-ethylaminoethyl, 3-ethylaminoethyl, etc., of which 3-aminopropyl is preferred, or an alkyl group, which may be substituted with an optionally substituted alkyloxycarbonylamino group, such as a group having the following formula o /-R ή

where R is a phenyl group forming a benzyloxycarbonylaminopropyl group.

The optionally substituted amino group of the present invention preferably includes an amino group (-NH ₂ ), an optionally substituted mono- or dialkylamino group (alkyl-NH-, (alkyl) ₂ N-), where alkyl has the definition given above for optionally substituted alkyl. Preferred are a mono- or dimethylamino group, a mono- or diethylamino group and a monopropylamino group. The most preferred amino group (-NH ₂ ) and monopropylamino group.

Moreover, in the context of the present invention, a carboxyl group means a [-(C=O)OH] group, and an aminocarbonyl group means a [NH ₂ -(C=O)-] group.

An optionally substituted alkoxy group includes an optionally substituted alkyl-O group, where the definition of alkyl group is as defined above. Preferred alkoxy groups are linear or branched alkoxy groups containing up to 6 carbon atoms, such as methoxy group, ethoxy group, n-propyloxy group, isopropyloxy group, n-butyloxy group, isobutyloxy group, sec-butyloxy group, t-butyloxy group, n-pentyloxy group, iso-pentyloxy group, sec- pentyloxy group, tert-pentyloxy group, 2-methylbutoxy group, n-hexyloxy group, iso-hexyloxy group, tert-hexyloxy group, sec-hexyloxy group, 2-methylpentyloxy group, 3-methylpentyloxy group, 1-ethylbutyloxy group, 2-ethylbutyloxy group, 1,1-dimethylbutyloxy group, 2, 2-dimethylbutyloxy group, 3,3-dimethylbutyloxy group, 1-ethyl-1-methylpropyloxy group, as well as cycloalkyloxy groups such as cyclopentyloxy group or cyclohexyloxy group. Methoxy group, ethoxy group, n-propyloxy group and isopropyloxy group are preferred. Methoxy and ethoxy group are more preferred. Particularly preferred is the methoxy group.

In the context of the present invention, the optionally substituted alkanediyl radical is preferably a divalent linear or branched alkanediyl radical containing from 1 to 6, preferably 1 to 4, more preferably 1, 2 or 3 carbon atoms, which may optionally have 1 to 3, preferably 1 or 2 substituents selected from the group consisting of halogen, hydroxyl (-OH), oxo group ((=O; forming a carbonyl or acyl group [-(C=O)-]) and an alkyl group as defined above, for example preferably methyl The following groups may be mentioned as preferred examples: methylene, ethane-1,2-diyl, ethane-1,1-diyl, propan-1,3-diyl, propan-1,1-diyl, propan-1,2 -diyl, propane-2,2-diyl, butane-1,4-diyl, butane-1,2-diyl, butane-1,3-diyl, butane-2,3-diyl, butane-1,1-diyl , butane-2,2-diyl, butane-3,3-diyl, pentane-1,5-diyl, etc.

- 11 044945

Particularly preferred are methylene, ethane-1,2-diyl, ethane-1,1-diyl, propan-1,3-diyl, propan2,2-diyl and butan-2,2-diyl. Most preferred are methylene, ethane-1,2-diyl and propane-1,3-diyl.

A preferred substituted alkanediyl radical is a hydroxy-substituted alkanediyl such as a hydroxy-substituted ethanediyl, an oxo-substituted alkanediyl such as an oxane-substituted methylene or an ethanediyl radical forming a carbonyl or acyl group, a halogen-substituted alkanediyl group such as an alkanediyl group substituted with one or two atoms halogen, selected from F and Cl, preferably 2,2-di-fluoroethanediyl, or an alkanediyl group substituted with a methyl group.

According to the present invention, it is also possible that A ¹ representing a linear or branched alkanediyl group as defined above, and R ² representing an optionally substituted alkyl group as defined above, together with the nitrogen atom to which they are attached, form an optionally substituted 4-6 membered ring, which may be substituted with 1-3 substituents listed above. Accordingly, A ¹ and R ² may together form a group having one of the following formulas:

And

The formation of a 4-membered (substituted or unsubstituted) ring is preferred, for example, in particular the group

The binding point on the left side of the formula indicates the site of direct binding to the heterocyclic 5-membered ring between the X ¹ and X ² positions in formula (I) of the present invention. The point of attachment on the right side of the formula indicates the point of attachment to the A ² group, which is an alkanediyl group as defined herein.

In formula (I) of the present invention, n is an integer from 1 to 3, including 1, 2 or 3, thus corresponding to a methylene group, an ethane-1,2-diyl group or a propan-1,3-diyl group. More preferably, n is 1 or 2, and even more preferably, n is 1, corresponding to a methylene group.

In the present invention, individual substituents in the above formula (I) may have the following meaning:

A) X ¹ represents N or O; And

X ² represents N, S or O;

provided that X ¹ and X ² are different;

thereby forming 5-membered heterocycles having the formulas

where * indicates the point of attachment to the aminocarbonyl group, and ** indicates the point of attachment to the A1 group.

B) n is an integer equal to 1, 2 or 3; preferably n is 1 or 2, more preferably n is 1.

C) R ¹ is selected from the group consisting of a hydrogen atom and an optionally substituted alkyl (defined above); preferably R ¹ represents a hydrogen atom or methyl, more preferably R ¹ represents a hydrogen atom.

D) R ² is selected from the group consisting of a hydrogen atom and an optionally substituted alkyl (defined above); preferably R ² represents a hydrogen atom or C1-C ₄ -alkyl, more preferably R ² represents a hydrogen atom or methyl, even more preferably R ² represents a hydrogen atom.

E) R ³ represents 1, 2 or 3 optional substituents, which may be independently selected from the group consisting of halogen (as defined above), cyano group,

- 12 044945 optionally substituted alkyl (defined above), optionally substituted alkoxy group (defined above), and carboxyl group (defined above);

preferably R ³ represents 1 or 2 optional substituents, which may be independently selected from the group consisting of halogen, cyano, alkyl (as defined above), which may be substituted with 1, 2 or 3 halogen atoms (as defined above), optionally substituted an alkoxy group (defined above), and a carboxyl group (defined above);

more preferably, R ³ represents 1 or 2 optional substituents, which may be independently selected from the group consisting of

F and Cl, cyano group, trifluoromethyl group, methoxy group, and carboxyl group;

even more preferably, R ³ represents a hydrogen atom, which means an unsubstituted terminal benzimidazolyl ring in formula (I) F) R ⁴ is selected from the group consisting of a hydrogen atom, halogen (as defined above), C ₁ -C ₃ -alkyl, and halogen-substituted alkyl (defined above);

preferably R ⁴ is selected from the group consisting of Cl, methyl, ethyl, isopropyl, and trifluoromethyl hydrogen;

more preferably, R ⁴ is selected from the group consisting of hydrogen, Cl, methyl, and trifluoromethyl;

more preferably, R ⁴ is selected from the group consisting of hydrogen, Cl, and methyl, even more preferably R ⁴ is a hydrogen atom.

G) A ¹ is alkanediyl, preferably A ¹ is methylene or ethane-1,2-diyl, more preferably A ¹ is ethane-1,2-diyl.

H) A ² is alkanediyl, preferably A ² is methylene, ethane-1,2-diyl or propane-1,3-diyl, more preferably A ² is methylene or ethane-1,2-diyl, even more preferably And ² is ethane-1,2-diyl.

I) either A ¹ and R ² together with the nitrogen atom to which they are attached form an optionally substituted 4-6 membered ring, as defined above;

where A ¹ and R ² together with the nitrogen atom to which they are attached preferably form an optionally substituted 4-membered ring as defined above, where A ¹ and R ² together with the nitrogen atom to which they are attached more preferably form an unsubstituted 4-membered ring (azetidinyl ring).

In the listed embodiments, the substituents in the compounds having formula (I) may in particular have the following meanings:

n has any of the meanings given in B) above, and the remaining substituents can have any of the meanings given in A) and C)-!).

R ¹ has any of the meanings given in C) above, and the remaining substituents may have any of the meanings given in A), B) and D)-I).

R ² has any of the meanings given in D) above, and the remaining substituents may have any of the meanings given in A) to C) and E) to H) or I).

R ³ has any of the meanings given in E) above, and the remaining substituents may have any of the meanings given in A)-D) and F)-I).

R ⁴ has any of the meanings specified in F) above, and the remaining substituents can have any of the meanings specified in A)-E) and G)-I).

A ¹ has any of the meanings given in G above), and the remaining substituents can have any of

- 13 044945 values indicated in A)-F) and H) or I).

A ² has any of the meanings given in H) above, and the remaining substituents may have any of the meanings given in A) to G) and I).

R ² and A ¹ have any of the meanings given in I), and the remaining substituents can have any of the meanings given in A) to C), E), F) and H).

A preferred embodiment of the present invention relates to new salts of compounds having the above general formula (I), wherein X ¹ represents N or O; and X ² represents N, S or O; provided that X ¹ and X ² are different; R ¹ represents a hydrogen atom; n is 1, 2 or 3;

And ¹ represents methylene or ethane-1,2-diyl;

A ² is methylene, ethane-1,2-diyl or propan-1,3-diyl;

R ² represents a hydrogen atom or C ₁ -C ₄ -alkyl; or

A ¹ and R ² together with the nitrogen atom to which they are attached form an optionally substituted 4-membered ring;

R ³ represents 1 or 2 optional substituents which may be independently selected from the group consisting of halogen, cyano group, alkyl which may be substituted by 1, 2 or 3 halogen atoms, an optionally substituted alkoxy group and a carboxyl group; R ⁴ is selected from the group consisting of Cl, methyl, ethyl, isopropyl, and trifluoromethyl hydrogen;

wherein said salts are selected from salts of compounds having formula (I) with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of the compound (C acid is from 1 to 2 to 1 to 3; and where the above x3HCl salts are excluded.

Another preferred embodiment of the present invention relates to new salts of compounds having the above general formula (I), wherein X ¹ represents N or O; and X ² represents N, S or O; provided that X ¹ and X ² are different; R ¹ represents a hydrogen atom; n is 1 or 2;

And ¹ represents methylene or ethane-1,2-diyl;

A ² is methylene, ethane-1,2-diyl or propan-1,3-diyl;

R ² represents a hydrogen atom or methyl; or

A ¹ and R ² , together with the nitrogen atom to which they are attached, form an unsubstituted 4-membered ring;

R ³ represents 1 or 2 optional substituents, which may be independently selected from the group consisting of F and Cl, cyano group, trifluoromethyl, methoxy group, and carboxyl group;

R ⁴ is selected from the group consisting of hydrogen, Cl, methyl, and trifluoromethyl;

wherein said salts are selected from salts of compounds having formula (I) with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of the compound (C acid is from 1 to 2 to 1 to 3; and where the above x3HCl salts are excluded.

Another preferred embodiment of the present invention relates to new salts of compounds having the above general formula (I), where

X ¹ represents N or O; And

X ² represents N, S or O;

provided that X ¹ and X ² are different;

R ¹ represents a hydrogen atom;

n is 1;

And ¹ represents methylene or ethane-1,2-diyl;

A ² is methylene, ethane-1,2-diyl or propan-1,3-diyl;

R ² represents a hydrogen atom; or

A ¹ and R ² , together with the nitrogen atom to which they are attached, form an unsubstituted 4-membered ring;

R ³ is a hydrogen atom, which corresponds to the formation of an unsubstituted terminal benzimidazolyl ring;

R ⁴ is selected from the group consisting of hydrogen, Cl, and methyl;

wherein said salts are selected from salts of compounds having formula (I) with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of the compound (C acid) is from 1 to 2 to 1 to 3; and

- 14 044945 where the above x3HCl salts are excluded.

Another preferred embodiment of the present invention relates to new salts of compounds having the above general formula (I), where

X ¹ represents N or O; And

X ² represents N, S or O;

provided that X ¹ and X ² are different;

R ¹ represents a hydrogen atom;

n is 1;

And ¹ represents methylene or ethane-1,2-diyl;

A ² is methylene, ethane-1,2-diyl or propan-1,3-diyl;

R ² represents a hydrogen atom; or

A ¹ and R ² , together with the nitrogen atom to which they are attached, form an unsubstituted 4-membered ring;

R ³ is a hydrogen atom, which corresponds to the formation of an unsubstituted terminal benzimidazolyl ring; And

R ⁴ represents a hydrogen atom;

wherein said salts are selected from salts of compounds having formula (I) with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of compound (I):acid is from 1 to 2 to 1 to 3; and where the above x3HCl salts are excluded.

Another preferred embodiment of the present invention relates to new salts of compounds having the above general formula (I), where n = 1;

R ³ = hydrogen atom;

R ⁴ = hydrogen atom;

A ¹ = ethane-1,2-diyl;

A ² = methylene, ethane-1,2-diyl or propan-1,3-diyl;

R ² = hydrogen atom;

or A ¹ and R ² together with the nitrogen atom to which they are attached form an optionally substituted 4-membered ring, forming compounds having the following formula (II) or (III)

(P)

(III), where in formula (II) and (III) m represents the integer 1, 2 or 3; And

X ¹ , X ² and R ¹ have the meanings defined above in any embodiment of the present invention involving compounds having formula (I).

In particular, in formulas (II) and (III), X ¹ and X ² have the meaning given above in A).

In formula (II), R ¹ and R ² preferably represent a hydrogen atom.

In formula (III), R ¹ is preferably a hydrogen atom, and m is preferably 2.

Another preferred embodiment of the present invention relates to new salts of compounds having the above general formula (II), where

X ¹ and X ² are selected from N and O, and are different from each other;

R ¹ = hydrogen atom;

R ² = hydrogen atom; and m = 2.

Hereinafter, compounds (I), (II) or (III) forming salts of the present invention are also referred to as base or free base. Compounds having formula (I), (II) or (III), in free base form, contain at least one basic group, such as amino groups, to which acidic groups can bind.

- 15 044945

According to the present invention, salts of compounds having formula (I), (II) or (III) according to any of the above-described embodiments of the present invention can be selected from salts having the ratio base (compound (I), (II) or ( Ш)): acid from 1:2 to 1:3, where salt-forming acids correspond to the selection listed above.

The present invention also covers mixed salts of a base (compound (I), (II) or (III)) with one or more of the above acids, which may have the same or different base/acid ratios according to the present invention. Acids provide a counterion to the cationic form of compound (I), (II) or (III). Accordingly, the selected acids of the present invention provide the following counterions:

- 16 044945

According to the present invention, the salts of compounds (I), (II) or (III) are distinguished by the selected base:acid ratio, i.e. compound (I), (II) or (III): the acids listed above, in the range of 1.0 - 2.0 (mol of base): 1.0 - 3.0 (mol of acid). In a particular embodiment, the selected acid:base ratio is 1.0-2.0 (mol base):1.0-2.0 (mol acid).

Particular examples include the following base:acid ratios, i.e. compound (I), (II) or (III): acids listed above:

1.0 (mol base) : 1.0 (mol acid);

1.0 (mol base) : 1.25 (mol acid):

1.0 (mol base) : 1.35 (mol acid);

1.0 (mol base) : 1.5 (mol acid);

1.0 (mol base) : 1.75 (mol acid);

1.0 (mol base) : 2.0 (mol acid); And

2.0 (moles of base) : 1.0 (moles of acid).

In the context of the present invention, a salt with an acid:base ratio of 1:1 is also called a monosalt or 1:1 salt. For example, mono-HCl salt is also referred to as 1HCl or 1HCl salt.

In the context of the present invention, a salt with an acid:base ratio of 1:2 is also called a di-salt or 1:2 salt. For example, di-HCl salt is also referred to as 2HCl or 2HCl salt.

In the context of the present invention, a salt with an acid:base ratio of 1:3 is also called a tri-salt, triple salt or 1:3 salt. For example, tri-HCl salt is also referred to as 3HCl or 3HCl salt.

A salt having a base:acid ratio of 1:1.25 is also called a 1:1.25 salt.

- 17 044945

A salt having a base:acid ratio of 1:1.35 is also called a 1:1.35 salt.

A salt having a base:acid ratio of 1:1.5 is also called a 1:1.5 salt.

A salt having a base:acid ratio of 1:1.75 is also called a 1:1.75 salt.

A salt having a base:acid ratio of 2:1 is also called a hemi salt or a 2:1 salt.

In a more preferred embodiment of the present invention, the salts of the compounds having the above formula (I) are selected from monosalts (1:1 salts) with one or more of the acids listed above.

Another embodiment of the present invention relates to the above-described salts of compounds having formula (I), (II) or (III), wherein the acids are selected from the group consisting of citric acid, hydrochloric acid, maleic acid, phosphoric acid and sulfuric acid.

Another embodiment of the present invention relates to the above-described salts of compounds having formula (I), (II) or (III), where the acids are selected from the group consisting of phosphoric acid and sulfuric acid.

Salts of the compounds of the present invention may be present in amorphous, polymorphic, crystalline and/or semi-crystalline (partially crystalline) form, as well as in the form of a salt solvate.

Preferably, the salts of the present invention are present in crystalline and/or semi-crystalline (partially crystalline) form and/or in the form of solvates thereof.

The preferred crystallinity of the salts or solvates of salts of the present invention can be determined using standard analytical methods, for example, in particular, using various x-ray techniques, which allow a clear and simple analysis of the salts of the compounds. In particular, the degree of crystallinity can be determined or confirmed using powder x-ray diffraction (reflection) techniques, as described, for example, below in the examples, or using powder x-ray diffraction (transmission) techniques, as described, for example, below in the examples (both methods abbreviated PXRD in this text). For crystalline solids having the same chemical composition, different crystal characteristics are generally referred to as polymorphism.

Preferably, the salts of the present invention have a degree of crystallinity greater than 30%, more preferably greater than 40%, even more preferably greater than 50%, for example at least 55-60%, as determined herein by PXRD.

The salts of the present invention may be present as solvates and/or hydrates, which may be formed by attraction, association, adsorption, adhesion, association or complexation of solvent molecules in the crystal lattice of the salts of the present invention. Solvent molecules that may be included in the crystal lattice may be solvent molecules used for crystallization, as well as water molecules from the environment.

Solvents used for crystallization include acetonitrile, dichloromethane, alcohols such as in particular methanol, ethanol, 2-propanol (isopropanol), aldehydes, ketones, especially acetone, ethers such as tetrahydrofuran (THF) or dioxane , esters, for example ethyl acetate, or alkanes, such as in particular pentane, hexane, heptane or cyclohexane, and water, as well as mixtures thereof. Preferred solvents used for crystallization are selected from the group consisting of acetonitrile, dichloromethane, methanol, ethanol, 2-propanol, ethyl acetate, THF, water, and mixtures thereof.

Particularly preferred solvents used for crystallization are selected from the group consisting of acetonitrile, methanol, ethanol, 2-propanol, ethyl acetate, THF, water, and mixtures thereof. Preferred water/solvent mixtures include mixtures of water and acetone, mixtures of water and ethanol, and mixtures of water and methanol, where mixtures of water and ethanol and mixtures of water and methanol are preferred.

Particularly preferred solvents used for crystallization are selected from the group consisting of acetonitrile, dichloromethane, ethanol, 2-propanol (isopropanol), acetone and ethyl acetate, as well as mixtures thereof with water, such as in particular mixtures of ethanol and water , and a mixture of acetone and water. Particularly preferred mixtures are the following mixtures of solvent and water (all solvent mixture ratios given herein are by volume):

acetone:water = 9:1 (v:v) acetone:water = 95:1 (v:v) ethanol:water = 4:1 (v:v) ethanol:water = 3:1 ( v:v) ethanol:water = 8:2 (v:v).

The extent to which a selected solvent or water is prone to form a solvate or hydrate during crystallization and subsequent process steps, or it produces a free base, is generally not predictable and depends on a combination of process conditions and various interactions between the particular compound (I), the counterion from a selected acid and a selected solvent or environmental moisture. Solvates or hydrates of salts may be preferred because the solvent or water molecules in the crystal structure are bound by strong intermolecular forces and may therefore constitute a building block of such crystals, which in turn may improve the stability of the salt. However, solvent and/or water molecules are also located in certain crystal lattices, which are connected by rather weak intermolecular forces. Such molecules are more or less integrated into the resulting crystal structure, but with less energetic effect. The solvent and/or water content of the solvates also depends on the drying conditions and environmental conditions (ie relative humidity). In the case of stable solvates or hydrates, there are usually clear stoichiometric relationships between the active substance (ie the salt of the present invention) and the solvent or water. In many cases, these ratios do not completely correspond to the stoichiometric ratios, usually it is somewhat less than theoretical due to certain defects in the crystals. The ratio of organic molecules and solvent or water molecules for more weakly bound water can vary to a noticeable extent, for example, up to di-, tri- or tetra-hydrates. On the other hand, in amorphous solids, the solvent and/or water content of the molecular structure is not stoichiometric; it can turn out to be stoichiometric only by chance. In some cases, it is not possible to determine the exact stoichiometry of the solvent or water molecule content because a layered structure is formed, and the content of the included solvent or water molecules cannot be determined with certainty.

The solvent and/or water content of amorphous solids, as well as crystalline solvates, can generally be determined by well-known methods, such as, for example, the well-known Karl Fischer titration method, dynamic vapor sorption (DVA) method, thermogravimetric analysis (TG) method -FT-IR), as described, for example, below in the examples. In addition, elemental analysis or structural analysis techniques such as 1H-NMR spectroscopy or Raman spectroscopy (Fourier-Raman spectroscopy) can provide information about the extent of solvate or hydrate formation, and/or can be used to confirm or validate titration measurements. Karl Fischer, chipboard or TG-IR-Fourier.

Examples of solvates and/or hydrates of the present invention include, for example, hemi-(0.5), mono-, sesqui-(1.5), di-, tri-, tetra-, penta-, hexa-, hepta-, octa-, nona -, deca-, etc. solvates or hydrates, respectively. An intermediate degree of solvation may also occur, such as solvation with 2.5, 3.5, 4.5, etc. solvent and/or water molecules.

Preferred examples of solvates and/or hydrates include hydrates containing about

1.5, 2.5, 3, 4 and 7 water molecules. More preferred examples of solvates and/or hydrates include hydrates containing about 0.5, 1.5, 2.5, 3, 4, 6 and 7 molecules of water. Anhydrous salts are also preferred. It is also possible that solvent and/or water molecules are present in the salt in non-stoichiometric quantities.

In addition, mixtures of water and solvent may be present in the salt, resulting in so-called mixed hydrate/solvate forms. Examples of such mixed hydrate/solvate forms include, but are not limited to, acetone/water, preferably in a ratio of 1 to 4:1; for example, in particular, 4:1; methanol/water, preferably with a ratio of 3 - 9:1; for example, in particular, 3:1,4:1 and 9:1; ethanol/water, preferably with a ratio of 1-4:1; for example, in particular, 3:1 and 4:1.

Any reference above or below to the salts of the present invention should be understood to also include the corresponding solvates, such as hydrates, solvates and mixed hydrate/solvate forms, and polymorphs, as well as amorphous forms, where appropriate and applicable.

The new salts of the present invention show good solubility, they are stable and maintain good quality also during storage and distribution.

The appropriate stability of the resulting crystalline or amorphous solid compounds in the forms of salts, solvates and hydrates (including mixed hydrate/solvate forms), as well as the corresponding solvates of salts or hydrates of salts, can be determined by standard experimental methods. Improved stability may include improved hygroscopic properties, improved enthalpy of fusion. An important characteristic of the quality of the pure active substance, both for physico-chemical procedures such as drying, sieving, grinding, and for processes for the preparation of finished pharmaceutical forms, which are carried out with pharmaceutically acceptable excipients, namely for processes of mixing, granulation, spray drying, tableting, is the absorption of water or loss of water by this active substance, depending on the temperature and relative humidity of the environment. In the case of some finished forms, free and bound water is certainly introduced along with excipients, and/or water is added to the processed mass according to the process of preparation of such finished form. In this case, the pharmaceutical active substance is in contact with free water for a fairly long period of time, depending on the temperature of the various processes (partial vapor pressure). It is therefore obvious that particularly stable pure substances are preferred from the point of view of the preparation of finished pharmaceutical forms and from the point of view of their applicability for all stages of the processes of preparation of the finished forms and for introduction into the composition of various dosage forms.

- 19 044945

The salts of the present invention may exist in isolated and substantially pure form, for example, with a purity of >65%, preferably >70%, more preferably >75%, more preferably >80%.

In the context of the present invention, the term salts includes the corresponding solvates, hydrates and mixed hydrate/solvate forms, etc., as well as various polymorphs thereof, such as, in particular, the particular examples of polymorphs described herein.

The salts of the present invention can be structurally characterized by conventional methods, such as, for example, elemental analysis, thermogravimetric analysis (TG-FT-IR), 1H-NMR spectroscopy and Raman spectroscopy (FT-Raman spectroscopy), differential scanning calorimetry (DSC) to determine melting temperature, as described, for example, below in the examples, as well as a combination of the listed methods, and in particular in combination with other mentioned methods for determining the degree of solvate/hydrate formation.

Another embodiment of the present invention relates to a process for preparing the salts described herein. The method of obtaining such salts can be described as follows.

The formation of salts is carried out in a solvent system in which the two reactants, namely compound (I), (II) or (III) in base form and the corresponding acid, are appreciably soluble. It is convenient to use a solvent or mixture of solvents in which the resulting salt is slightly soluble or completely insoluble to ensure crystallization and precipitation of the salt. One option for forming the salt of the present invention would be to use a solvent in which the salt in question is very soluble and then add to the resulting solution an antisolvent, that is, a solvent in which the resulting salt has very low solubility. Another option for salt formation involves concentrating the salt solution, for example by heating, if necessary under reduced pressure, or by slowly evaporating the solvent, for example, at room temperature, or by adding seed crystals, or by achieving the water activity necessary for hydrate formation. In these cases, the above solvents can be used.

To obtain hydrates, dissolution and crystallization processes or crystallization processes with water equilibration can be used.

The processes of dissolution and crystallization can be described by the following stages:

(i) compound (I), (II) or (III) as a free base is dissolved in an organic solvent, (ii) an acid selected from those listed above, preferably in the form of an aqueous solution, is added to the solution prepared in step (i), (iii) the resulting solution is left to stand for crystallization to begin, (iv) the precipitated crystals are filtered and dried to obtain a salt.

In dissolution process (i), the solvent used is preferably acetonitrile, dichloromethane, methanol, ethanol, 2-propanol, ethyl acetate, THF, water and mixtures thereof, more preferably acetonitrile, methanol, ethanol, 2-propanol, ethyl acetate, THF, water or mixtures thereof, for example, in particular a mixture with water, for example a mixture of ethanol and water, a mixture of methanol and water, or a mixture of acetone and water. If necessary, the solvent can be heated to above room temperature, for example 25-60°C, more preferably 30-50°C.

In step (ii), the aqueous acid solution used is preferably a 5-30%, more preferably a 5-25%, for example a 10% solution of the corresponding acid. Specifically, the acid:base ratio is 1:1 (mol:mol). If phosphoric acid or sulfuric acid is used, a base:acid ratio of 10:1 (mol:mol) can also be used.

In step (iii), the resulting solution is preferably left to stand for the solvent to slowly evaporate. This is preferably carried out by cooling to room temperature or below, more preferably to a temperature of -10 to 20°C, even more preferably to a temperature of -5 to 10°C, most preferably from 0 to 5°C. Alternatively, concentration of the solution can also occur by heating to above room temperature, for example, to a temperature of >25 to 100°C, more preferably from 30 to 70°C. Typically left for 8-48 hours, preferably 17-36 hours, more preferably 20-30 hours.

In step (iv), drying is preferably carried out at elevated temperature, more preferably at 20-50°C, most preferably at 30-40°C. In any case, drying must be carried out at temperatures below the melting point of the corresponding salt. The pressure is preferably selected in the range from 1 to 100 mbar, preferably from 10 to 50 mbar, more preferably from 20 to 40 mbar, for example 30 mbar. Drying is usually carried out until constant weight is achieved. Depending on the drying conditions, drying can take from 5 to 48 hours, preferably from 10 to 24 hours, for example from 15 to 20 hours.

It is also possible to accelerate crystallization by adding a crystallization initiator such as

- 20 044945 for example, at least one seed crystal.

In a more preferred embodiment of the present invention, the 3HCl (x3HCl) salts of compounds falling under the above general formulas (I), (II) or (III) are excluded.

A particular embodiment of the present invention relates to salts of compounds having formula (I) described in any of the above embodiments, wherein the compounds having formula (I) are selected from the group consisting of the following compounds:

More preferably, the compounds having formula (I) are selected from the group consisting of the following compounds:

- 21 044945

Even more preferably, the present invention relates to the salts described in any of the above embodiments, wherein the compound having formula (I) is

F

Another particularly preferred embodiment of the present invention relates to salts of the compounds having formula (I) described in any of the above embodiments, where the acids are selected from the group consisting of phosphoric acid and sulfuric acid.

Another particularly preferred embodiment of the present invention relates to salts of the compounds having formula (I) described in any of the above embodiments, wherein the crystallization solvents are selected from the group consisting of acetonitrile, dichloromethane, ethanol, 2-propanol (isopropanol), acetone and ethyl acetate, as well as from mixtures thereof with water, such as, in particular, mixtures of ethanol and water and mixtures of acetone and water. Particularly preferred mixtures are the following mixtures of solvent and water: acetone:water = 9:1 (v:v) acetone:water = 95:1 (v:v) ethanol:water = 4:1 (v:v) .) ethanol:water = 3:1 (v:v) ethanol:water = 8:2 (v:v).

Another particularly preferred embodiment of the present invention relates to salts of compounds having formula (I) described in any of the above embodiments, where the acid is phosphoric acid and the phosphate salt has a ratio of compound (I):acid = 1-2:1 , preferably the ratio of compound (I):acid = 1:1 or 2:1.

More preferably, such preferred phosphate salts are prepared by crystallization using a solvent from the group consisting of acetonitrile, ethanol, 2-propanol (isopropanol), acetone and ethyl acetate, as well as mixtures thereof with water, such as, in particular, mixtures of ethanol and water, and a mixture of acetone and water. A particularly preferred mixture is ethanol:water = 8:2.

Another particularly preferred embodiment of the present invention relates to salts of the compounds having formula (I) described in any of the above embodiments, where the acid is sulfuric acid and the sulfate salt has a ratio of compound (I):acid = 1:1.

More preferably, such preferred sulfate salts are prepared by crystallization using a solvent from the group consisting of acetonitrile, dichloromethane, ethanol, 2-propanol (isopropanol) and acetone, as well as mixtures thereof with water, such as, in particular, mixtures of ethanol and water, and a mixture of acetone and water. A particularly preferred mixture is selected from the following mixtures: acetone:water = 9:1 (v:v) acetone:water = 95:1 (v:v) ethanol:water = 4:1 (v:v) ethanol:water = 3:1 (v:v).

It is also particularly preferred that the preferred phosphate and sulfate salts described above are salts of compounds having formula (I) selected from Compound Examples Nos. 1, 2, 4, 40, 94, 118, 126, 127, 193, 206, 208, 233 shown in the table above. More preferably, compounds having formula (I) are selected from Examples of Compounds Nos. 1, 40, 94, 127, 208.

- 22 044945

Even more preferably, compounds having formula (I) are selected from Examples of Compounds No. 1 and

127, where compound 127 is most preferred.

Accordingly, the following salts are particularly preferred:

(1:1 salt) (1:1 salt) sphatic salt of compound 127 (2:1 salt, hemiphosphate) salt of compound 127 salt of compound 127

Another particularly preferred embodiment of the present invention relates to phosphate salts of compounds corresponding to compound 127, with a ratio of compound (1):acid = 1:1, which is characterized by the polymorphic form PM2, described in detail below in the examples.

Another particularly preferred embodiment of the present invention relates to sulfate salts of compounds corresponding to compound 127, with a ratio of compound (1):acid = 1:1, which is characterized by the polymorphic form PM1, described in detail below in the examples.

Surprisingly, it has been found that the compounds described in the present invention have good and even improved long-term stability, including reduced or no solvent release and/or weight loss with increasing temperature, these compounds are less hygroscopic or non-hygroscopic, they retain their solid-state structure even over long periods of time. storage at various ambient temperatures and/or humidity, the crystalline form is resistant to vacuum drying, the compounds show good reproducibility with high purity and low amounts of by-products or degradation products in the preparation method used, and maintain their solubility profile even after prolonged storage at various ambient temperature and/or humidity. The inventors of the present invention unexpectedly discovered that, in particular, the preferred sulfate and phosphate salts of compound 127 (1:1 salts) described above, in particular the polymorphs PM1 (sulfate salt) and PM2 (phosphate salt) described in detail herein, had the indicated characteristics that provide an advantage. This makes these polymorphs particularly well suited as active ingredients in pharmaceutical preparations for the prevention and treatment described above. The particular preferred polymorphs PM1 (1:1 sulfate salt) and PM2 (1:1 phosphate salt) contain less water than the other polymorphs tested herein, which provides an advantage in achieving long-term stability.

Depending on their structure, the salts of the invention may exist in stereoisomeric forms (enantiomers, diastereomers) in the presence of asymmetric carbon atoms. The present invention includes all enantiomers and diastereomers, as well as mixtures thereof. Enantiomerically pure forms can optionally be obtained by well-known optical resolution methods, such as fractional crystallization of their diastereomers obtained by reaction with optically active compounds. Since the compounds of the present invention may exist in tautomeric forms, the present invention covers the use of all tautomeric forms. The salts of the present invention may be present as mixtures of various possible isomeric forms, in particular stereoisomers such as E- and Z-, syn- and anti-, as well as optical isomers. The present invention includes the E-23 044945 isomers and Z-isomers, as well as optical isomers and any mixtures of these isomers.

The present invention also relates to new polymorphs of new salts of compounds having the above formulas (I), (II) or (III).

Polymorphs are formed when the same compositions crystallize with different crystal lattice characteristics, resulting in different thermodynamic properties and stability specific to the particular polymorph.

One particular embodiment of the present invention relates to polymorphs of the citric acid salt of compound 127, which is characterized by a powder X-ray diffraction pattern (PXRD pattern) containing characteristic crystal peaks expressed in degrees 2-theta, 4.5 and 5.3 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees. Preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 24.3, 21.6, 17.1, 5.9, 25.3, 8.1, 15.1, 20.1 or 12.6 ± 0.25 degrees, or ± 0.20 degrees, or ±0.10 degrees, or ±0.05 degrees.

More preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 24.3, 21.6, 17.1, 5.9, 25.3, 8.1, 15.1, 20.1 or 12.6.

More preferably, in such a polymorph embodiment, the PXRD pattern contains characteristic crystal peaks, expressed in degrees 2-theta, at each of 24.5, 5.3, 24.3, 21.6, and 17.1, and optionally one or more, two or more, three or more at each of the values 5.9, 25.3, 8.1, 15.1, 20.1 or 12.6 ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

Preferably, said citric acid salt polymorphs of compound 127 are in the form of a 1:1 salt.

Another particular embodiment of the present invention relates to polymorphs of the salt of compound 127 with maleic acid, which is characterized by a powder X-ray diffraction pattern (PXRD pattern) containing characteristic crystal peaks expressed in degrees 2-theta, 19.0 and 24.5 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees. Preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 25.1, 17.5, 18.7, 25.7, 18.3, 21.9, 9.6 or 6.1 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

More preferably, in such a polymorph embodiment, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 25.1, 17.5, 18.7, 25.7, 18.3, 21.9, 9.6 or 6.1.

More preferably, in such a polymorph embodiment, the PXRD pattern contains characteristic crystal peaks, expressed in degrees 2-theta, at each of 19.0, 24.5, 25.1, 17.5 and 18.7, and optionally one or more, two or more, three or more at each from values 25.7, 18.3, 21.9, 9.6 or 6.1 ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

Preferably, said maleic acid salt polymorphs of compound 127 are in the form of a 1:1.75 salt.

Another particular embodiment of the present invention relates to polymorphs of the salt of compound 127 with phosphoric acid, which is characterized by a powder X-ray diffraction pattern (PXRD pattern) containing characteristic crystal peaks expressed in degrees 2-theta, 27.2 and 4.6 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees. Preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 16.8, 22.0, 24.5, 5.4, 8.9, 13.1, 12.3, 19.7 or 15.9 ± 0.25 degrees, or ± 0.20 degrees, or ±0.10 degrees, or ±0.05 degrees.

More preferably, in such a polymorph embodiment, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 16.8, 22.0, 24.5, 5.4, 8.9, 13.1, 12.3, 19.7 or 15.9.

More preferably, in such a polymorph embodiment, the PXRD pattern contains characteristic crystal peaks, expressed in degrees 2-theta, at each of 27.2, 4.6, 16.8, 22.0 and 24.5, and optionally one or more, two or more, three or more at each from values 5.4, 8.9, 13.1, 12.3, 19.7 or 15.9 ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

Preferably, said phosphoric acid salt polymorphs of compound 127 are in the form of a 2:1 salt.

Another particular embodiment of the present invention relates to polymorphs of the salt of compound 127 with phosphoric acid, which is characterized by a powder X-ray diffraction pattern (PXRD pattern) containing characteristic crystal peaks expressed in degrees 2-theta, 26.1 and 16.5 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees. Preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 15.5, 18.4, 17.4, 14.7, 25.4, 20.4, 13.2 or 22.1 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

- 24 044945

More preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 15.5, 18.4, 17.4,

14.7, 25.4, 20.4, 13.2 or 22.1.

More preferably, in such a polymorph embodiment, the PXRD pattern contains characteristic crystal peaks, expressed in degrees 2-theta, at each of 26.1, 16.5, 15.5, 18.4 and 17.4, and optionally one or more, two or more, three or more at each from values 14.7, 25.4, 20.4, 13.2 or 22.1 ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

Preferably, said phosphoric acid salt polymorphs of compound 127 are in the form of a 1:1 salt.

Another particular embodiment of the present invention relates to polymorphs of the salt of compound 127 with sulfuric acid, which is characterized by a powder X-ray diffraction pattern (PXRD pattern) containing characteristic crystal peaks expressed in degrees 2-theta, 25.4 and 18.1 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees. Preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 4.5, 25.1, 16.8, 18.5, 18.6, 14.9, 15.6 or 17.6 ± 0.25 degrees, or ± 0.20 degrees, or ±0.10 degrees, or ±0.05 degrees.

More preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 4.5, 25.1, 16.8, 18.5, 18.6, 14.9, 15.6 or 17.6.

More preferably, in such a polymorph embodiment, the PXRD pattern contains characteristic crystal peaks, expressed in degrees 2-theta, at each of 25.4, 18.1, 4.5, 25.1 and 16.8, and optionally one or more, two or more, three or more at each from values 18.5, 18.6, 14.9, 15.6 or 17.6 ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

Preferably, said sulfuric acid salt polymorphs of compound 127 are in the form of a 1:1 salt.

Another particular embodiment of the present invention relates to polymorphs of the sulfuric acid salt of compound 127, which is characterized by a powder x-ray diffraction pattern (PXRD pattern) containing characteristic crystal peaks expressed in degrees 2-theta, 25.5 and 4.5 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees. Preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 18.1, 18.4, 16.8, 6.2, 14.9, 25.2, 15.6 or 13.1 ± 0.25 degrees, or ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

More preferably, in such an embodiment of the polymorph, the PXRD pattern contains one or more additional peaks expressed in degrees 2-theta selected from values of about 18.1, 18.4, 16.8, 6.2, 14.9, 25.2, 15.6 or 13.1.

More preferably, in such a polymorph embodiment, the PXRD pattern contains characteristic crystal peaks, expressed in degrees 2-theta, at each of 25.5, 4.5, 18.1, 18.4 and 16.8, and optionally one or more, two or more, three or more at each from values 6.2, 14.9, 25.2, 15.6 or 13.1 ± 0.20 degrees, or ± 0.10 degrees, or ± 0.05 degrees.

Preferably, said sulfuric acid salt polymorphs of compound 127 are in the form of a 1:1 salt.

In particular, the present invention includes polymorphs of the following salts of compound 127 described herein having the following peaks in the PXRD diagram.

- 25 044945

Salt of compound 127 with citric acid (1:1 salt)

2-Theta (°) d value (A) Relative intensity (%) 5.3 16.54 87 5.9 14.91 48 8.1 10.97 42 12.2 7.26 39 12.6 7.01 40 13.1 6.77 37 15.0 5.89 41 15.6 5.67 36 16.3 5.44 39 17.1 5.18 53 18.4 4.81 32 18.8 4.71 32 20.1 4.42 41 21.6 4.12 56 23.4 3.80 29 24.3 3.65 77 24.5 3.62 100 25.3 3.52 44 25.4 3.51 39 25.9 3.44 29 26.8 3.33 24 27.4 3.25 21 36.6 2.46 14 37.2 2.41 15 37.9 2.37 14

Salt of compound 127 with maleic acid (1:1.75 salt)

2-Theta (°) d value (A) Relative intensity (%) 6.1 14.52 42 9.6 9.17 43 11.0 8.04 35 12.9 6.84 39 15.3 5.80 34 16.6 5.32 31 17.5 5.06 47 17.8 4.98 31 18.3 4.84 46 18.7 4.75 47 19.0 4.66 100 19.3 4.60 40 19.6 4.53 33 20.0 4.44 32 20.5 4.32 28 21.2 4.19 29 21.7 4.10 29 21.9 4.06 44 22.5 3.94 25 23.3 3.81 28 23.5 3.78 24 24.5 3.63 70 24.9 3.57 36 25.1 3.54 50 25.7 3.47 47 26.2 3.40 24 26.5 3.36 21 27.8 3.21 26 28.4 3.14 19 29.2 3.06 19 30.1 2.97 18 30.7 2.91 15 31.1 2.87 14 32.7 2.74 12 34.2 2.62 12 35.0 2.56 13 35.5 2.53 16 38.8 2.32 12

-26044945

Salt of compound 127 with phosphoric acid (2:1 salt)

2-Theta (°) d value (A) Relative intensity (%) 4.6 19.39 94 5.4 16.37 38 7.8 11.36 24 8.9 9.91 38 9.1 9.68 26 10.8 8.16 24 11.0 8.07 25 11.4 7.73 24 12.3 7.19 31 13.1 6.75 35 13.7 6.46 27 13.9 6.35 24 14.0 6.30 26 14.5 6.10 23 14.7 6.03 27 15.0 5.91 23 15.5 5.72 25 15.7 5.64 28 15.9 5.58 thirty 16.1 5.50 27 16.8 5.27 75 17.3 5.12 23 17.9 4.96 21 18.3 4.83 24 19.4 4.58 27 19.7 4.50 31 19.9 4.46 25 20.1 4.42 24 20.2 4.40 22 20.9 4.24 21 21.2 4.19 24 21.6 4.11 22 21.7 4.09 23 22.0 4.03 44 22.8 3.89 20 23.0 3.87 17 23.4 3.79 21 24.5 3.63 39 24.9 3.58 23 25.4 3.50 17 25.6 3.47 14 26.1 3.42 13 26.4 3.37 16 27.2 3.27 100 27.6 3.23 22 28.2 3.16 29 28.5 3.13 15 29.4 3.04 12 29.8 3.00 13 30.2 2.95 14 30.3 2.94 15 30.7 2.91 10 30.8 2.90 AND 31.0 2.88 AND 31.2 2.87 AND 31.4 2.84 AND 32.3 2.77 9 32.8 2.73 AND 33.2 2.70 9 34.5 2.60 8

In particular,

- 27 044945

Salt of compound 127 with phosphoric acid (1:1 salt)

2-Theta (°) d value (A) Relative intensity (%) 4.90 18.02 40 6.95 12.71 30.7 11.23 7.87 30.2 12.00 7.37 39.8 13.17 6.72 50.3 14.70 6.02 65.1 15.49 5.72 86.8 15.96 5.55 27.5 16.46 5.38 93.9 16.98 5.22 47.6 17.39 5.09 76.4 18.39 4.82 77.4 19.65 4.51 39.2 20.00 4.44 26.2 20.42 4.35 57.8 21.62 4,I 30.6 22.06 4.03 50.1 22.59 3.93 23.3 23.14 3.84 28.3 23.34 3.81 23.6 24.07 3.69 26.5 24.97 3.56 38.5 25.37 3.51 60 26.06 3.42 100 26.83 3.32 42.9 27.41 3.25 17.1 27.85 3.20 14.7 28.62 3.12 30.8 29.04 3.07 24.7 30.97 2.89 13.5 31.29 2.86 12.8 31.54 2.83 11.7 33.31 2.69 13.2 33.60 2.67 11.8 33.80 2.65 9.9 34.35 2.61 9.6 35.00 2.56 12.9 35.30 2.54 10.8 35.54 2.52 16.4 35.74 2.51 10.8 36.44 2.46 9.5

- 28 044945

Salt of compound 127 with sulfuric acid (1:1 salt)

2-Theta (°) d value (A) Relative intensity (%) 4.5 19.61 62 6.1 14.38 26 11.1 7.99 17 11.8 7.49 19 13.1 6.75 26 13.9 6.35 20 14.1 6.26 18 14.9 5.96 32 15.6 5.68 31 16.1 5.50 17 16.8 5.27 41 17.6 5.04 27 18.1 4.89 77 18.5 4.80 37 18.6 4.77 34 19.3 4.59 19 19.7 4.51 18 20.1 4.42 16 20.4 4.34 19 21.1 4.22 21 22.5 3.95 20 22.7 3.91 21 22.9 3.88 18 23.4 3.79 16 25.1 3.54 45 25.4 3.50 100 25.9 3.43 18 26.4 3.37 25 26.8 3.32 26 27.5 3.24 25 28.1 3.17 16 28.6 3.11 14 29.2 3.05 12 29.6 3.01 15 31.2 2.87 16 33.3 2.69 9 33.9 2.64 9 35.4 2.54 8 36.4 2.47 7

In particular,

- 29 044945

Salt of compound 127 with sulfuric acid (1:1 salt)

2-Theta (°) d value (A) Relative intensity (%) 4.45 19.84 100 6.16 14.34 69.8 13.11 6.75 51.7 14.86 5.96 54.9 15.58 5.68 52 16.75 5.29 71.7 18.11 4.89 81.9 18.44 4.81 72.9 20.91 4.24 44.6 21.08 4.21 45.5 23.38 3.80 34.7 23.70 3.75 38.6 25.18 3.53 53.5 25.48 3.49 86.9 26.44 3.37 39.4 27.48 3.24 35.1 32.66 2.74 19.2 36.68 2.45 15.8

According to the present invention, it is particularly preferred that >70 wt%, preferably >75 wt%, >85 wt%, >90 wt%, >95 wt% of the corresponding new salts of the present invention (i.e. the active substance ) of the total weight of said new salt was in the form of such a specific polymorph. Accordingly, a particular embodiment of the present invention relates to compositions, drugs or pharmaceuticals described below, wherein >70 wt%, preferably >75 wt%, >85 wt%, >90 wt%, >95 wt% corresponding new salts of the active substance (based on the total weight of the specified new active substance) is in the form of a specific polymorph.

All compounds (free base or salts, including solvates, hydrates, mixed hydrate/solvate forms and polymorphs, etc.) described in the invention are ferroportin inhibitors. All of the new salts described in the invention retain their effect as a ferroportin inhibitor and can also enhance the inhibitory effect on ferroportin and/or improve the pharmacokinetic profile of the compounds and/or improve the physicochemical properties of the compounds, facilitating their introduction into finished pharmaceutical forms, and/or have the advantage of allowing isolate them in crystalline form, which improves the physicochemical properties of the compounds, facilitating the introduction of these compounds into finished pharmaceutical forms or making them easier to handle or improving their stability. Thus, the new salts of the present invention are suitable for use as a medicine, for example, in particular, for use as ferroportin inhibitors.

As explained above, ferroportin is an iron transport protein responsible for absorbing released iron through the intestines and transporting it into the circulatory system, ensuring the supply of iron to the appropriate organs and tissues. Inactivation or suppression of ferroportin makes it impossible to export iron, thereby reducing iron absorption in the intestine. In the context of the present invention, inhibition of ferroportin includes inhibition of iron transport from cells into the circulatory system and inhibition of iron absorption in the intestine. Suppression of iron transport and/or iron return can be achieved through various mechanisms, including, for example, suppression of iron transport activity of ferroportin and corresponding suppression of iron return, triggering internalization, degradation and/or reduction of ferroportin levels, administration of hepcidin agonists, i.e. compounds that compete with hepcidin or inhibit the binding of hepcidin to ferroportin.

Ferroportin inhibition can be assessed by measuring the inhibition of ferroportin-mediated iron transport in an iron induced response assay (BLAzer test), described in more detail in the examples below. In addition, ferroportin inhibition can be assessed by measuring internalization and/or degradation of ferroportin using the Ferroportin Internalization and Degradation Assay (FACS) or the Ferroportin Ubiquitination and Degradation Assay, each of which is described in more detail. described below in examples. In addition, ferroportin inhibition can be assessed by measuring hepcidin agonist activity, for example by assessing the ability to bind to hepcidin in the Hepcidin Internalization Assay (J774), described in more detail in the Examples below. In addition, inhibition of ferroportin can be determined by confirming inhibition of hepcidin binding to ferroportin, for example, in the Biophysical Ferroportin Hepcidin Binding Assay (Biophysical Ferroportin-30044945

Hepcidin Binding Assay (Hep Bind FP)), described in more detail below in the examples. In addition, inhibition of ferroportin can be assessed by measuring the activity of a compound in terms of its ability to block the export of iron to ferroportin, for example, in the iron excretion inhibition test described in more detail in the Examples below.

Thus, the inhibition of ferroportin according to the present invention can, in particular, be determined by the manifestation of ferroportin inhibitory activity in at least one of the above tests, namely:

inhibition of iron transport carried out by ferroportin in the iron-induced response assay (BLAzer test): IC ₅₀ value [μM] not more than 100 (<100), preferably not more than 50 (<50), more preferably below 50 (<50).

Ferroportin Internalization and Degradation Assay (FACS): EC ₅₀ [µM] value is not more than 100 (<100), preferably not more than 50 (<50), more preferably less than 50 (<50).

Ferroportin ubiquitination and degradation assay: visually assessed Western blot effect + comparable to hepcidin, +/- intermediate effect and +/+/- stronger intermediate effect, + or +/+/- effect preferred, + effect most preferred.

Hepcidin Internalization Test (J774): IC ₅₀ value not more than 100 (<100), preferably not more than 50 (<50), more preferably less than 50 (<50).

Biophysical test for binding of ferroportin to hepcidin: IC ₅₀ value is not more than 100 (<100), preferably not more than 50 (<50), more preferably less than 50 (<50).

Suppression of iron excretion: IC ₅₀ value is not more than 100 (<100), preferably not more than 50 (<50), more preferably less than 50 (<50).

Ferroportin inhibition can also be determined in in vivo models, as described in more detail in the Examples below. Suitable in vivo models may include, for example, testing hypoferremia in naïve mice by measuring decreased blood iron levels; study of the prevention of iron absorption in anemic rats by measuring blood iron inhibition; a study of correction of hypoferremia in beta2-microglobulin-deficient mice by measuring decreased blood iron levels; a study to prevent iron overload in beta2microglobulin-deficient mice by measuring total iron content in the spleen or liver; investigating improvements in anemia, ineffective erythropoiesis, and iron overload in a mouse model of β-thalassemia mean.

The activity of salts of the compounds of the present invention as ferroportin inhibitors can, in particular, be determined by the methods described below in the examples.

As already described above, inhibition of ferroportin can be carried out, for example, by hepcidin, which is thus the most important factor in regulating iron absorption, inhibiting ferroportin and blocking the transfer of iron from cells to the circulatory system. The inventors of the present invention have unexpectedly discovered that several salts of the compounds described herein act as hepcidin mimetics or hepcidin agonists, which is also included in the inhibition of ferroportin in the context of the present invention.

Accordingly, salts of the compounds of the present invention can also be used to inhibit the transport of iron from cells into the circulatory system and inhibit the absorption of iron in the intestine, as well as hepcidin mimetics or hepcidin agonists.

Due to the activity of the salts described in the present invention as inhibitors of ferroportin, the salts of the present invention are also particularly well suited for use in inhibiting the transport of iron carried out by ferroportin, and thereby for use in the prevention and/or treatment of iron metabolic disorders leading to elevated iron levels. iron, diseases associated with or caused by increased iron levels, increased iron absorption or iron overload, such as, in particular, tissue iron overload, diseases associated with ineffective erythropoiesis, or diseases caused by decreased hepcidin levels. In addition, the compounds of the present invention are suitable for use in adjuvant therapy by limiting the amount of iron available to pathogenic microorganisms, such as the bacterium Vibrio vulnificus, thereby preventing or treating infections caused by these pathogenic microorganisms.

In the present context, diseases associated with, related to, caused by, or resulting in increased iron levels, increased iron absorption, iron overload (eg, tissue iron overload), or ineffective erythropoiesis include thalassemia, hemoglobinopathy, such as hemoglobin E (HbE) disease, hemoglobin disease H (HbH), hemochromatosis, hemolytic anemia such as sickle cell anemia and congenital dyserythropoietic anemia.

The activity of the salts of the present invention in the treatment of drepanocytic anemia (sickle cell anemia) can be determined using a mouse model, such as, for example, described in Yulin Zhao et al. in MEK1/2 inhibitors reverse acute vascular occlusion in mouse models of sickle cell

- 31 044945 disease; The FASEB Journal Vol. 30, No. 3, pp 1171-1186, 2016. This mouse model can be suitably adapted to determine the activity of the salts of the present invention in the treatment of drepanocytic anemia. Similarly, the activity of the compounds described in the above-mentioned unpublished international patent applications PCT/EP2016/075305 and PCT/EP2016/075306 concerning compounds having activity as ferroportin inhibitors, in the form of free bases and/or in the form of pharmaceutically acceptable salts in In general, the treatment of drepanocytic anemia can be tested using the specified mouse model, likely with proper adaptation to optimized testing conditions, which is a routine task for a skilled person in the field.

Diseases associated with, related to, caused by or resulting in increased iron levels, increased iron absorption, iron overload (eg, tissue iron overload) also include neurodegenerative diseases, such as, for example, Alzheimer's disease and Parkinson's disease, where the compounds described herein are considered as effective by limiting the accumulation or increase of iron in tissues or cells.

Salts of the compounds of the present invention are also suitable for use in the prevention and/or treatment of the formation of radicals, reactive oxygen species (ROS) and oxidative stress caused by excess iron or iron overload, as well as for the prevention and/or treatment of damage to the heart, liver and endocrine system. systems caused by excess iron or iron overload, as well as for the prevention and/or treatment of inflammation initiated by excess iron or iron overload.

Diseases associated with ineffective erythropoiesis include, but are not limited to, myelodysplastic syndrome (MDS) and polycythemia vera, as well as congenital dyserythropoietic anemia.

Other diseases, disorders and/or disease states include iron overload caused by mutations in genes involved in regulating systemic iron stores, such as hepcidin (Hamp1), hemochromatosis protein (HFE), hemojuvelin (HJV) and transferrin receptor 2 (TFR2) , such as, in particular, diseases associated with mutations of the HFE and HJV gene, diseases associated with chronic hemolysis, sickle cell anemia, red blood cell membrane disorders, glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency), erythropoietic porphyria, Friedreich's ataxia, as well as subtypes of iron overload such as transfusion iron overload, iron toxicity, pulmonary hemosiderosis, osteopenia, insulin resistance, African iron overload, Hallervorden-Spatz disease, hyperferritinemia, ceruloplasmin deficiency, neonatal hemochromatosis and red blood cell disorders including thalassemia, including alpha thalassemia , betathalassemia and delta thalassemia, sickle cell anemia and myelodysplastic syndrome.

Other diseases and/or disorders and/or disease states associated with elevated iron levels include, but are not limited to, diseases caused by elevated iron levels including ataxia, Friedreich's ataxia, age-related macular degeneration, age-related cataracts, age-related retinal diseases and neurodegenerative diseases. diseases such as pantothenate kinase-associated neurodegeneration, restless legs syndrome and Huntington's disease.

Salts of the compounds of the present invention may also be suitable for use in the prevention and treatment of diseases caused by hepcidin deficiency.

In light of the foregoing, another aspect of the present invention relates to a medicament containing one or more of the above-described salts of the compounds, such as, in particular, a medicament for the prophylaxis and treatment of any of the above-described indications, conditions, disorders or diseases.

Another subject of the present invention relates to pharmaceutical compositions and medicinal products containing one or more salts of the compounds of the present invention described above, and also, optionally, one or more pharmacologically acceptable carriers and/or auxiliary compounds and/or solvents. Another aspect of the present invention relates to pharmaceutical compositions and medicaments containing one or more of the above-described salts of the compounds of the present invention, and optionally one or more additional pharmaceutically effective compounds. Said pharmaceutical compositions contain, for example, up to 99 wt.%, or up to 90 wt.%, or up to 80 wt.%, or up to 70 wt.% salts of the compounds of the present invention, the balance being pharmaceutically acceptable carriers and/or excipients and/or solvents and/or optional additional pharmaceutically effective compounds.

In the context of the invention, pharmaceutically acceptable carriers, excipients or solvents are conventionally used pharmaceutical carriers, excipients or solvents, including various organic or inorganic carriers and/or excipients commonly used in pharmaceuticals, in particular for the preparation of solid medicinal products. Examples include fillers (such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders

- 32 044945 va, such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; tablet disintegrating agents such as starch, hydrolyzed starch, carboxymethylcellulose, carboxymethylcellulose calcium, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; flavorings such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (eg methylaparben, ethylparaben, propylparaben, butylparaben); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series, such as, for example, diethylenetriamine pentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats and oils such as beeswax, cocoa butter; polyethylene glycol; white Vaseline, etc.

Liquid drugs, such as solutions, suspensions and gels, typically contain a liquid carrier such as water and/or pharmaceutically acceptable organic solvents. In addition, such liquid preparations may also contain pH regulators, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (eg methylcellulose), coloring agents and/or flavoring agents. The compositions may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the compositions can be adjusted using sodium chloride and other pharmaceutically acceptable substances, such as, for example, dextrose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic and organic solutes. The viscosity of the liquid compositions can be controlled using pharmaceutically acceptable thickeners such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer and the like. The preferred concentration of thickener depends on the substance chosen.

Pharmaceutically acceptable preservatives can be used to increase the shelf life of liquid compositions. Benzyl alcohol is a suitable agent, although a variety of preservatives can also be used, including, for example, parabens, thimerosal, chlorobutanol and benzalkonium chloride.

The above-mentioned pharmaceutical compositions can be used, for example, for intravenous, intraperitoneal, intramuscular, intravaginal, intrabuccal, transdermal, subcutaneous, mucocutaneous, oral, rectal, transdermal, topical, intradermal, intragastric administration, and available, for example, in the form of pills, tablets, enteric-coated tablets, film-coated tablets, multilayer tablets, sustained-release preparations for oral, subcutaneous or cutaneous use (especially in patch form), delayed-release preparations, dragees, suppositories, gels, creams, syrup, granulates , suppositories, emulsions, dispersions, microcapsules, micropreparations, nanopreparations, liposomal preparations, capsules, enteric-coated capsules, powders, powders for inhalation, microcrystalline preparations, sprays for inhalation, powders, drops, nasal drops, nasal sprays, aerosols, ampoules, solutions, juices, suspensions, solutions for infusion or solutions for injections, etc.

Another aspect of the present invention relates to medicinal products or combination preparations containing one or more salts of the compounds of the present invention and at least one additional pharmaceutically active compound, such as, in particular, a compound for the prevention and treatment of iron overload and related symptoms, preferably an iron-chelating compound, or a compound for the prevention and treatment of any of the above conditions, disorders or diseases, such as, in particular, a pharmaceutically active compound for the prevention and treatment of thalassemia, hemochromatosis, sickle cell anemia, neurodegenerative diseases (such as Alzheimer's disease or Parkinson's disease ) and associated symptoms.

Another aspect of the present invention relates to the use of the above-described salts of the compounds in combination therapy (fixed-ratio dose or free combination of dosages for sequential administration) with one or two other active ingredients (drug compounds). Such combination therapy includes co-administration of salts of the compounds of the present invention and at least one additional pharmaceutically active compound (drug). Fixed-ratio combination therapy involves administering salts of the compounds of the present invention with at least one additional pharmaceutically active compound in a fixed-ratio dose. Combination therapy in the form of a free combination of dosages for sequential administration includes co-administration of the compounds of the present invention and at least one additional pharmaceutically active compound at random dosages of the respective compounds, either by simultaneous administration of the individual compounds, or by sequential administration of the individual compounds, distributed

- 33 044945 in time. Said at least one additional pharmaceutically active compound (drug) includes, in particular, drugs for reducing iron oversaturation (for example, Tmprss6-ASO) or iron chelators, in particular curcumin, SSP-004184, deferithrin, deferasirox, deferoxamine and/ or deferiprone, or antioxidants such as N-acetylcysteine, antidiabetic agents such as GLP-1 receptor agonists, antibiotics such as vancomycin (Van) or tobramycin, drugs to treat malaria, anticancer drugs, antifungals, drugs to treat neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (eg, dopamine agonists such as levodopa), antivirals such as interferon-a or ribavirin, or immunosuppressants (cyclosporine A or cyclosporine A derivatives), iron supplements, vitamin supplements, stimulants red blood cell production (eg, erythropoietin, EPO), anti-inflammatory drugs, antithrombolytics, statins, vasoconstrictors, and inotropes.

Another aspect of the present invention relates to the use of the combinations described above for the prevention and/or treatment of diseases caused by hepcidin deficiency or disorders of iron metabolism, such as, in particular, conditions of iron overload, such as, in particular, thalassemia, sickle cell anemia and hemochromatosis, and other diseases as described in this text.

Another aspect of the present invention relates to the use of salts of the compounds described herein, alone or as part of a combination therapy described herein, in combination with blood transfusion.

Potential synergistic or additive effects of the salts of the present invention with other therapeutic agents (second agent) can be assessed through combination studies in mouse models of thalassemia intermedia (Hbb ^th3/+ or Hbb ^th1/th1 Jackson Laboratories) or thalassemia major (C57-FCL ^{th3/ th3} , which examine the effect of the salts of the present invention alone (i.e., administering salt alone) or in combination with additional compound(s) on anemia, hematopoiesis, iron overload, reactive oxygen species (ROS) production, splenomegaly and other biomarkers in thalassemia models In addition to the combination therapy already described in the previous paragraph, the combination therapy of the present invention also includes the use of the salts of the present invention in combination with one of the following second agents:

Modified activin receptor type IIA or IIB fusion proteins (such as those described in Suragani RN, et al. Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia. Blood. 2014 Jun 19;123(25): 3864-72 and Dussiot M, et al. An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia. Nat Med. 2014 Apr;20(4):398-407), which act as a ligand trap for members of the transforming factor superfamily. growth beta (TGFe), such as RAP-011 or RAP-536 (mouse analogues of ACE-011, Sotatercept or ACE-536, Luspatercept (described in patent application WO 2010019261 or claimed in US patent US 8361957), respectively, Acceleron/ Celgene) or other antagonists of members of the TGFe superfamily (antibodies, antibody fragments, non-antibody drugs, or cells that produce activin receptor ligand decoys).

Inhibitors of JAK1/2 or JAK2, including but not limited to rutoxilinib (Novartis as claimed in US Patents US 7,598,257 and US 8,415,362) or fedratinib (Sanofi), such as those described by Casu C, et al. Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of βthalassemia intermedia and major.; Haematologica, 2017.

pan-HDAC inhibitor such as panobinostat (LC Laboratories, USA, and claimed in US patents US 6552065 and US 6833384) or HDAC3 inhibitor RGFP966 (Selleckchem - such as described in Pasricha S.R. et al. Hepcidin is regulated by promoter-associated histone acetylation and HDAC3. Nat Commun. 2017 Sep 1;8(1):403).

Matriptase-2 (also known as Tmprss6) antagonists, such as lipid nanoparticle (LNP) Tmprss6 siRNAs or antisense oligonucleotides (ASOs) targeting murine Tmprss6 (such as those described in Guo S. et al. Reducing TMPRSS6 ameliorates hemochromatosis and β-thalassemia in mice. J. Clin Invest. 2013 Apr;123(4):1531-41 or in the work of Schmidt P. J. et al. An RNAi therapeutic targeting Tmprss6 decreases iron overload in Hfe(-/-) mice and ameliorates anemia and iron overload in murine β-thalassemia intermedia. Blood. 2013 Feb 14;121(7):1200-8).

Exogenous apotransferrin (such as described in Li H. et al. Transferrin therapy ameliorates disease in beta-thalassemic mice. Nat Med. 2010 Feb; 16(2): 177-82).

Hepcidin-inducing steroids (HIS), such as epithiostanol, progesterone and mifepristone, or progesterone receptor membrane component-1 (PGRMC1) antagonists, ref. 7.

Erythroferrone antagonists, such as antibodies or ligand decoys.

Recombinant erythropoietin (EPO). The erythropoietins available for use as therapeutic agents of the present invention are produced by recombinant DNA technology in cell culture and include Epogen/Procrit (epoetin-alpha) and Aranesp (dareroetin-alpha) or Myrcera

- 34 044945 (epoetin beta and methoxy polyethylene glycol).

Glycine transporter 1 (GlyT1) inhibitors such as bitopertine (Roche AG).

The salts of the present invention can be administered orally either as the sole active ingredient twice daily at dosages of 10, 30 and 60 mg/kg, or in combination with one or more of the above compounds (second agents). More specifically, the second agent is administered as a separate form or administered together with the salts of the present invention, as described below:

RAP-011 or RAP-536 can be administered subcutaneously twice weekly at 1, 10, or 30 mg/kg for up to 8 weeks.

JAK1/2 inhibitors can be administered orally twice daily with or without the salts of the present invention.

Rutoxilinib (60 or 180 mg/kg) or fedratinib (40 or 120 mg/kg) can be administered orally once daily for 2 weeks, with or without the salts of the present invention.

Panobinostat or RGFP966 can be administered once daily at a dosage of 10 or 20 mg/kg with or without the salts of the present invention.

Apotransferrin is administered by injection intraperitoneally at a dosage of 100 or 300 mg/kg per day for 8 weeks.

Mifepristone (30 or 100 mg/kg) is administered by injection intraperitoneally once daily for 2 weeks.

Erythroferrone-specific antibodies or ligand decoys can be administered twice weekly by subcutaneous injection.

Erythropoietin can be injected intraperitoneally at a dosage of 200 IU once daily for 2 weeks.

Glycine transporter 1 (GlyT1) inhibitors, such as bitopertine (Roche AG), can also be administered in a suitable manner.

The salts, drugs or combination preparations of the present invention can be administered orally, parenterally as well as intravenously.

For these purposes, salts of the compounds of the present invention are preferably formulated into drugs or pharmaceutical compositions in the form of pills, tablets such as enteric tablets, film-coated tablets and multilayer tablets, sustained-release oral preparations, depot preparations, dragees, granules , emulsions, dispersions, microcapsules, microformulations, nanoformulations, liposomal preparations, capsules such as enteric capsules, powders, microcrystalline preparations, epipastic preparations, drops, ampoules, solutions, suspensions, solutions for infusion or solutions for injection, or in the form drugs suitable for inhalation.

In a preferred embodiment of the present invention, the salts of the compounds are administered in the form of tablets or capsules. They can be, for example, acid-resistant forms or forms with a pH-sensitive coating.

Salts of the compounds of the present invention as active substances can be administered, for example, in a single dose of 0.001 to 500 mg/kg body weight, for example, 1-4 times a day. However, the dosage may be increased or decreased depending on the age, body weight, condition of the patient, severity of the disease and or route of administration.

Accordingly, another subject of the present invention relates to the above-described salts of the compounds, drugs, compositions and combination preparations for the preparation of drugs, in particular for the prevention and treatment of any of the above-described indications, conditions, disorders or diseases, in particular for oral or parenteral administration.

Another subject of the present invention relates to a method for the prevention and treatment, in particular for the prevention and/or treatment of disorders of iron metabolism associated with or leading to increased iron levels, and in particular iron overload, diseases associated or leading to increased iron levels or iron overload, iron storage disorders associated with or resulting in elevated iron levels, and diseases associated with ineffective erythropoiesis, wherein said method comprises administering to a patient (human or animal) in need of such treatment a salt, drug, composition or combination drug as described above.

In this context, diseases associated with, associated with, caused by or resulting in elevated iron levels or iron overload are as defined above.

Another subject of the present invention relates to the use of the above-described salts for the preparation of a medicinal product, in particular for the prevention and treatment of any of the above-described indications, conditions, disorders or diseases.

Description of the figures

Fig. 1: Formula (I) according to the present invention.

Fig. 2: Visualized example of a dynamic vapor sorption (DVA) program applied.

- 35 044945

Fig. 3.1: Structure of compound 127 in free base form, showing calculated pKa values.

Fig. 3.2: 1H-NMR spectrum of SP236-FB-P1 in DMSO-d ₆ .

Fig. 3.3: Fourier-Raman spectrum view of SP236-FB-P1 in the range from 50 to 3500 cm ^-1 .

Fig. 3.4: Fingerprint zone in the Fourier-Raman spectrum of the SP236-FB-P1 compound, ranging from 50 to 1800 cm ^-1 .

Fig. 4: PXRD diagram of SP236-FB-P1.

Fig. 5.1: Comparison of PXRD patterns of SP236-CIT-P1, SP236-CIT-P1(2), SP236-CIT-P2 and SP236-CITP3.

Fig. 5.2: TG-FTIR thermogram SP236-CIT-P2.

Fig. 5.3: DSC thermogram of SP236-CIT-P3.

Fig. 5.4: Comparison of FT-Raman spectra of SP236-CIT-P3 and SP236-FB-P1 in the range from 50 to 3500 cm ^-1 .

Fig. 5.5: Comparison of FT-Raman spectra of SP236-CIT-P3 and SP236-FB-P1 in the range from 50 to 1800 cm ^-1 .

Fig. 5.6: 1H-NMR spectrum of SP236-CIT-P2 in DMSO-d ₆ .

Fig. 5.7: Plot of sample mass (%) and relative humidity (%) versus time for SP236-CIT-P3, showing sample mass (left y-axis) and relative humidity set by the program (right y-axis).

Fig. 5.8: Water vapor sorption isotherm for SP236-CIT-P3.

Fig. 5.9: Comparison of PXRD patterns of SP236-MLE-P1, SP236-MLE-P2 and SP236-MLE-P3.

Fig. 5.10: TG-IR-FT thermogram SP236-MLE-P1.

Fig. 5.11: DSC thermogram of SP236-MLE-P3.

Fig. 5.12: Comparison of FT-Raman spectra of SP236-MLE-P3 and SP236-FB-F1 in the range from 50 to 3500 cm ^-1 .

Fig. 5.13: Comparison of FT-Raman spectra of SP236-MLE-P3 and SP236-FB-F1 in the range from 50 to 1800 cm ^-1 .

Fig. 5.14: 1H-NMR spectrum of SP236-MLE-P1 in DMSO-d ₆ .

Fig. 5.15: Plot of sample mass (%) and relative humidity (%) versus time for SP236-MLE-P3, showing sample mass (left y-axis) and relative humidity set by the program (right y-axis).

Fig. 5.16: Water vapor sorption isotherm for SP236-MLE-P3.

Fig. 5.17: Comparison of PXRD patterns of SP236-PO4-P1 and SP236-PO4-P2.

Fig. 5.18: TG-FTIR thermogram of SP236-PO4-P2.

Fig. 5.19: 1H-NMR spectrum of SP236-PO4-P2 in DMSO-d ₆ .

Fig. 5.20: ³¹ P NMR spectrum of SP236-PO4-P2 in DMSO-d ₆ .

Fig. 5.21: Comparison of PXRD patterns of SP236-PO4-P2, SP236-PO4-P5, SP236-PO4-P6, SP236-PO4-P7 and SP236-PO4-P8.

Fig. 5.22: Comparison of the Fourier-Raman spectra of SP236-PO4-P8 and SP236-FB-P1 in the range from 50 to 3500 cm ^-1 .

Fig. 5.23: Comparison of the Fourier-Raman spectra of SP236-PO4-P8 and SP236-FB-P1 in the range from 50 to 1800 cm ^-1 .

Fig. 5.24: TG-FTIR thermogram SP236-PO4-P8.

Fig. 5.25: DSC thermogram of SP236-PO4-P6.

Fig. 5.26: DSC thermogram of SP236-PO4-P8.

Fig. 5.27: Plot of sample mass (%) and relative humidity (%) versus time for SP236-PO4-P8, showing sample mass (left y-axis) and relative humidity set by the program (right y-axis).

Fig. 5.28: Water vapor sorption isotherm for SP236-PO4-P8.

Fig. 5.29: Comparison of PXRD patterns of SP236-SO4-P1 and SP236-SO4-P3.

Fig. 5.30: 1H-NMR spectrum of SP236-SO4-P3 in DMSO-d ₆ .

Fig. 5.31: Comparison of PXRD patterns of SP236-SO4-P4, SP236-SO4-P5 and SP236-SO4-P6.

Fig. 5.32: TG-FTIR thermogram of SP236-SO4-P4.

Fig. 5.33: Plot of sample mass (%) and relative humidity (%) versus time for SP236-SO4-P6, showing sample mass (left y-axis) and relative humidity set by the program (right y-axis).

Fig. 5.34: water vapor sorption isotherm for SP236-SO4-P6.

Fig. 5.35: DSC thermogram of SP236-SO4-P6.

Fig. 5.36: 1H-NMR spectrum of SP236-SO4-P4 in DMSO-d ₆ .

Fig. 5.37: Comparison of the Fourier-Raman spectra of SP236-SO4-P6 and SP236-FB-P1 in the range from 50 to 3500 cm ^-1 .

Fig. 5.38: comparison of Fourier-Raman spectra of SP236-SO4-P6 and SP236-FB-P1 in the range of 50

- 36 044945 to 1800 cm ^-1 .

Fig. 5.39: enlarged fragment of the HPLC chromatogram for SP236-FB-P1.

Fig. 5.40: enlarged fragment of the HPLC chromatogram for SP236-CIT-P3.

Fig. 5.41: enlarged fragment of the HPLC chromatogram for SP236-MLE-P3.

Fig. 5.42: enlarged fragment of the HPLC chromatogram for SP236-PO4-P8.

Fig. 5.43: enlarged fragment of the HPLC chromatogram for SP236-SO4-P6.

Fig. 6.1: PXRD diagram of SP236-BNZ-P2.

Fig. 6.2: TG-FTIR thermogram SP236-BNZ-P2.

Fig. 6.3: 1H-NMR spectrum of SP236-BNZ-P2 in DMSO-d ₆ .

Fig. 6.4: Comparison of PXRD diagrams of SP236-FUM-P1 and SP236-FUM-P2.

Fig. 6.5: TG-FTIR thermogram SP236-FUM-P2.

Fig. 6.6: 1H-NMR spectrum of SP236-FUM-P2 in DMSO-d ₆ .

Fig. 6.7: Comparison of PXRD patterns of SP236-MLA-P1 and SP236-MLA-P2.

Fig. 6.8: 1H-NMR spectrum of SP236-MLA-P1 in DMSO-d ₆ .

Fig. 6.9: PXRD diagram of SP236-SUC-P2.

Fig. 6.10: 1H-NMR spectrum of SP236-SUC-P2 in DMSO-d ₆ .

Fig. 6.11: TG-FTIR thermogram SP236-SUC-P2.

Fig. 6.12: Comparison of PXRD diagrams of SP236-LTAR-P1 and SP236-LTAR-P2.

Fig. 6.13: TG-FTIR thermogram SP236-LTAR-P1.

Fig. 6.14: TG-FTIR thermogram SP236-LTAR-P2.

Fig. 6.15: 1H-NMR spectrum of SP236-LTAR-P1 in DMSO-d ₆ .

Fig. 6.16: 1H-NMR spectrum of SP236-LTAR-P2 in DMSO-d ₆ .

Fig. 6.17: Comparison of PXRD diagrams of SP236-TOS-P1 and SP236-TOS-P2.

Fig. 6.18: 1H-NMR spectrum of SP236-TOS-P2 in DMSO-d ₆ .

Fig. 7.1: HPLC analysis of the HCl monosalt of compound 127 from Preparation Example 7.2.

Fig. 7.2: DSC thermogram of the HCl monosalt of compound 127 from Preparation Example 7.2.

Fig. 8.1: summary of PXRD diagrams of polymorphs PM1 - PM6 of the compound PP566-SO4-P1 (from bottom to top: PP566-SO4-P2 (PM1), P5 (PM2), P6 (PM3), P8 (PM4), P10 (PM5) and P11 ( RM6)).

Fig. 8.2: PXRD diagram of the PM1 polymorph (PP566-SO4-P2).

Fig. 8.3: 1H-NMR spectrum of the PM1 polymorph (PP566-SO4-P2).

Fig. 8.4: DSC thermogram of PM1 (PP566-SO4-P2).

Fig. 8.5: DSP behavior of the PM1 polymorph of the PP566-SO4-P1 compound.

Fig. 9.1: summary of PXRD diagrams of polymorphs PM1 - PM11 of the compound PP566-PO4-P1 (from bottom to top PP566-PO4-P4(RM1), P4-DRY(PM9), P5(PM2), P8(PM3), P10(PM4), P11 (PM5), P13(PM6), P13DRY(PM10), P15(PM7), P15-DRY (PM11) and P19(PM8)).

Fig. 9.2: PXRD diagram of the PM2 polymorph (PP566-PO4-P5).

Fig. 9.3: 1H-NMR spectrum of the PM2 polymorph (PP566-PO4-P5).

Fig. 9.4: TG-IR-Fourier thermogram of PM2 (PP566-PO4-P5).

Fig. 9.5: TG-IR-Fourier thermogram of PM2 (PP566-PO4-P12).

Fig. 9.6: summary of PXRD diagrams of PM2 polymorphs of samples PP566-PO4-P2, P5, P6, P9, P12 (from bottom to top: PP566-PO4-P2, P5, P6, P9 and P12).

Fig. 9.7: Chipboard behavior of PP566-PO4-P2 (PP566-PO4-P12).

Fig. 10: immunoblotting of immunoprecipitates with anti-Fpn antibody MTP1.

Fig. 11: Inhibition of iron efflux by hepcidin (IC50: 0.086 µM) and compound 127 (IC50: 0.080 µM).

Fig. 12: reduction in blood iron levels caused by hepcidin and ferroportin inhibitor compound 94 (compound No. 94); from fig. 12A: Blood iron kinetics in naïve C57BL/6 mice injected with synthetic hepcidin (5 mg/kg) intraperitoneally (i.p.) for the indicated period of time; and from fig. 12B: Blood iron levels in naïve C57BL/6 mice treated with the indicated amounts of hepcidin (i.p., i.p.) or compound 94 (orally) for 3 h.

Fig. 13: complete correction of elevated iron levels in b2m-/- mice by administration of the ferroportin inhibitor compound 40/methylcellulose (A.) and compound 94/Cremophor EL (B.) for 3 hours.

Examples

The present invention is illustrated in more detail in the following examples. The examples serve solely to provide a more complete understanding of the invention, and those skilled in the art may extend these specific examples to other salts claimed, for example, in particular, other salts described in the present invention formed with compounds having formula (I) depicted in fig. 1.

- 37 044945

Further in the text, samples are indicated under identification codes in the format SP236-XYZ-Pw, where

XYZ indicates the salt/crystal coformer (i.e. type of acid) and Pw indicates the specific sample/experiment (w = 1, 2, ... n).

The free base of compound 127 was used as the starting compound.

I. Preparation of various salts of compound 127.

1. Abbreviations.

DCM - dichloromethane;

DMSO - dimethyl sulfoxide;

DSC - differential scanning calorimetry;

EAF - dynamic vapor sorption;

EtoAc - ethyl acetate;

EtOH - ethanol;

Fourier-Raman - Fourier-Raman spectroscopy;

1H-NMR - proton nuclear magnetic resonance;

i-PrOH - isopropanol;

MeCN - acetonitrile;

MeOH - methanol;

n-BuOH - 1-butanol;

relative humidity/RH - relative humidity;

r.t./RT - room temperature (22-25°C);

Tg - glass transition temperature;

TG-IR-Fourier - thermogravimetry in conjunction with IR-Fourier spectroscopy;

THF - tetrahydrofuran;

PXRD - powder x-ray diffraction.

2. General details of experiments.

DSC: Differential scanning calorimetry was performed on a TA Instruments Q2000 instrument (closed or open, gold or aluminum sample cells, with or without hole). The heating rate was usually 10 K/min. The melting point is in most cases understood as the temperature at which the peak begins.

Dynamic vapor sorption: EAF experiments were carried out on the SPS11-100n Sorptions Prufsystem from ProUmid (formerly Projekt Messtechnik), August-Nagel-Str. 23, 89079 Ulm (Germany) or on the DVS-1 from Surface Measurement Systems. Approximately 5 - 20 mg of sample was placed in an aluminum cell. A humidity change rate of 5% per hour was used. An example of the experimental program used is shown in Fig. 2. Diagrams showing effective water content are constructed using mass loss data from thermogravimetric analysis (TGA). In some cases, a double cycle was used.

In polymorph studies, the sample was placed on an aluminum holder on a microbalance and allowed to equilibrate at 50% relative humidity (RH) before running preset humidity programs:

(1) 2 hours at 50% RH.

(2) 50^0% RH (5%/h); 5 hours at 0% RH.

(3) 0 ^ 95% RH (5%/h); 5 hours at 95% RH.

(4) 95^50% RH (5%/h); 2 hours at 50% RH.

Classification of Hygroscopicity: Hygroscopicity was classified based on the increase in mass at 85% RH relative to the initial mass, as follows: deliquescent (adsorbs enough water to form a liquid), very hygroscopic (>15% increase in mass), hygroscopic (<15% increase in mass, but >2%), slightly hygroscopic (weight increase <2% but >0.2%) or non-hygroscopic (weight increase <0.2%).

Elemental analysis: elemental analysis was carried out on a vario EL cube analyzer manufactured by Elementar. The analyzer uses combustion to convert elements into simple gases, such as CO ₂ , H ₂ O, N ₂ . The resulting gases are separated on selective collection columns and quantified as a function of thermal conductivity. Oxygen is converted to carbon monoxide by pyrolysis and can also be quantified as a function of thermal conductivity.

1H-NMR: Bruker DPX300 spectrometer; operating frequency for protons 300.13 MHz; 30° excitation pulse; pulse delay 1 s; accumulation of 16 scans; deuterated DMSO as solvent; the solvent peak was used as a reference; chemical shift values are given relative to TMS.

HPLC: Agilent Series 1100 HPLC system with Agilent 1260 Infinity degasser, Chromeleon Version 6.8 software.

Karl Fischer titration: Karl Fischer titration was carried out according to well-known methods, for example according to ISO 760-1978: Determination of water content by the method

- 38 044945

Karl Fischer (General method).

pKa measurement: Sirius T3 titration device. Photometric or potentiometric analysis was used using co-solvents for samples with low water solubility.

Powder X-ray diffraction (reflection): experiments on a Bruker D8 Advance powder X-ray diffractometer were carried out in reflection mode (Bragg-Brentano). 2θ values were usually obtained within the measurement error of ±0.1-0.2°. The samples were usually prepared without any special treatment other than applying slight pressure to obtain a flat surface. Silicon monocrystalline sample holder for polymorph screening, width 0.5 mm. Typically, uncoated samples were examined. The voltage in the tube is 40 kV, the current is 40 mA. The PXRD diffractometer is equipped with a LynxEye detector. A different divergence slit with a 3° window was used. Step size 0.02 °2θ, step time 37 s. The samples were rotated at a speed of 0.5 rps during the experiment. Sample preparation and experiments were carried out in ambient air.

Powder X-ray diffraction (transmission): Stoe Stadi P instrument equipped with Mythen1K detector; Cu-Ka1 radiation; standard experimental conditions: transmission; tube parameters: 40 kV and 40 mA; curved Ge monochromator; step size 0.02°2θ, step time 12 or 48 s, scanning range 1.5-50.5°2θ; detector mode: step-by-step scanning; detector pitch 1°2θ; standard sample preparation: a 10-20 mg sample was placed between two sheets of acetate foil; sample holder: Stoe sample holder for transmission; the sample was rotated during the experiment. Sample preparation and experiments were carried out in ambient air.

In polymorph studies, each sample (25-40 mg powder) was placed between two sheets of cellulose acetate, which were separated by a metal separator (thickness 0.4 mm, internal diameter 12 mm). This sandwich element was transferred to a special sample holder for highly active compounds (SCell), which was again sealed with acetate film. No special treatment was used during sample preparation. All experiments and measurements were performed in ambient air, and each sample was rotated during the experiments.

Raman spectroscopy: FT-Raman spectra were recorded on a Bruker MultiRAM FT-Raman system with a near-infrared Nd:YAG laser at 1064 nm and a liquid nitrogen-cooled germanium detector. 64 scans with a resolution of 2 cm ^-1 were accumulated in the range from 3500 to 50 cm ^-1 ; however, only data above 100 cm ^-1 were analyzed due to filter cutoff effects. The laser power rating is typically 100 or 300 mV.

Solubility: Approximate solubility values were determined by incremental addition of solvent to approximately 10 mg of compound. If a substance did not dissolve when a total of at least 10 mL of solvent was added, the solubility was reported as <1 mg/mL. Due to the experimental error inherent in this method, solubility values should be estimated as a rough approximation and used only for the design of crystallization experiments.

TG-FTIR: thermogravimetric measurements were carried out on a Netzsch ThermoMicrobalance TG 209 instrument combined with a Bruker FTIR Spectrometer Vector 22 FTIR spectrometer (sample cells with a hole, N2 atmosphere, heating rate 10 K/min).

Approximate solubility: Approximate solubility values were determined by incremental addition of solvent to approximately 10 mg of compound. If a substance did not dissolve when a total of at least 10 mL of solvent was added, the solubility was reported as <1 mg/mL. Due to the experimental error inherent in this method, solubility values should be estimated as a rough approximation and used only for the design of crystallization experiments.

3. Study of the starting substance.

Initial Connection (Free Base/FB):

connection 127 (SP236-FB-P1).

Calculation of pKa of the original compound:

theoretical pKa values were calculated using the ACD/pKa DB Vers program. 10.00, Release 10.00. The obtained values are shown along with the structure of the parent compound (free base) in FIG. 3.1.

1H-NMR spectroscopy of the starting compound:

The NMR spectrum of SP236-FB-P1 was recorded in DMSO-d ₆ as shown in FIG. 3.2. The spectrum contains at least one broad signal with a chemical shift of ~δ12 ppm, but the spectrum as a whole appears consistent with the given chemical structure. Signals of residual ethanol and dichloromethane were also observed in the NMR spectrum.

Raman spectroscopy:

The FT-Raman spectrum of SP236-FB-P1 was recorded in the range from 50 to 3500 cm ^-1 as shown in FIG. 3.3, with an enlarged view of the fingerprint area from 50 to 1800 cm ^-1 as shown in FIG. 3.4.

Powder X-ray diffraction:

The PXRD diagram of the SP236-FB-P1 connection was recorded in pass-through mode and is shown in FIG.

- 39 044945

4, it confirms that the sample (in free base form) is amorphous in nature.

Approximate solubility of the starting compound:

The approximate solubility of SP236-FB-P1 was determined in a number of different solvents and solvent mixtures to help better design salt cocrystallization experiments. The results are shown below)

Solvent Solubility (mg/ml) Solvent mixture Solubility (mg/ml) Acetonitrile S>200 Dioxane S > 100 DMSO S > 100 Ethanol S > 100 Ethyl acetate s>100 Ethyl acetate - cyclohexane 3:1 5<S<7 Heptane S< 1 Methanol-water 1:1 S > 100 Methanol S>200 2-propanol 80 < S < 120 2-propanol-water 1:1 39<S<37 THF S>200 THF-heptane 3:1 S>200 Water S~ 15

4. Crystallization experiments.

Crystallization conditions.

In all experiments, a free base:acid ratio of 1:1 (moles) was used; in the case of PO ₄ and SO4, two experiments were also carried out with a free base:acid ratio of 10:1. Many experiments resulted in crystalline products, as shown by PXRD diagrams, which are discussed in more detail below. Those experiments that resulted in only amorphous products are not reported in this text in further detail (ie, LLAC and MES).

Selected acids and solvents for crystallization: ________________________

Acid Reduction Solvent used Result Benzoic acid BNZ 2-propanol Ethyl acetate Amorphous Crystal PXRD Lemon acid CIT Ethanol Methanol Crystal PXRD Crystal PXRD Fumaric acid FUM THF 2-propanol Semi-crystalline PXRD Crystal PXRD Lactic acid, L- LLAC 2-propanol Methanol Amorphous Amorphous Malic acid, L- M.L.A. 2-propanol THF Semi-crystalline PXRD Mostly amorphous Maleic acid MLE 2-propanol THF Crystal PXRD Crystal PXRD Methanesulfonic acid MES THF acetonitrile Amorphous Amorphous Phosphoric acid PO4 Acetonitrile (104) 2-propanol (10:1) Acetonitrile (1:1) Crystalline PXRD Crystalline PXRD Amorphous Amorphous 2-propanol (1:1) succinic acid sue Ethanol THF Amorphous Semi-crystalline PXRD Sulfuric acid SO4 2-propanol (10:1) Acetonitrile (U:1) 2-propanol (1:1) Acetonitrile (1:1) Crystalline PXRD No solid product Crystalline PXRD Crystalline PXRD Tartaric acid, L- LTAR Ethanol Methanol Crystal PXRD Crystal PXRD T oluene sulfonic acid T.O.S. 2-propanol THF Semi-crystalline PXRD Crystal PXRD

The following describes in more detail the preparation and study of selected salts according to the above conditions.

5. Some salts of compound 127.

5.1. Salt of compound 127 with citric acid.

A crystallization experiment using citric acid in ethanol initially resulted in an amorphous substance which crystallized when heated to 30°C with occasional sonication (SP236-CIT-P1(2)). The PXRD diagram corresponds to the crystalline substance obtained in the experiment with methanol (Fig. 5.1, SP236-CIT-P2). The production of this crystalline form could also be reproduced at ~600 mg scale in the SP236-CIT-P3 experiment. Sample -P2 contains ~0.9% water and methanol, which it loses at ~150°C (Fig. 5.2). Another 8.1% of water is lost when heated to 200°C. DSC of sample SP236-CIT-P3 shows that this salt melts with an onset temperature of 153°C (Fig. 5.3). The FT-Raman spectrum of SP236-CIT-P3 is compared with the free base spectra in FIG. 5.4 and fig. 5.5, and there are clear differences between the two spectra. The 1H-NMR spectrum of SP236-CIT-P2 recorded in DMSO-d ₆ contains an additional signal of ~δ 2.6 ppm, having an integrated intensity of 3.8, which indicates the formation of a 1:1 free base:acid salt (Fig. 5.6). Considering this ratio to be correct, the 8.1% water observed in the TG-FTIR experiment indicates a 1:1 salt trihydrate. Interestingly, the EAF shows a decrease in the relative weight of the sample from the start of the measurement and the sample eventually becomes anhydrous at 0% relative humidity (Figs. 5.7 and 5.8). It then begins to adsorb water as soon as the relative humidity increases, and the difference between the relative mass of the sample at 0 and 95% RH is ~8%, which is in good agreement with the TG-FTIR results. The results of elemental analysis also correspond well to 1:1 salt, but determination of water content by Karl Fischer titration indicates that the sample is anhydrous: ___________________

Element/substance Theoretically for 1:1 salt SP236-CIT-P3, found WITH 54.0 53.0 n 4.9 5.1 N 14.0 13.7 ABOUT 24.0 24.2 H ₂ O - 0.5 (according to Karl Fischer)

5.2. Salt of compound 127 with maleic acid.

Crystallization screens using maleic acid in 2-propanol and THF yielded crystalline solids whose PXRD patterns matched well with each other (Figure 5.9). Scale-up of this synthesis by approximately 500 mg in 2-propanol yielded the same crystal form for sample SP236-MLE-P3. TG-FTIR analysis showed that the sample was essentially anhydrous, but experienced large mass loss and decomposition starting at about 170°C (Fig. 5.10). DSC of sample SP236-MLE-P3 in a hermetically sealed gold cell showed a melting point of ~161°C (Fig. 5.11). The FT-Raman spectrum of SP236-MLE-P3 was recorded and showed some differences from the free base spectrum (Figure 5.12 and Figure 5.13). ^{The 1} H NMR spectrum of SP236-MLE-P1 contains a signal attributable to maleic acid at δ 6.1 with an integrated intensity of 3.5, corresponding to a free base:acid ratio of 1:1.75 (Fig. 5.14). The chipboard shows ~1% mass loss when the relative humidity drops to 0%, and then water adsorption when the relative humidity rises again (Fig. 5.15 and Fig. 5.16). The maximum mass increase of ~6.5% is achieved at 95% relative humidity, which corresponds to ~2.5 water molecules per mole of salt (assuming a free base:MLE ratio of 1:1.75). The elemental analysis of SP236-MLE-P3 matches well with the 1:1.75 salt, and the 0.4% water content determined by Karl Fischer titration is in agreement with the TG-FTIR results:

Element Theoretically for free base: MLE SP236-MLE-P3, found 1:1 1:1.75 1:2 WITH 57.2 55.0 54.4 54.7 n 4.8 4.6 4.6 4.8 N 16.0 13.7 13.1 13.6 ABOUT 18.3 23.5 25.0 23.8 H ₂ O 0.4 (according to Karl Fischer)

5.3. Salt of compound 127 with phosphoric acid.

Crystallization screening results using phosphoric acid with acetonitrile (SP236-PO4-P1) and 2-propanol (SP236-PO4-P2) as solvents yielded two different crystalline solids as determined by PXRD (Figure 5.17). It is important to note that these two experiments used a free base:acid molar ratio of 10:1. TG-FTIR of sample -P2 showed a mass loss of 1.0% at 130°C due to loss of 2-propanol, with decomposition starting above 200°C (Figure 5.18). This sample was also examined by 1H and ^31P NMR, the second of which showed the presence of a phosphate ion (Fig. 5.19 and Fig. 5.20). These two experiments were performed using the same procedure, but with a 1:1 free base:acid molar ratio. These experiments produced only amorphous solids, which were not further investigated. Several more experiments were carried out to further study this system and gain a deeper understanding of this synthesis. In SP236-PO4-P5, experiment -P2 was repeated to confirm its reproducibility. The PXRD diagram of sample -P5 coincides with that of -P2 (Fig. 5.21), and the analysis for phos

- 41 044945 for indicates that sample -P5 is a hemiphosphate (i.e., it has a 2:1 ratio of free base: phosphate salt (Table 5.3). In the experiment SP236-PO4-P6, aqueous phosphoric acid was added in portions of 0.1 molar equivalent until a 2:1 free base:acid ratio is achieved.The PXRD diagram confirms the same crystalline form obtained in -P2 and -P5 (Figure 5.21), and the presence of 2.83 mass percent phosphorus again confirms the formation of hemiphosphate.Experiment SP236 -PO4-P7 was carried out similarly to -P6, however phosphoric acid was added to achieve a 1:1 free base:acid ratio to attempt to obtain the monophosphate salt. PXRD analysis of the resulting solid product again showed the production of hemiphosphate (Fig. 5.21).Scaling up this synthesis to ~ 600 mg in the experiment SP236-PO4-P8 was also successful and gave the same crystal form.The FT-Raman spectrum of this sample is compared with the spectrum of the free base in Fig. 5.22 and fig. 5.23, and significant differences are visible. Unexpectedly, the TG-FTIR spectrum of SP236-PO4-P8 showed the presence of noticeably more water and 2-propanol in the sample, despite the same PXRD pattern (Fig. 5.24). Thus, it appears that this phosphate salt has isomorphic solvated/hydrated and non-solvated/hydrated forms. DSC of samples SP236-PO4-P6 and -P8 in hermetically sealed gold cells showed highly reproducible melting points with starting points of 79 and 80°C (Fig. 5.25 and Fig. 5.26). These values correspond well to the melting point of 2-propanol and indicate simultaneous release of the solvent and melting of the solid. DSC experiments will most likely also need to be carried out in open cells. The chipboard of sample SP236-PO4-P8 showed an immediate weight loss when decreasing relative humidity in the range of 50 to 0%, which is incomplete during the first cycle. Additional mass loss is observed during the second EAF cycle, and the difference between the highest and lowest relative mass of the sample of ~12.5% is in good agreement with the mass loss observed by TG-FTIR. A water content of 12.5% corresponds to ~7 molecules of water per 2:1 salt. The elemental analyzes of samples SP236-PO4-P5 and -P8 are largely the same, although the carbon content varies significantly: ______________________________

Element/substance Theoretically for a free base: PO4 Found 2:1 2:1 4Η ₂ Ο SP236-PO4-P5 SP236-PO4-P8 WITH 55.1 51.1 52.1 50.7 n 5.0 5.4 5.7 5.9 N 18.4 17.0 16.4 15.9 ABOUT 14.0 19.5 - 20.2 R 3.4 3.1 2.96 2.92 H ₂ O - 7.3 - 8.0

It is also important to note the water content of 8 wt.%, which was determined by the Karl Fischer titration method. This value is noticeably lower than the 12 wt.% water adsorbed according to the EAF, but may indicate that some amount of 2-propanol is retained.

5.4. Salt of compound 127 with sulfuric acid.

The first screening experiments with sulfuric acid were performed at a molar ratio of 10:1 free base:acid. Crystallization in 2-propanol (SP236-SO4-P1) gave a crystalline solid, the experiment was reproduced on a larger scale (-P3), while the experiment with acetonitrile (-P2) did not give any solid products (Fig. 5.29). The 1H-NMR spectrum of the resulting crystalline substance showed that the molecule had not decomposed at all (Fig. 5.30). Two additional experiments were carried out in a similar manner, but this time a free base:acid molar ratio of 1:1 was used. These results are more interesting because the desired ratio of starting compounds was initially used. Crystallization experiments in both solvents yielded very similar crystal forms as determined by PXRD, and the experiment in 2-propanol was reproducible on a scale of ~600 mg (Figure 5.31). TG-FTIR analysis showed that sample -P4 contains ~5.5 wt.% water, which it loses in two different stepwise losses of 4 and 1.5%, and begins to decompose at temperatures above 200°C (Fig. 5.32). A water content of 4% corresponds to ~1 molecule of water in the salt, while 1.5 wt.% corresponds to 0.5 molecules of water in the salt (based on the salt with a 1:1 free base:sulfate ratio). The chipboard shows a constant decrease in mass as the relative humidity decreases from 50 to 0%, followed by an immediate gain in mass as the relative humidity rises again (Figs. 5.33 and 5.34). The difference between the minimum and maximum relative weight of the sample in the chipboard is approximately 5 to 5.5%, which is in good agreement with the results of TG-FTIR analysis and indicates a total content of 1.5 water molecules per salt (based on 1:1 salt). The onset melting point of 173°C was determined for sample SP236-SO4-P6 by DSC in a sealed gold cell (Fig. 5.35). The 1H NMR spectrum of SP236-SO4-P4 does not show any decomposition of the molecule (Fig. 5.36), and FT-Raman spectroscopy of the sulfate salt shows significant differences compared to the spectrum of the free base (Figs. 5.37 and 5.38). The results of elemental analysis are given in table. 5.4 are in good agreement with the content of 1.5 water molecules, although the values of the oxygen and water contents determined by the Karl Fischer titration method come into some conflict

Element Theoretically for salts 1:1 Found Anhydrous +1.5H ₂ O +2.5H ₂ O SP236-SO4-P4 SP236-SO4-P6 WITH 49.79 47.27 45.73 47.0 45.5 n 4.58 4.91 5.12 4.6 4.6 Ν 16.60 15.76 15.24 15.7 15.2 ABOUT 19.0 22.5 24.66 - 19.0 S 6.33 6.01 5.81 6.1 5.7 H ₂ O - 5.06 5.56 (TG-IR-Fourier) 8.0 (KF)

5.5. Review of water solubility and HPLC purity of selected salts.

The water solubility and HPLC purity of each of the selected salts were determined and the results are presented in the table below:

Sample Solubility (mg/ml) pH of saturated solution Cleanliness (relative area %) SP236-CIT-P3 23.6 4.3 88.2 SP236-MLE-P3 14.2 4.2 82.2 SP236-PO4-P8 29.8 7.0 93.1 SP236-SO4-P6 8.2 3.2 77.9

Measured water solubility values range from ~8 mg/ml for sulfate salt to ~30 mg/ml for phosphate salt. It should be noted, however, that SP236-PO4-P8 is a salt with a 2:1 free base:phosphate ratio and therefore produces two free base molecules. The pH value of the saturated solution for this sample is also significantly higher than for other salts (pH ~7 versus pH 3.2-4.3). The purity of each salt was also determined by the relative percentage of the area of the main peak compared to the area of all other detected peaks, and the purity ranges from 78% for SP238-SO4-P6 to 93% for SP236-PO4-P8, enlarged HPLC chromatograms of the tested samples are shown in fig. 5.39 (SP236-FB-P1), 5.40 (SP236-CIT-P3), 5.41 (SP236-MLE-P3), 5.42 (SP236-PO4-P8) and 5.43 (SP236-SO4-P6).

6. Screening experiments with other salt-forming agents.

6.1. Benzoic acid.

Two crystallization experiments were carried out with benzoic acid. No solid was obtained using 2-propanol as solvent (SP236-BNZ-P1), but a crystalline solid was obtained in the ethyl acetate experiment (SP236-BNZ-P2, Figure 6.1). The sample still contains ~0.6% ethyl acetate, which is visible by TG-FTIR, and begins to decompose with the loss of benzoic acid at temperatures above ~200°C (Fig. 6.2). The 1H-NMR spectrum shows an additional 5 aromatic protons and indicates a salt with a 1:1 free base:BNZ ratio (Figure 6.3).

6.2. Fumaric acid.

A white precipitate formed when crystallized with fumaric acid using THF as solvent (SP236-FUM-P1), but the solid was found to be only partially crystalline by PXRD. The 2-propanol experiment yields a much more crystalline sample after keeping the reaction mixture at 25 to 30°C (Figure 6.4, SP236-FUM-P2). TG-FTIR of the latter sample showed a loss of ~2.6% of 2-propanol at ~140°C (Figure 6.5), and the Ή-NMR spectrum showed a free base:acid ratio of 1:1.35, as judged by the signal at δ 6.6 m. d.

6.3. L-Malic acid.

Crystallization with L-malic acid in 2-propanol initially gave an oily solid, which crystallized when kept at 25 to 30°C (SP236-MLA-P1, Fig. 6.7). A similar experiment in THF yielded only amorphous solids (SP236-MLA-P2). The 1H NMR spectrum of the first sample contained signals attributable to L-malic acid at 8 2.4 and 3.9, which show a free base:acid ratio of 1:1 (Fig. 6.8).

6.4. Succinic acid.

Crystallization with L-succinic acid in THF gave a partially crystalline solid which showed the presence of succinate in a 1:1 ratio with the free base by 1 HNMR (SP236-SUC-P2, Fig. 6.9 and Fig. 6.10). TG-FTIR shows a mass loss of 3.1% at 110°C due to loss of THF, decomposition begins above 150°C (Fig. 6.11). An experiment with ethanol as solvent (SP236-SUC-P1) yielded only a viscous solid, which was not further investigated.

6.5. L-Tartaric acid.

Crystallization with L-tartaric acid was carried out using ethanol (-P1) and methanol (-P2) as solvents. PXRD patterns of the resulting solids indicated that both samples were crystalline and may be structurally similar (Figure 6.12). The products of these two crystallizations contain similar mass percentages of solvent/water (i.e. ~5.3%, see Figs. 6.13 and 6.14), and their 1H-NMR spectra show a free base:LTAR ratio of 1:1 in signal at δ~ 4 ppm

- 43 044945 (see Figs. 6.15 and 6.16).

6.6. Toluenesulfonic acid.

Crystallization with toluenesulfonic acid was carried out in 2-propanol (-P1) and THF (-P2). PXRD showed that the resulting solid forms were similar, but the THF experiment produced a much more crystalline sample (Figure 6.17). Unfortunately, the yields of these crystallizations were very low, and only a small amount of fine, highly electrifying solid was obtained. The 1H-NMR spectrum of sample -P2 was recorded, which showed a free base:TOS ratio of 1:1.5, with ~10 mole percent THF remaining in the sample (Fig. 6.18).

7. Preparation of HCl monosalt of compound 127.

7.1. Salt formation starting from a free base.

The mono-HCl salt exhibits much higher solubility in ethanol than the ZHC1 salt (which is excluded from the scope of the present invention).

Accordingly, the monosalt yields are lower. To increase the yield, it can be used as a solvent for the crystallization of an ethanol-water mixture.

To obtain the mono-HCl salt from the free base, 1.4 g (3.4 mmol) of compound 127 in the free base form was dissolved in 86 ml of ethanol and heated to 50°C. 0.61 g (1.05 eq.) of 32% HCl was added dropwise, and the solution was cooled to 0-5°C for 2 hours. The resulting suspension was filtered and washed with 10 ml of 2-propanol. The resulting wet product was dried for at least 10 hours in vacuum (<100 mbar) at 45°C.

Yield is 0.51 g (34% of theoretical yield) as a white solid.

7.2. Salt formation starting from the GNS1 salt of compound 127.

Since mono-HCl salt has reduced solubility in water, it precipitates at pH>5.

To obtain mono-HCl salt from 3HC1 salt, 5 g (9.7 mmol) of compound 127 in the form of 3HC1 salt was dissolved in 50 ml of water at 20-25°C. Then the pH was adjusted to pH 5-6 with a 30% NaOH solution and the suspension was stirred for 10 minutes. The suspension was filtered and washed with 10 ml of 2-propanol. The resulting wet product was dried for at least 10 hours in vacuum (<100 mbar) at 45°C.

Yield 3.7 g (85% of theoretical yield) as a white solid.

The product, which is a monosol, was characterized by standard titrimetric determination of Cl content ^- according to the applicant’s internal procedure INS005324IPV-DE03v.2:

no/f / t V f 0.03545 Cl % (m/m) = — —е--V - volume of AgNO3 0.01M;

f - AgNO ₃ standard 0.01M 1.004;

E - initial weight [g].__________

Sample E V С1% average Sample 1-1 31.34 mg 6.9877 ml 7.94% m/m C1 7.94% m/m Sample 1-2 30.21 mg 6.7354 ml 7.94% m/m C1 Sample 2-1 31.98 mg 7.1941 ml 8.01% m/m C1 7.99% m/m Sample 2-2 29.55 mg 6.6148 ml 7.97% m/m C1

The theoretically calculated value is 7.97%, which confirms the formation of a monosalt. Elemental analysis

Sample From 1%1 N [%] N [%] Prot[%] Theoretically calculated for mono-salt 56.69 4.98 18.89 Sample 1-1 55.47 5.589 18.36 0.000 Sample 1-2 55.71 5.684 18.40 0.000 average 55.59 5.636 18.38 0.000 Standard deviation, abs. 0.17 0.068 0.03 0.000 Standard deviation, rel. G%1 0.3 1.199 0.16 0.000 Delta [%] 0.23 0.096 0.04 0.000

Crystallization is observed at pH 5-5.5.

The delta most likely results from the presence of residual water. In fig. 7.1 shows confirmation of monosalt formation by HPLC. FIG. Figure 7.2 shows confirmation of the formation of a monosalt by DSC. II. Study of Polymorphs of Selected Salts of Compound 127 The following samples are listed under identification codes in the form PP566-XYZ-Pw, where XYZ denotes the salt/co-crystal former (i.e. type of acid), being either SO4 for the sulfate salt or PO4 for the phosphate salt, and Pw denotes the specific sample/experiment (w = 1, 2, ... n).

Polymorphs are numbered PMx, i.e. PM1, PM2, PM3...etc.

1. Polymorphs of sulfuric acid salts with compound 127.

The example below describes the study of various sulfuric acid salt polymorphs of compound 127 and the determination of the stable form (or hydrate) of the sulfate salt of compound 127 in the solid state. All polymorphs with sulfuric acid studied in this text represent

- 44 044945 is a 1:1 salt of compound 127.

1.1. Characteristics of the parent compound (PP566-SO4-P1).

Powder X-ray diffraction.

The PXRD pattern of the compound PP566-SO4-P1 was recorded in reflection mode (not shown) and showed that the sample was amorphous in nature.

TG-FTIR analysis.

TG-FTIR analysis showed that the amorphous sulfate salt PP566-SO4-P1 contains about 5 wt.% and begins to decompose at a temperature of about 160°C (not shown).

DSC analysis.

For PP566-SO4-P1, the first small endothermic peak was observed at 53°C, with AH = 4.7 J/g. At 78°C a sharp exothermic peak with AH = 52 J/g was observed, most likely crystallization. An additional small thermal peak was observed at 59°C. This signal apparently corresponds to the glass transition transition of the amorphous fraction, which, however, has not been conclusively confirmed. At 164°C, a broad endothermic peak is observed, possibly due to the melting of a new crystalline phase along with the decomposition of the compound.

1H-NMR Spectroscopy.

The chemical structure of the compound PP566-SO4-P1 was confirmed by 1H-NMR. The spectrum showed a broad peak centered at 10 ppm, attributed to hydrogen bonding agents or possibly an incompletely deprotonated acid (most likely HSO ₄ ) (spectrum not shown).

Chipboard research.

The behavior of the connection was analyzed by the DSP method. The compound absorbs water very quickly at 50% RH (about 4.5 wt%) and reaches a plateau, indicating the formation of a crystalline hydrate. At 0% RH, the sample loses about 5 wt% of its initial weight (9% of its weight at 50% RH) but does not reach a plateau, confirming that some water may still be present in the compound and the substance may eventually reach anhydrous condition. However, this hypothetical state is extremely unstable, since already at 5% RH it begins to absorb water again, gaining more than 10 wt.% at 55% RH, when it undergoes a sharp drop of about 3 wt.%, speaking of humidity-induced recrystallization, in during which a less hydrated structure is formed. This process continues until about 65% RH, when the compound reaches a minimum, then the substance slowly gains water to 80% RH (about 2 wt%); however, when the critical RH value is reached, after a few minutes the sample gains more than 13 wt.% and reaches a plateau that appears very stable even if the relative humidity is lowered to 50% RH, and this indicates the formation of a more highly hydrated compound that is stable at humidity above 50% (not shown).

A PXRD experiment was carried out after the EAF cycle, the results of which confirmed the crystallinity of the substance (diagram not shown in this text).

1.2. Study of polymorphs.

The polymorphism spectrum of the compound PP566-SO4-P1 was examined by suspending the compound in various solvents and solvent mixtures to study a wide range of physical states and water activities. At least 6 crystal forms have been identified (PM1 to PM6), but the existence of more can be assumed. The resulting solid forms were tested under vacuum drying conditions at 45°C. An overview of the results is given in the table below:

- 45 044945

Sample Solvent/Conditions Result PP566-SO4-P1 Initial connection amorphous PP566-SO4-P2 MeCN PM1 PP566-SO4-P3 dioxane amorphous PP566-SO4-P4 DXM PM1 PP566-SO4-P5 EtOH PM2 PP566-SO4-P5-DRY vacuum drying 45°C/30 mbar PM2 PP566-SO4-P6 EtOAc RMZ PP566-SO4-P6-DRY vacuum drying 45°C/30 mbar RMZ PP566-SO4-P7 heptane amorphous PP566-SO4-P8 MeOH PM4 PP566-SO4-P9 i-PrOH PM1 PP566-SO4-P9-DRY vacuum drying 45°C/30 mbar PM1 PP566-SO4-P10 ΤΓΦ PM5 PP566-SO4-P10-DRY vacuum drying 45°C/30 mbar PM5 PP566-SO4-P11 water PM6 PP566-SO4-P12 acetone PM1 PP566-SO4-P13 MeOH:water 3:1 a _w =0.6 PM4 PP566-SO4-P14 MeOH:water 8:2 a _w =0.4 PM4 PP566-SO4-P15 MeOH:water 9:1 a _w =0.3 PM4 PP566-SO4-P16 MeOH:water 95:5 a _w =0.2 PM4 PP566-SO4-P16-DRY vacuum drying 45°C/30 mbar PM4 PP566-SO4-P17 acetone:water 8:2 a _w =0.8 PM6 PP566-SO4-P17-DRY vacuum drying 45°C/30 mbar amorphous PP566-SO4-P18 acetone:water 9:1 a _w =0.7 PM1 PP566-SO4-P19 acetone:water 95:5 a _w =0.5 PM1 PP566-SO4-P1 after chipboard Water vapor - higher/lower PM6 PP566-SO4-P20 EUN:water 4:1, 50°C a _w =0.6 PM1 PP566-SO4-P21 EUN:water 3:1, 50°C a _w =0.7 PM1 PP566-SO4-P22 EUN:water 4:1, 5°C a _w =0.6 PM1 PP566-SO4-P23 EUN:water 3:1, 5°C a _w =0.7 PM1

An overview of the PXRD data of the resulting forms PM1 - PM6 is shown in FIG. 8.1.

1.3. Polymorphic form of PM1.

The PM1 polymorph was prepared from MeCN, i-PrOH, DCM, acetone, 95:5 acetone:water, and 9:1 acetone:water. PXRD contains broad peaks, indicating low crystallinity (Fig. 8.2), but ¹ H-NMR shows that the chemical structure is maintained (Fig. 8.3). TG-FTIR for sample PP566-SO4-P2 shows a water loss of 2.5 wt.%, which begins at about 50°C and up to 150°C, indicating a hemihydrate (Fig. 8.4). The fact that the PM1 form was obtained from the sample in a 9:1 acetone-water mixture confirms that PM1 is stable up to a water activity of 0.7. Unexpectedly, the PM1 form was obtained in experiments with anhydrous solvents. In these cases, the water is most likely taken from the parent compound, which contains about 5% water, as can be seen from the corresponding TG-FTIR data (results not presented in this text). The PM1 form is resistant to vacuum drying, and crystallinity was maintained even after overnight drying at 45°C and p<30 mbar (experiment PP566-SO4-P9-DRY). This form was also reproducibly and independently obtained in an independent experiment. This form was further studied by DSP, TG-FT-IR, DSC and NMR.

EAF was carried out in the form of two cycles of humidity changes (Fig. 8.5). As with the PM1 form (as an amorphous starting material, results not shown in this text), this material exhibits extremely complex behavior in the thermogram. A few wt.% were lost when the humidity dropped to 0% RH, and the thermogram reached a minimum, when the RH increased, the sample returned to close to the original weight and slowly gained water (2-3 wt.%), however, when 80 was reached % RH, the substance quickly adsorbs about 15 wt.% water without reaching a plateau; the increase would probably continue if the substance was stored for a longer period at approximately 95% RH. The hydration level appears stable when the humidity returns to 50% and then around 0% suddenly drops 18 wt%. During the second cycle, mass gain occurs faster and is similar to the same process in the amorphous phase (results not presented in this text), an unexpected sharp decrease indicates recrystallization. Then the substance shows a sharp increase in mass up to almost 20 wt.%, then stable until the end of the cycle at 50 wt.%. This behavior provides some insight into the mechanism of hydrate formation and confirms that when a salt reaches a high level of hydration and is then subjected to drying conditions, the crystal lattice breaks down and enters an amorphous phase; this is confirmed by the behavior of the PM6 form (see below). The PM1 form also appears stable when suspended in 4:1 and 3:1 EtOH:water mixtures at 5 and 50°C.

In conclusion, the PM1 form remains stable under strictly controlled conditions

- 46 044945 humidity (about 50%, and in any case below 70%RH), which was confirmed by keeping 10 mg

PM1 at 53%RH for 10 days, followed by PXRD analysis (experiment PP566-SO4-P24). The polymorphic form PM1 has been preserved.

Theoretical and experimental hydrates_______________________________________________

Degree of hydration Water content (%) Hemihydrate 1.8 Monohydrate 3.7 found 2.5

1.4. Polymorphic form of PM2.

The PM2 polymorph is highly crystalline and was prepared only from EtOH. ¹ NMR and TG-FTIR correspond to mono-EtOH solvate (results not presented in this text). Interestingly, only traces of water were detected in TG-FTIR analysis, indicating that EtOH (about 8.3 wt%) replaced water in the crystal lattice and promoted the formation of a highly ordered system. This is consistent with a dramatic loss of solvent at temperatures between 120 and 150°C, well above the boiling point of ethanol. This form is also resistant to vacuum drying and the PXRD remains unchanged after overnight drying at 30 mbar and 45°C, as confirmed by the PP566-SO4-P5-DRY experiment.

1.5. Polymorphic form of PM3.

The polymorphic form PM3 was obtained from a suspension in EtOAc and turned out to be weakly crystalline. Form PM3 shares several features in terms of linewidth with form PM1, but the positions of the peaks in the PXRD diagram are significantly different (results not shown in this text). 1H-NMR confirms the chemical structure of this compound (results not shown in this text), and TG-FTIR analysis confirms the solvated nature of this form, showing the release of EtOAc at temperatures up to 150°C (results not shown in this text). This form is also resistant to vacuum drying and the PXRD remains unchanged after overnight drying at 30 mbar and 45°C, as confirmed by the PP566-SO4-P6-DRY experiment.

1.6. Polymorphic form of PM4.

The PM4 polymorph is a highly crystalline form that was obtained from a MeOH suspension experiment and from several MeOH:water mixtures. The solvated nature of this form is confirmed by the results of 1H-NMR and TG-FT-IR (results not presented in this text). Although some water is present, most of it is lost at about 100°C and therefore a mixed hydrate/solvate is unlikely. On the other hand, the release of MeOH begins at a temperature of about 110°C, more than 35°C above the boiling point of the solvent, and ends at 170°C, indicating that MeOH is tightly bound in the crystal lattice. The PM4 form is stable up to a water activity of at least 0.6, as shown in experiments PP566-SO4-P13 to P16.

1.7. Polymorphic form of PM5.

The PM5 polymorph was obtained from an equilibrium suspension in THF and shows a good degree of crystallinity with sharp reflections (results not shown in this text). This compound is a THF solvate with a minor amount of water, which has not been quantified. 1H-NMR analysis showed that the chemical structure of this salt compound is preserved, and THF is also visible at 1.76 ppm, but a signal at 3.63 ppm. overlaps with other signals (Fig. 9.16). The amount of THF was estimated approximately by TG-FTIR as a signal overlapping with water and its content was less than 3.9 wt%. The solvated nature of the compound was confirmed by the fact that THF was observed in the thermogram at temperatures up to 160°C, indicating that it was tightly bound to the crystal lattice (Fig. 9.17). This can be confirmed by the fact that the compound is also stable to vacuum drying and the PXRD pattern remains unchanged after overnight drying at 30 mbar and 45°C, as confirmed by the PP566-SO4-P10-DRY experiment.

1.8. Polymorphic form of PM6.

The PM6 polymorph is the most hydrated form obtained in the experiments described and was obtained from an equilibrium suspension in water. This form exhibits a high degree of crystallinity (results not presented in this text) and appears stable over a reasonable period of time even when the RH is reduced to 50%. Similar to other polymorphic forms studied in this application, the chemical structure of the compound remained unchanged (1H-NMR, results not shown in this text). The water contained in the crystal lattice is 19.6 wt.%, which is close to the hexahydrate, and is released at a temperature of approximately 150°C (results not presented in this text), which corresponds to the value obtained by the EAF method (about 19 wt.%) . This form undergoes transformation to an amorphous phase when vacuum dried overnight, as confirmed by the PP566-SO4P17-DRY experiment.

2. Polymorphs of phosphoric acid salts with compound 127.

- 47 044945

The experiment below describes the study of various phosphate salt polymorphs of compound 127 and the determination of the stable phosphate salt form (or hydrate) of the compound

127 in solid state. The phosphoric acid polymorphs PM1 and PM3-PM11 studied in this application are 2:1 salts of compound 127. The phosphoric acid polymorph PM2 studied in this application is a 1:1 salt of compound 127.

2.1. Characteristics of the starting compound (PP566-PO4-P1).

Powder X-ray diffraction.

A PXRD pattern of PP566-PO4-P1 was recorded in reflection mode and confirmed that the sample was partially crystalline or mesomorphic (results not shown in this text).

TG-FTIR analysis.

TG-FTIR analysis showed that the phosphate complex contains about 1.3 wt.% isopropanol. PP566-PO4-P1 begins to decompose at a temperature of about 120°C. (the results are not presented in this text).

DSC analysis.

PP566-PO4-P1 exhibits complex thermal behavior. The glass transition temperature is observed at about 47°C, with a heat absorption change of about 0.8 J/g-°C, followed by an endothermic peak at about 57°C. The sample is partially crystalline or composed of mesomorphic (glassy liquid crystal) material (results not presented in this text).

¹ H-NMR spectroscopy.

The chemical structure of the compound PP566-PO4-P1 was confirmed by 1H-NMR. A small amount of isopropanol was observed in the phosphate spectrum, consistent with TG-FTIR analysis. The spectrum contains a broad signal centered at 5.7 ppm, attributed to hydrogen bond formers or, possibly, to incompletely deprotonated acids (H ₂ PO ₄ ^- , HPO ₄ ^2- ).

Chipboard analysis.

The behavior of the substance was studied using the DSP method. The PP566-PO4-P1 compound forms several hydrates. The sample gains water for a few minutes and then plateaus at 50% RH, followed by the formation of a possibly anhydrous phase at 0% RH that is very sensitive to water (begins to gain mass at 5% RH). Finally a plateau is observed at 95% RH, but this most hydrated state is unstable at lower RH values and loses water, entering a new plateau at 50% RH (but not the same as the original plateau observed at the beginning of the experiment). The final water content at 50% RH is about 10%.

PXRD analysis was carried out after the EAF cycle and showed the presence of the hydrate form of PM5 (results not presented in this text).

Elemental analysis.

The parent compound was analyzed for CHNF and phosphorus content was determined by ICP-OES. The stoichiometry was close to the ratio of compound 127:PO ₄ = 2:1.

Summary of elemental analysis results___________________________________________

Complex WITH (%) N(%) N (%) F (%) R (%) 1:1 (theoretically) 50.1 4.2 16.7 3.6 6.1 2:1 (theoretically) 55.1 4.6 18.3 3.9 3.4 Found 54.0 5.0 18.0 4.0 3.4

2.2. Study of polymorphs.

The polymorphism spectrum of the compound PP566-PO4-P1 was studied by suspending the substance in various solvents and solvent mixtures to study a wide range of physical states and water activity. At least 11 crystal forms have been identified (PM1 to PM11), but the existence of more can be assumed. An overview of the results is given in the table below:

Sample Solvent/Conditions Result PP566-PO4-R1 Initial connection Mesomorphic substance PP566-PO4-P2 MeCN mixture of PM2 + unknown peaks PP566-PO4-RZ dioxane amorphous PP566-PO4-P4 DXM new phase PM1

- 48 044945

PP566-PO4-P4-DRY Vacuum drying 45°C/30 mbar new phase PM9 PP566-PO4-P5 EtOH new phase PM2 PP566-PO4-P5-DRY Vacuum drying 45°C/30 mbar new phase PM2 PP566-PO4-P6 EtOAc new phase PM2 PP566-PO4-P7 heptane Mesomorphic substance PP566-PO4-P8 MeOH new phase of RMZ PP566-PO4-P8-DRY Vacuum drying 45°C/30 mbar new phase of RMZ PP566-PO4-P9 iso-PgOH new phase PM2 PP566-PO4-P10 THF new phase PM4 PP566-PO4-R11 water new phase PM5 PP566-PO4-R12 acetone new phase PM2 PP566-PO4-R13 MeOH:water3:1 a _w =0.6 new phase PM6 PP566-PO4-P13-DRY Vacuum drying 45°C/30 mbar new phase PM10 PP566-PO4-R14 MeOH:water 8:2 a _w =0.4 new phase PM6 PP566-PO4-R15 MeOH:water 9:1 a _w =0.3 new phase PM7 PP566-PO4-P15-DRY Vacuum drying 45°C/30 mbar new phase PM11 PP566-PO4-R16 MeOH:water 95:5 a _w =0.2 mixture of PM7 + PMZ PP566-PO4-R17 acetone:water 8:2 a _w =0.8 mixture of PM8 + PM5 PP566-PO4-R18 acetone:water 9:1 a _w =0.7 mixture of PM8 + PM5 PP566-PO4-R19 acetone:water 95:5 a _w =0.5 new phase PM8 PP566-PO4-P19-DRY Vacuum drying 45°C/30 mbar new phase PM8 PP566-PO4-R1 after chipboard Water vapor - lower/higher new phase PM5 PP566-PO4-P20 EUN:water 8:2, 50°C a _w =0.6 amorphous PP566-PO4-R21 EUN:water 3:1, 50°C a _w =0.7 amorphous PP566-PO4-P22 EUN:water 8:2, 5°C a _w =0.6 new phase PM2 PP566-PO4-P23 EUN:water 3:1, 5°C a _w =0.7 amorphous PP566-PO4-P24 acetone new phase PM2

An overview of the PXRD data of the resulting forms PM1 - PM11 is shown in FIG. 9.1.

2.3. Polymorphic form of PM1.

The PM1 polymorph was obtained from DCM and showed moderate crystallinity (results not shown in this text), 1H-NMR shows that the chemical structure is retained (results not shown in this text). Signal in 1H-NMR spectrum at 5.57 ppm. indicates the presence of DXM. TG-FTIR analysis showed a loss of DCM of 14.2 wt.%, which begins at approximately 30°C, likely physisorbed DCM; however, mass loss continued until 150°C, indicating a solvate (results not shown in this text). This form becomes less crystalline when vacuum dried for 12 hours at 45°C.

2.4. Polymorphic form of PM2.

The PM2 polymorph is highly crystalline and was prepared only from EtOH, as well as from several other solvents, suggesting that it may represent an anhydrous phase (Figs. 9.2 and 9.6). even though the 1H-NMR and TG-FT-IR data for experiment PP566PO4-P5 suggest that this may be a hemi-EtOH solvate (Figs. 9.3 and 9.4, respectively), since ethanol is released at temperatures up to 170°C, TG-FTIR analysis was carried out on another sample where the PM2 form was obtained (experiment PP566-PO4-P12, suspension from acetone), and the thermogram showed no release of solvent, as well as no loss of mass up to the decomposition temperature about 150°C. This indicates that the structure of a given compound can accept different solvents in the crystal lattice and maintain the same solid-state structure. This crystalline form is resistant to vacuum drying overnight at 45°C. DSP analysis was also carried out for this form. The substance gains approximately 0.7 wt.% at 95% RH, reaching a plateau, and returns to a slightly lower weight after completion of the cycle (Fig. 9.7). This substance is only slightly hygroscopic.

The substance was also subjected to elemental analysis and the results are consistent with a 1:1 salt: Elemental analysis results for PP566-PO4-P24

Complex With (%) N(%) N (%) F (%) R (%) 1:1 (theoretically) 50.1 4.2 16.7 3.6 6.1 2:1 (theoretically) 55.1 4.6 18.3 3.9 3.4 Found 49.1 4.6 16.1 3.6 5.9

2.5. Polymorphic form of PM3.

The PM3 polymorph is a highly crystalline form that was obtained from an experiment in a MeOH suspension and in a mixture with the PM7 form (see below) in a MeOH:water = 95:5 mixture. The solvated nature of this form is confirmed by the results of 1H-NMR and TG-FT-IR (results not presented in this text). The release of MeOH begins at a temperature of about 90°C, more than 35°C above the boiling point of the solvent, and ends at approximately

- 49 044945

120°C at a sharp stage, indicating that MeOH is tightly bound in the crystal lattice. It is interesting to note that the formation of this phase has a very narrow range of water activity and produces a mixed solvate/hydrate form (PM7 form) somewhat below _aw = 0.2, and no trace of this form is observed at _aw = 0.3.

2.6. Polymorphic form of PM4.

The PM4 polymorph was obtained from an equilibrium suspension in THF and showed low crystallinity with broad reflectances (results not shown in this text). This compound is a THF solvate. The 1H-NMR spectrum shows that the chemical structure of the active pharmaceutical ingredient remains unchanged and THF is also visible at 1.76 ppm. and at 3.63 ppm (the results are not presented in this text). The amount of THF can be estimated by TG-FTIR analysis and is about 2.8 wt%. The solvate nature of this phase is confirmed by the fact that THF can be observed in the thermogram up to 180°C along with decomposition, which confirms that it is tightly bound in the crystal lattice (results not presented in this text).

2.7. Polymorphic form of PM5.

The PM5 polymorph is the most hydrated form obtained in the experiments described. It was obtained from an equilibrium suspension in water by storing the substance at 95% RH (see ADI of the original substance). This form exhibits a high degree of crystallinity (results not presented in this text). Similar to other forms obtained, the chemical structure of the active pharmaceutical ingredient remained unchanged (1H-NMR, results not shown in this text). The water contained in the crystal lattice cannot be quantified by the TG-FTIR method, since insufficient material was obtained in the PP566-PO4-P11 experiment, but this parameter can be deduced from the results of DSP (results not presented in this text), where it is about 11 wt.%.

2.8. Polymorphic form of PM6.

The polymorphic form of PM6 is a hydrate with a lower water content than in PM5, obtained from a mixture of MeOH:water = 3:1 and 4:1. This form shows a high degree of crystallinity (results not presented in this text). As with other forms obtained, the chemical structure of the compound was not modified by 1H-NMR; the results are not presented in this text). Water is released during the TG-FTIR experiment in two stages (results not presented in this text). No traces of methanol were observed in either TG-FTIR analysis or NMR spectrum. This form is formed at moderate water activity (about 0.4 - 0.6).

2.9. Polymorphic form of PM7.

The PM7 polymorph is a mixed hydrate/solvate obtained from a 9:1 methanol:water mixture (a _w = 0.3) and is a purely crystalline phase (results not presented in this text) in which both methanol and water are bound to the crystal lattice . This form is formed at relatively low water activity. TG-FTIR analysis data show the release of both solvents in two separate stages (results not presented in this text).

2.10. Polymorphic form of PM8.

The PM8 polymorph appears to be a mixed solvate/hydrate and is formed at water activities between 0.5 and 0.7 (results not presented in this text). Interestingly, no acetone solvate is observed when the compound is stirred in pure acetone at room temperature (experiment PP566-PO4-P12).

2.11. Polymorphic form of PM9.

The polymorphic form PM9 was obtained after exposure of the substance PP566-PO4-P4 at 30 mbar and 45°C for approximately 12 hours. This form appears to be a desolvated form from PM1, the DCM solvate. This form is purely crystalline and contains broad peaks in the diffraction pattern (results not shown in this text).

2.12. Polymorphic form of PM10.

The polymorphic form PM10 was obtained after keeping the substance PP566-PO4-P13 at 30 mbar and 45°C for approximately 12 hours (results not presented in this text). Interestingly, this form has several features in common with the hydrate form PM5 rather than PM6 (results not presented in this text), but is less crystalline and has a slight shift towards higher 2-Theta values, indicating somewhat smaller unit cells (this leading to the possibility that it may be a lower hydrate). This conclusion is confirmed by TG-FTIR data (the results are not presented in this text).

2.13. Polymorphic form of PM11.

The polymorphic form PM11 was obtained after keeping the substance PP566-PO4-P15 at 30 mbar and 45°C for approximately 12 hours (results not presented in this text). This form is purely crystalline, and TG-FTIR analysis showed the presence of water (results not presented in this text).

III. Pharmacological tests.

The pharmacological tests described below were performed with selected compounds in the corresponding free base and/or HCl ternary salt form. Since the compound having formula (I) mainly constitutes the active substance, comparable activity results were expected for the corresponding salts of the present invention. The experimental results described below confirm that the new salts (including their solvates, hydrates and polymorphs, etc.) of the present invention retain their effect as a ferroportin inhibitor and may also enhance the inhibitory effect on ferroportin and/or improve the pharmacokinetic profile of the compounds and/or improve the physicochemical properties of the compounds, facilitating their introduction into finished pharmaceutical forms, and/or have the advantage of allowing them to be isolated in crystalline form, which improves the physicochemical properties of the compounds, facilitating the introduction of these compounds into finished pharmaceutical forms or making them easier to handle or improving their stability.

Specifically, in the tests described below, compounds were tested in triple salt form (ZNS1) and/or free base form, as follows:________________

Compound Base ZNS1 salt 1 + + 2 + - 4 + - 40 + + 94 + + 118 - + 126 + + 127 + + 193 + - 206 + - 233 + - 234 + - 208 + - 225 + +

1. Hepcidin internalization test (J774).

This cell-based assay quantifies the binding of hepcidin to ferroportin (Fpn) by microscopically detecting the internalization of fluorescently labeled hepcidin in J774 cells. J774 is a murine macrophage cell line that has been shown to express endogenously Fpn when incubated with iron (Knutson et al., 2005). Binding of hepcidin to Fpn triggers the internalization and degradation of hepcidin and Fpn. However, the TMR (6-carboxytetramethylrhodamine) fluorophore attached to hepcidin remains associated with the cell after degradation of the hepcidin peptide backbone. Therefore, microscopic detection of fluorescence from the cell-associated TMR serves as a measure of hepcidin binding to Fpn and internalization of hepcidin and Fpn. If the binding of the TMR-hepcidin complex to Fpn is prevented, cellular TMR fluorescence remains low (Diirrenberger et al., 2013). The effect of low molecular weight Fpn inhibitory compounds in this test was assessed in vitro as described below.

J774 cells, which were collected from cultures at approximately 80% confluency, were seeded at a concentration of 8x10 ⁵ cells/ml in complete medium (DMEM, 10% FBS, 1% penicillin-streptomycin) containing 200 μM Fe(III)NTA (nitrilotriacetic acid) , 100 μl per well in 96-well MicroClear plates (Greiner; Cat. 655090), and grown at 37°C with 5% CO ₂ . After overnight incubation, cells were washed 4 times with prewarmed DMEM without phenol red, 30 μl/well of DMEM without phenol red was added after the final wash, and 10 μl/well of serial dilutions of test compounds were added in triplicate. J774 cells were pre-incubated with test compounds at 37°C with 5% CO ₂ for 15 min, after which TMR-hepcidin was added to a final concentration of 25 nM. Cells were incubated in a total volume of 50 μl at 37°C with 5% _CO2 for 2 hours, then Hoechst 33342 dye was added at a final concentration of 0.5 μg/ml to stain the nuclei, and then incubated for 10 min at 37°C with 5% CO ₂ Cells were washed 3 times with phosphate-buffered saline (PBS) and fixed in 100 μl of 4% paraformaldehyde in PBS for 15 min at room temperature. After removing the paraformaldehyde solution, the cells were washed 3 times with phosphate-buffered saline, leaving 100 μl per well, and the plates were covered with film. Fluorescence patterns of TMR (530-550 nm excitation/575-625 nm emission/400 ms exposure time) and Hoechst 33342 (360-370 nm excitation/420-460 nm emission/10 ms exposure time) were recorded using a ScanR spectrophotometer (Olympus) for tablet reading, equipped with a 20x aperture lens. Four images per well were recorded, covering approximately 1500 cells per well. The resulting images were analyzed using ScanR image analysis software. Image analysis included detection of nuclei (Hoechst 33342 fluorescence), identification of cell-associated regions, application of a virtual channel and determination of a threshold for rolling ball background reduction, followed by an average sum algorithm to measure the TMR of cell-associated fluorescence as a quantitative evaluation parameter internalized TMR-hepcidin. The values of 1С ₅ о were calculated from the obtained data of the average sum

-51 044945 we use statistical processing of 1od(inhibitor) vs. curves. answer in Prism 5 (GraphPad Software Inc., version 5.02). For each data set, the fit of the log(inhibitor) vs. model was compared. response (three parameters) and model fit log(inhibitor) vs. answer - Variable slope (four parameters), and used IC ₅₀ data for the preferred model. IC ₅₀ data for Fpn inhibitors tested in the hepcidin internalization test are listed in Table. 1. The IC ₅₀ value for unlabeled hepcidin in this test is 0.015 ± 0.011 µM.

In table 1 shows the average IC ₅₀ data for Fpn inhibitors tested in the hepcidin internalization assay for several measurements.

Table 1

Conn. J774 IC50 (µM) 1 0.012 2 0.035 4 0.155 40 0.049 94 0.012 118 0.103 126 0.096 127 0.009 193 0.287 206 0.18 208 0.012 233 16

2. Biophysical test for the binding of ferroportin to hepcidin.

This biophysical test was designed to more directly confirm the inhibition of hepcidin binding to ferroportin (Fpn). Incubation of TMR-hepcidin with purified human Fpn isolated from yeast cells Pichia pastoris expressing human Fpn with the Terminal Affinity Marker FLAG (Bonaccorsi di Patti, 2014) results in an increase in the fluorescent polarization (FP) of the TMR-hepcidin ligand. Small molecule Fpn inhibitors were tested for inhibition of TMR-hepcidin binding to Fpn, detected by a dose-dependent reduction in TMR AF signal, as detailed below.

A mixture of 1.3 μM human Fpn and 30 nM TMR-hepcidin in FP assay buffer containing 50 mM Tris-HCl pH 7.3, 200 mM NaCl, 0.02% DDM, 0.1% BSA was distributed into a 384-well black plate with small round-bottomed wells (Corning, Cat. 3677) 16 µl per well. 8 μl of serial dilutions of test compounds were added in duplicate, with final concentrations of Fpn and TMR-hepcidin of 1 μM and 20 nM, respectively. The plates were incubated for 90 min at room temperature, and parallel (S) and perpendicular (P) fluorescence was measured using a Synergy H1 fluorescence imaging plate reader (BioTek). FP values were calculated as mP using the formula below

Fnapann - Rperpend tr = ------------ xYO

Rparall + Rperpend

IC50 values were determined from calculated mP values as described for the hepcidin internalization test and tabulated. 2. The IC50 value for unlabeled hepcidin in this test is 0.37 ± 0.067 µM.

In table 2 shows the multi-measurement average (AVE) IC ₅₀ data for the Fpn inhibitors tested in the ferroportin-hepcidin binding biophysical assay.

table 2

Conn. FP IC50 (µM) 1 0.016 2 0.017 40 0.068 94 0.044 118 0.25 126 0.12 127 0.023 193 0.074 206 0.036 208 0.019 233 6.776

3. Inhibition of iron transport by ferroportin in an iron-induced response assay.

Intracellular iron concentrations in this test are indirectly measured by monitoring

- 52 044945 activity of a beta-lactamase (BLA) reporter gene hybridized to the human ferritin promoter and the associated iron control factor regulatory element (IRE) contained in the 5' untranslated region of ferritin mRNA. Expression of ferroportin (Fpn) in such a cell line results in iron efflux and decreased iron levels, which is reflected by decreased reporter gene activity. On the other hand, inhibition of Fpn-mediated iron efflux leads to an increase in cellular iron concentration, which is detected as increased reporter gene activity. Small molecule Fpn inhibitor compounds were tested for dose-dependent effects in an in vitro iron response assay as described below.

HEK-293 cell line #354 was generated by stably integrating (i) a human Fpn-GFP chimeric construct inserted into a derivative of the doxycycline-inducible pTRE-Tight-BI plasmid (Clontech, Cat. 631068), and (ii) a human ferritin-promoter construct. BLA reporter gene in the HEK-293 derivative Tet-ON Advanced cell line (Clontech). To create the ferritin-BLA reporter gene construct, a 1.4 kb fragment of the human ferritin H promoter was amplified by PCR from human genomic DNA (forward primer 5'CAGGTTTGTGAGCATCCTGAA-3'; reverse primer 5'-GGCGGCGACTAAGGAGAGG-3') and introduced upstream of the BLA gene present in the pcDNA™6.2/cGeneBLAzer™-DEST plasmid (Invitrogen, Cat. 12578-043), thereby replacing the original CMV promoter, and placed the IRE that regulates the translation of the ferritin gene approximately 170 bp upstream of the start codon of the reporter gene. #354 cells were collected from approximately 80% confluent cultures and seeded at ^1.8x105 cells/ml in DMEM/F12 GlutaMAX™ (Invitrogen, Cat. 31331-028) containing 10% FBS (Clontech, Cat. 631106 ), 1% penicillin-streptomycin, 200 μg/ml Hygromycin B (Invitrogen, Cat. 10687ОУ), blasticidin 5 μg/ml (Invitrogen, Cat. R210-01), 4 μg/ml doxycycline (Clontech, Cat. 631311), in a volume of 50 μl per well into 384-well plates coated with poly-b-lysine (PDL) and incubated at 37°C with 5% CO ₂ . After incubation overnight, serial dilutions of test compounds (10 μl/well) were added in quadruplicate and the plates were further incubated overnight at 37°C with 5% CO ₂ . Cells were washed 3 times with Hanks' balanced salt solution (HBSS), leaving 25 μl per well. BLA activity was determined by adding 5 μl/well of GeneBlazer reagent CCF4-AM (Invitrogen, Cat. K1085) to the cells. After incubating the plates in the dark at 18°C for 60 min, blue and green fluorescence signals were measured on a scanning spectrophotometer for plate reading Safire2 (Tesap) with an exciting beam wavelength of 410 nm and emission at 458 nm (blue) and 522 nm (green ). The blue/green fluorescence intensity ratio was calculated as a measure of BLA activity, and the EC ₅ o value was determined from the calculated blue/green fluorescence ratio as described for the hepcidin internalization assay. EC ₅₀ data for the tested Fpn inhibitors are given in table. 3. The _EC50 value for hepcidin in this test is 0.096 ± 0.063 µM (n=37).

In table 3 - Average (AVE) EC ₅₀ values for Fpn inhibitors tested in the iron response assay are given for several measurements.

Table 3

Conn. BLAzer EC50 (µM) 1 0.93 2 1.03 4 1.259 40 1.45 94 0.53 118 2.69 126 1.26 127 0.42 193 3.64 206 3.26 208 0.50

4. Analysis of internalization and degradation of ferroportin.

HEK-293 cell line #354 (described in Example 3) was used to measure the ability of compounds to induce internalization and degradation of ferroportin (Fpn) by fluorescence-activated cell sorting (FACS). Incubation of HEK-293 #354 cells in doxycycline-containing media induced expression of the human Fpn-GFP chimeric protein on the cell surface. Data from 10 independent experiments showed that growing HEK#354 cells for 48 hours in the presence of 4 μg/ml doxycycline induced an average of 42.6% ± 6.4% Fpn-GFP-positive cells. Small molecule Fpn inhibitory compounds were tested for dose-dependent effects on Fpn-GFP mean fluorescence intensity (MFI) in the HEK-293 #354 cell line as described below.

-53 044945

HEK#354 cells were collected from cultures with approximately 80% confluency, seeded at a concentration of 0.6x106 cells/ml in DMEM/F12 GlutaMAX™ medium (Invitrogen, Cat. 31331-028) containing 10% FBS (Clontech, Cat. 631106), 1% penicillin-streptomycin (Invitrogen, Cat. 15140-122), 200 μg/ml Hygromycin B (Invitrogen, Cat. 10687-010), Blasticidin 5 μg/ml, (Invitrogen, Cat. R210-01), 4 μg/ ml doxycycline (Clontech, Cat. 631311), at 50 μl per well in 384-well plates (Greiner; Cat. 781091) and grown at 37°C with 5% CO2. After incubation overnight, 10 μl/well of serial dilutions of test compounds were added in quadruplicate and the plates were further incubated at 37°C with 5% CO2 overnight. Cells were washed once with FACS buffer (phosphate buffered saline containing 1% FBS, 2 mM EDTA and 0.05% _NaN3 ), collected in FACS buffer with 0.5 μg/ml propidium iodide (Sigma, Cat. P4864) and analyzed on a flow cytometer (CANTOtm II, BD Biosciences) equipped with a high-performance sampler. Live HEK#354 cells were separated as a propidium iodide negative population and analyzed for FpnGFP expression. The Fpn-GFP MFI was calculated for >2000 live cells for each compound dilution using FlowJo (Tree Star's, Oregon), and the potential of Fpn inhibitors to induce Fpn-GFP internalization and degradation was calculated as described for the hepcidin internalization assay. The EC ₅₀ values for the Fpn inhibitors tested in the FACS ferroportin internalization and degradation assay are shown in Table 1. 4. The average EC ₅₀ value for hepcidin in this assay is 0.004 ± 0.002 µM.

In table 4 - Average (AVE) EC ₅₀ values for Fpn inhibitors tested in the ferroportin internalization and degradation assay are given for several measurements.

Table 4

Conn. EC50 (µM) 1 0.22 2 0.63 4 1.198 40 0.81 94 0.22 118 4.908 126 0.757 127 0.081 193 3.946 193-B 1.391 206 2.072 208 0.15

5. Analysis of ubiquitination and degradation of ferroportin.

Contact of ferroportin (Fpn)-expressing cells with hepcidin is known to initiate ubiquitination and subsequent internalization and degradation of Fpn (Qiao, 2012). The ability of Fpn inhibitors to induce Fpn ubiquitination and degradation was examined by immunoprecipitation assay using the J774 mouse macrophage cell line, which expresses Fpn upon iron treatment.

J774 cells (DSMZ, Cat. ACC170) were seeded at a concentration of 0.8x106 cells/ml in 15 ml of medium (DMEM Gibco Cat. 11971-025, 10% heat-inactivated FBS Gibco Cat. 10500-064, 1% penicillin-streptomycin Gibco Cat. 15140-122) containing 200 µM Fe(III)-NTA in 10 cm Petri dishes (Greiner Cat. 664160) and incubated overnight at 37°C with 5% CO2. Cells were incubated with synthetic human hepcidin (Bachem, Cat. H-5926) or Fpn inhibitory compounds for 10 or 120 min. Cells were washed and lysed with ice-cold lysis buffer (Pierce, Life Technologies, Cat. 87787) containing 1X HALT protease inhibitor cocktail (Life technologies, Cat. 78429) and 10 mM iodoacetamide (Sigma, Cat. 16125) to stabilize ubiquitinated proteins. Immunoprecipitation was performed using Pierce Classic IP Kit (Life Technologies, Cat. 26146) according to the manufacturer's protocol. Briefly, 2 mg of protein in 1.25 ml IP lysis buffer was incubated by mixing for 1 h at 4°C with control agarose beads to preclear the lysate and reduce nonspecific signal. The unbound lysate was then incubated overnight with 12 μg (per reaction) of affinity-purified anti-Fpn antibody F308, which was raised against the GST chimeric protein of amino acids 224-308 of mouse Fpn. Immune complexes were captured by dropping Pierce Protein A/G Plus Agarose beads (Life Technologies, Cat. 20423) at a rate of 14 μl per reaction, and the resulting suspension was incubated for 1.5 h at 4°C with gentle inversion mixing. The beads were washed and the immune complexes were directly eluted with 75 μl NuPAGE LDS SDS buffer (Life Technologies, Cat. NP0007) containing DTT (Life Technologies, Cat. NP0009).

After immunoprecipitation, samples were analyzed by Western blotting using serum containing rabbit anti-mouse MTP1 antibodies (Alpha Diagnostic International, Cat. MTP11-A) and mouse monoclonal antibodies to mono- and polyubiquitinated conjugates (Enzo

- 54 044945

Lifesciences, Cat. BML-PW8810) to detect ferroportin and ubiquitin, respectively. Horseradish peroxidase-conjugated mouse monoclonal antibodies against rabbit IgG light chain (Abcam, Cat. ab99697) and anti-mouse IgG H&L (Abcam, Cat. ab6789) were used as secondary antibodies.

A series of eleven Fpn inhibitors were tested by this method and compared with hepcidin. As shown in FIG. 10 and in table. 5, treatment of cells with Fpn inhibitors leads to rapid ubiquitination within 10 min (Fig. 10, upper part) and Fpn degradation after 2 hours (Fig. 10, lower part). The extent of Fpn degradation by Fpn inhibitors was comparable to the effect of hepcidin. However, hepcidin treatment produced higher molecular weight ubiquitinated Fpn compared with Fpn inhibitor treatment, suggesting poly-ubiquitination by hepcidin, as opposed to mono-ubiquitination by Fpn inhibitor.

In table 5 is a summary of the results of Fpn inhibitors tested in the Fpn ubiquitination and degradation assay. The effect of treatment with Fpn inhibitors on Fpn degradation and Fpn ubiquitination was assessed visually by Western blotting (+ comparable to hepcidin; - no effect; +/medium effect).

Table 5

Conn. Concentration (µM) Fpn ubiquitination (10 min.) Fpn decomposition (120 min.) 1 0.12 + + 40 1.9 + + 94 0.3 + + 126 0.8 +/- + 127 0.1 + + 208 0.2 + + Hepcidin 0.15 + +

Fig. 10. The Fpn inhibitor initiates the ubiquitination and degradation of Fpn expressed in a murine macrophage cell line. J774 cells were incubated overnight with Fe(III)-NTA to initiate Fpn production. Cells were then treated with 10-fold IC ₅₀ concentrations determined in the hepcidin internalization assay (see Table 1), hepcidin (Hepcidin, 150 nM) or Fpn inhibitors: compound No. 208 (210 nM), compound No. 167 (1.5 μM), compound #127 (120 nM), compound #152 (40 nM) for 10 or 120 min before collection and immunoprecipitation with anti-Fpn antibody F308. Cells treated without the addition of active substances were harvested after 120 min (control).

Immunoblotting of the immunoprecipitates with the anti-Fpn antibody MTP1 revealed the disappearance of ferroportin 120 min after treatment with Fpn inhibitors to a degree comparable to the hepcidin-treated sample (top). Rapid ubiquitination of Fpn was observed 10 min after treatment of cells with Fpn inhibitors and hepcidin. Protein molecular weight standards are shown on the left in kDa.

6. Suppression of iron excretion by ferroportin inhibitors.

The activity of hepcidin and ferroportin inhibitor compounds in blocking iron export by ferroportin was tested in T47D cells (ECACC, Cat. 85102201) as described below.

Cells were placed in 24-well plates (Greiner, Cat. 662160) at 350,000 cells per well and incubated overnight with 100 µM ⁵⁸ Fe ( ⁵⁸ Fe(II)-sulfate, Vifor Pharma Batch No. ROR 3085) in medium containing 500 µM L-ascorbic acid (Sigma Aldrich, Cat. 795437). Cells were washed once with 500 μl of iron uptake buffer (IUB; PIPES 40 mM, Cat. P1851, glucose monohydrate 10 mM, Cat. 49158, sodium chloride 260 mM, Cat. 71379, potassium chloride 20 mM, Cat. P9541, magnesium sulfate 2 mM , Cat. 63138, Sigma Aldrich), then once with removal buffer (2 min incubation, BPDS 100 µM, Cat. 11890 and Na ₂ S ₂ O ₄₅₀₀ µM, Cat. 157953, Sigma Aldrich, at IUB), and again twice times IUB.

Serial dilutions of hepcidin (Bachem) or ferroportin inhibitors (4 µM-0.0064 µM, 5-fold dilution) were added in a total volume of 0.6 ml per well. Cells were incubated at 37°C with 5% CO2 for 20 hours. Supernatants were collected and ^58Fe content was determined using inductively coupled plasma mass spectrometry (ICP-MS, Thermo Scientific, Element 2). Pellets were separated to measure protein concentration. The results were plotted as nanograms ^{of 58} Fe in supernatant per milligram of protein in cell lysates. Compound 127 inhibited iron excretion to a similar degree to the endogenous Fpn ligand hepcidin (Fig. 11).

Fig. 11. Representative example of suppression of iron excretion by hepcidin ( _IC50 : 0.086 µM) and compound 127 (IC50: 0.080 µM).

7. Hypoferremia in intact mice.

Injection of synthetic hepcidin into intact mice (wild type) resulted in a decrease in plasma iron levels (by 40-50% relative to the control group) with a maximum effect 3-4 hours after administration (Rivera, 2005; Fig. 12A). These data suggest that injected hepcidin binds to and initiates the internalization of ferroportin (Fpn) in duodenal enterocytes and splenocytes, causing a rapid fall in plasma iron levels. Similarly, orally administered small molecule Fpn inhibitors reduced plasma iron levels in WT

C57BL/6 mice in a dose-dependent manner, with efficacy comparable to hepcidin. These data indicate that naïve mice can be used as a simple and reliable model to test the acute efficacy of Fpn inhibitors in vivo.

Female C57BL/6 mice (Janvier, France) at 9 weeks of age were fed a standard diet (Harlan Provimi Kliba 3436) and administered orally (po) compounds or an appropriate amount of vehicle in a volume of 10 ml/kg body weight. Fpn inhibitors were formulated in 0.5% methylcellulose in water or 20% cremophor EL/water and administered orally to mice at 10, 30, or 100 mg/kg body weight. After 3 h, mice were preterminally euthanized in isoflurane chambers, and blood samples were collected from the retroorbital plexus. Mice were sacrificed by cervical dislocation, and the spleen, liver, and duodenum were collected for further biomarker analysis. All experiments were carried out in accordance with a license confirmed by competent veterinary services. Plasma was separated by centrifugation of blood in gel microcontainers, and plasma iron levels were determined using the MULTIGENT Iron assay (Abbott Diagnostics, 6K95). There were eight mice in each group, and one-way ANOVA with Bonferroni correction for multiple comparisons was used to analyze statistical differences between experimental groups. The effectiveness of some Fpn inhibitors for WT C57BL/6 mice is given in table. 6.

Fig. 12. Reduction of plasma iron levels caused by hepcidin and the ferroportin inhibitor compound 94 (compound 94).

A. Kinetics of iron levels in naïve C57BL/6 mice injected with synthetic hepcidin (5 mg/kg) intraperitoneally (i.p.) for the indicated times. * - ***- shows a statistically significant decrease in plasma iron levels compared to mice injected with phosphate-buffered saline (PBS).

B. Plasma iron levels in naïve C57BL/6 mice treated with the indicated amounts of hepcidin (i.p.) or compound 94 (p.o.) for 3 h.

Table 6 - effectiveness of Fpn inhibitors tested in a model of hypoferremia in native mice.

A decrease in plasma iron levels induced by certain ferroportin inhibitors administered p.o. to native WT C57BL/6 mice at a dosage of 10, 30 and 100 mg/kg. The relative decrease in plasma iron levels 3 hours after administration was calculated by subtracting the mean plasma iron concentrations for animals treated with the Fpn inhibitor from those for animals treated with vehicle alone. The resulting difference in mean iron values between the control group and the test compound group was then divided by the mean plasma iron for the control group (which received vehicle alone) and tabulated as a percentage.

___________________________________________Table 6

Decrease in plasma iron level after 3 hours (%) Conn. Dose 10 mg/kg Dose 30 mg/kg Dose 100 mg/kg 1 0 28 51 2 9 26 50 40 10 thirty 50 94 thirty 50 80 118 8 24 49 126 7 23 62 127 17 47 54 193 13 AND 31 208 50 65 73

8. Prevention of iron absorption in anemic rats.

To evaluate the in vivo effectiveness of ferroportin (Fpn) inhibitors in blocking iron absorption, a series of Fpn inhibitors were tested in a rat model of anemia for iron absorption. Wistar rats (3-4 weeks old, n=5, Janvier Labs) were kept on a low iron diet (Provimi-Kliba, Cat. 2039) until their hemoglobin (Hb) value reached 7-8 g/dL one day before the administration of Fpn inhibitors. One hour before oral administration of 0.5 mg/kg ferrous sulfate, test compounds were orally administered as a formulation in methylcellulose or Cremophor solution. Blood samples were taken from the tail vein one hour before iron administration (-1 hour), immediately after administration of Fpn inhibitors (0 hour) and after 1 hour (1 hour), three hours (3 hours) and sometimes up to 6 hours (6 hours). h) after administration of the test compounds. Plasma iron levels were measured (Abbott Diagnostics, Cat. 6K95) and the suppression of plasma iron levels three hours after administration of test compounds was calculated as a measure of the effectiveness of Fpn inhibitors in blocking iron absorption (Table 7). As shown in FIG. 4, oral administration of the Fpn inhibitor - Illustrative Example No. 55 - at a dosage of 3 mg/kg, 10 mg/kg, or 30 mg/kg reduced plasma iron levels by 54 and 89%, respectively, after three hours. after iron administration, compared with plasma iron levels in control animals before iron administration, adjusted for background plasma iron concentrations in non-iron dosed control animals.

In table Figure 7 shows Fpn inhibitors tested in a rat model of anemia to inhibit iron absorption. Shown are the relative suppression (%) of the increase in plasma iron concentrations, adjusted to mean background plasma iron concentrations, in the control group that did not dose with iron compared with the control groups that received only vehicle before iron administration. Shown are mean values for groups (n=5) administered the indicated dosages of Fpn inhibitor. Shown are statistically significant (two-way ANOVA with Bonferroni test) differences between the test group (test compound administered) and the control group (vehicle only administered) (*** p < 0.001; ** p < 0.01, * p < 0.05).

Table 7

Suppression of the increase in iron concentration in the blood (%) after 3 hours Conn. Dose Dose Dose Dose Dose 1 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg 100 mg/kg 1 But. 2.1 42.6** 64 9*** But. 2 But. -3 29** 57*** But. 40 But. But. 32** 53*** 97*** 94 59*** 0 70*** But. But. 127 But. -8 47*** 79*** But. 208 But. 59*** 86*** But.

9. Correction of hyperferremia in mice with beta2-microglobulin deficiency.

Mutations in genes involved in regulating systemic iron stores, such as hepcidin (Hampl), cause iron overload in mice and humans. HFE, HJV, and TFR2 molecules on hepatocytes are required to signal adequate hepcidin production, and their deficiency results in pathophysiologically low hepcidin concentrations and excess iron absorption. HFE mutations are the most common cause of congenital hemochromatosis (CH) in Caucasians. HFE is a member of the MHC class of isomembrane molecule that binds to beta2microglobulin and is involved in the transcriptional regulation of hepcidin through the bone morphogenetic protein receptor (BMPR). HFE−/− mice have reduced hepcidin levels and develop hyperferremia and high liver iron content, making them a suitable animal model for studying iron overload in humans (Zhou, 1998). Beta2microglobulin-deficient mice (b2m−/−) develop hyperferremia and hemochromatosis, similar to HFE−/− animals, since beta2microglobulin is required for cell surface expression of HFE and FIFE function (Rothenberg and Voland, 1996). Due to the unavailability of FIFE-/- mice, b2m-/- mice were used as a model of iron overload. A pilot study confirmed that HFE-/- and L2m-/- mice have similar parameters related to iron metabolism.

Female and male homozygous b2m-/- mice were obtained from Jackson Laboratories (B6.129P2B2mtm1Unc/J, Stock Number: 002087) at 6 to 7 weeks of age and maintained on a standard diet (Harlan Provimi Kliba 3436) without restriction of access to food and water. . Age- and sex-matched WT C57BL/6 mice were obtained from Charles River. To study the acute effects of ferroportin (Fpn) inhibitors on iron overload, b2m-/- mice were administered test compounds or an appropriate amount of vehicle at a volume of 10 ml/kg body weight. Fpn inhibitor compounds were formulated in 0.5% methylcellulose/water or 20% cremophor EL/water and administered orally to mice at a dosage of 50 mg/kg body weight. WT control animals received vehicle only. Three hours later, mice were preterminally euthanized in isoflurane chambers and blood samples were collected from the retroorbital plexus. Mice were sacrificed by cervical dislocation, and the spleen, liver, and duodenum were collected for further biomarker analysis. All experiments were carried out in accordance with a license confirmed by competent veterinary services. Plasma was separated by centrifugation of blood in gel microcontainers (BD Biosciences), and plasma iron levels were determined using the MULTIGENT Iron assay (Abbott Diagnostics, 6K95). There were 4–9 mice in each group, and one-way ANOVA with Bonferroni correction for multiple comparisons was used to analyze statistical differences between experimental groups.

To examine the effect of the Fpn inhibitors Compound 40 and Compound 94 under conditions of iron oversaturation, b2m-/- mice or WT control animals were treated with Fpn inhibitors or vehicle for 3 h. Due to their genetic deficiency, b2m-/- mice treated vehicle alone showed significantly higher plasma iron levels compared to WT mice (Fig. 13, group mean 60 μM in A and 56 μM in B). Administration of compound 40 or compound 94 to b2m-/mice at a dosage of 50 mg/kg for 3 hours corrected the elevated plasma iron levels to values observed in control WT animals. These data demonstrate the acute efficacy of small molecule ferroportin inhibitors in a relevant disease model. Correction of plasma iron levels has been observed in additional studies, as summarized in Table 1. 8.

Fig. 13. Complete correction of the increased iron content in the blood plasma in b2m-/- mice with the introduction of ferroportin inhibitors - compound 40/methylcellulose (A.) and compound 94/cremophor EL (B.) - within 3 hours.

In table 8 - Fpn inhibitors tested in a mouse model of beta2-microglobulin deficiency to reduce elevated plasma iron levels.

Blood was collected 1 (#) or 3 (##) hours after oral administration of the indicated dosages of Fpn inhibitors to beta2-microglobulin-deficient mice, and plasma iron concentrations were determined. The relative decrease (%) in plasma iron is shown and was calculated by subtracting the mean iron values for animals treated with the Fpn inhibitor from those found for animals treated with vehicle alone. The difference in mean iron levels between the control (vehicle only) and test (test compounds) groups was then divided by the mean iron in the control group and expressed as a percentage. Values for females ($) and males (^) are reported separately because there was a marked sex difference in performance. Shown are the statistically significant differences (two-way ANOVA with Bonferroni test) between the groups administered the test compound and the control groups (vehicle only) (***p<0.001; **p<0.01, *p<0.05).

___________________________________ Table 8,

Decreased iron content in the blood (%)

Conn. Dose 20 mg/kg Dose 60 mg/kg 1 ? 31** 52** V 31** 59** 2 ? 27 57** d 29 66** 40 ^# + 0 13 35** 32** 40 ^# + But. 10 V But. 58** 94## ? But. 47 But. 67 127 + 47*** 74*** 21 83** 208 ^## + 9 49*** 44 67**

10. Prevention of iron overload in beta2-microglobulin-deficient mice.

As a result of decreased hepcidin and increased iron absorption, beta2-microglobulin-deficient mice (b2m-/-) fed a standard diet accumulate excess iron in the liver, heart, and pancreas. A pilot study showed that liver iron accumulation in b2m−/− mice begins at 3–4 weeks of age and that liver iron content reaches 4-fold that of wild-type (WT) mice at 6 weeks of age. Additionally, placing 3-week-old b2m-/- mice on a low-iron diet (LID) immediately after completion of breastfeeding prevented liver iron accumulation at 6-7 weeks of age. The effectiveness of Fpn inhibitors in preventing hepatic iron accumulation in b2m-/- mice was examined. 3-week-old L2 −/− mice maintained on a low-iron diet (LID) were treated with Fpn inhibitor or vehicle alone (methylcellulose; 10 ml/kg). Mice had access to drinking water supplemented with 1 mM ⁵⁸ Fe(II)-sulfate and 10 mM ascorbic acid. Administration of Fpn inhibitor or vehicle followed by access to iron-containing water was continued for 14 days. Mice were euthanized and the iron content of the liver and spleen was analyzed by ICP-OES (all iron isotopes were determined), and the liver tissue was further analyzed for ⁵⁸ Fe concentration (ICP-MS). The data summarized in table. 9 show that oral administration of Fpn inhibitors for two weeks prevented hepatic iron accumulation in L2m −/− mice and increased splenic iron concentrations, indicating inhibition of ferroportin in both the intestine and spleen.

These data demonstrate the effectiveness of the small molecule inhibitor ferroportin in preventing hepatic iron accumulation in L2-/- mice, validating the concept in a relevant disease model.

Table 9 - Fpn inhibitors tested in a mouse model of beta2-microglobulin deficiency to suppress hepatic iron overload.

Livers and spleens were collected after 14 days of administration (orally; twice daily) of the indicated doses of Fpn inhibitors to beta2-microglobulin-deficient mice. Total iron concentration in liver and spleen tissues was determined using the ICP-OES method, and ⁵⁸ Fe concentration in the liver was determined using the ICP-MS method. Relative changes (%) in tissue iron were calculated by normalizing the difference between the mean tissue iron values for Fpn inhibitor-treated and vehicle-only animals by the mean of the control group (vehicle-only). Values for females (5) and males (^) are given separately because there was a marked sex difference in efficiency. Shown are the statistically significant differences (two-way ANOVA with Bonferroni test) between the groups administered the test compound and the control groups (vehicle only) (***p<0.001;**p<0.01,*p<0.05). But. - undefined; n.d. not available.

Table 9

Conn. Increase in total iron levels in the spleen (%) Decrease in total iron levels in the liver (%) Decrease in liver ⁵⁸ Fe level (%) Dose (mg/kg) 20 60 20 60 20 60 1 + 21 65 -1 15 4 59 b 28 49 16 25 -8 22 2 13 1 26 45 60** 77*** V 18 -20 10 28 24 70 40 50* 85*** 32 67* 44 80* V 25 24 31 69*** 53* 81*** 40 + But. 9 But. 66 But. 67 X But. 36 But. 85** But. 95** 94 ? But. 65 But. 57 But. N.d. V But. 41 But. 79 But. N.d. 127 71* 51 -38 2 34 63*** b -7 -16 50** 65*** 71*** 73*** 208 + 56** 150*** 15 8 71* 87** b 21 43 41 84** 58 94**

11. Improvement in anemia, ineffective erythropoiesis and iron overload in a mouse model of β-thalassemia intermedia.

β-thalassemia is an inherited anemia caused by mutations in the hemoglobin β-globin gene, resulting in atypical red blood cells with a shortened lifespan. The most severe form, thalassemia major, requires blood transfusions, which lead to the development of secondary iron overload. Patients with thalassemia intermedia have mild transfusion-independent anemia but still develop iron overload due to ineffective erythropoiesis and chronic suppression of hepcidin production.

As shown in the previous examples, oral ferroportin (Fpn) inhibitors, like hepcidin, block ferroportin-mediated iron export from cells in vitro and, when administered to wild-type mice, transiently lower plasma iron levels. Based on these results and published studies (Schmidt PJ, et al., Blood 2013, Guo S., et al., JCI, 2013 and Casu S. et al., Blood, 2016), Fpn inhibitors were tested for their ability to prevent iron accumulation and improve erythropoiesis in thalassemia intermediate by limiting iron absorption and recycling iron from senescent red blood cells. The effectiveness of Fpn inhibitors was studied using a mouse model of transfusion-independent β-thalassemia. Mice with heterozygous deletion of the β1 and β2 globin genes (called Hbb th3/+ mice) develop transfusion-independent β-thalassemia, ineffective erythropoiesis, splenomegaly, and secondary iron overload in the spleen, liver, and kidneys. Heterozygous Hbb th3/+ mice were obtained from Jackson Laboratories (B6;129P-Hbb-b1tm1Unc Hbb-b2tm1Unc/J, Stock Number: 002683) at 8–18 weeks of age and maintained on a low-iron diet (Harlan Provimi) during the experiment Kliba 2039, 13.4 ppm Fe) without restricting access to food. Hbb th3/+ mice were administered twice daily with either 20 or 60 mg/kg test compound or methylcellulose (10 ml/kg, Sigma, Cat. 274429) as vehicle. Between these doses, mice had access to drinking water supplemented with 1 mM ⁵⁸ Fe(II)-sulfate (Vifor Pharma, Batch No. ROR 3096) and 10 mM ascorbic acid (Sigma, Cat. 795437) for 6 h. The concentration of ⁵⁸ Fe(II)-sulfate added to drinking water was adjusted to replace iron intake in standard rodent food containing 250 ppm iron. Access to water without ⁵⁸ Fe(II)-sulfate and ascorbic acid was provided during the remaining 18 hours. Administration of Fpn inhibitors or vehicle followed by access to iron-containing water was repeated for 20-46 days.

Similar to what was previously shown for wild-type and b2m-/- mice, Fpn inhibitors administered for 3 hours effectively reduced plasma iron levels also in Hbb th3/+ mice (Table

- 59 044945

10), demonstrating the ability of these small molecules to cause iron limitation.

Hbb th3/+ mice are anemic, with hemoglobin levels in the range of 70-80 g/L. Oral administration of Fpn inhibitors to Hbb th3/+ mice for two weeks significantly increased hemoglobin levels compared to vehicle-only controls (Table 10). The change in hemoglobin levels in the group administered the test compounds, compared to the control group, reached 19-22 g/L at the end of the study. Additionally, hematological parameters in peripheral blood were also measured using an automatic blood cell analyzer. Administration of Fpn inhibitors to Hbb th3/+ mice increased red blood cell count, increased hematocrit, and decreased reticulocyte concentration and red blood cell distribution width (RDW), indicating improved erythropoiesis. In addition, Hbb th3/+ mice treated with Fpn inhibitors had significantly lower blood leukocyte counts compared to controls, further demonstrating the beneficial effects of Fpn inhibitors in correcting pathologically altered parameters in this disease model. Thus, Fpn inhibitors had a significant ameliorative effect on anemia and corrected blood composition in a mouse model of thalassemia intermedia.

Ineffective erythropoiesis in Hbb th3/+ mice causes overgrowth of erythroid precursors in the spleen, leading to splenomegaly. Administration of Fpn inhibitors to Hbb th3/+ mice resulted in a significant reduction in spleen weight, demonstrating the potential of Fpn inhibitors to reverse splenomegaly (Table 10).

The effect of Fpn inhibitors on erythropoiesis was examined by analyzing the percentage of differentiating erythroid progenitors in the bone marrow and spleen using flow cytometry and the markers Ter119 (eBioscience, Cat. 17-5921) and CD44 (BioLegend, Cat. 103028). Bone marrow or spleen cells isolated from Hbb th3/+ mice treated with Fpn inhibitors contained a significantly lower percentage of early erythroid precursors proerythroblasts, basophilic and prolychromatic erythroblasts, and a higher percentage of mature erythrocytes, compared with control Hbb th3/+ mice , who were administered only the vehicle (Table 10). These data showed that Fpn inhibitors reduced the severity of ineffective erythropoiesis in Hbb th3/+ mice, and these results are consistent with the improvement in hematological blood parameters.

Plasma erythropoietin levels are elevated in Hbb th3/+ mice and thalassemia patients due to a response to anemia, hypoxia, and ineffective erythropoiesis (Guo et al. JCI, 2013). Hbb th3/+ mice treated with Fpn inhibitors had significantly lower plasma erythropoietin (DuoSet ELISA R&D Systems, Cat. DY959) compared with vehicle-only controls, most likely due to partially corrected anemia and improved erythropoiesis (Table 10).

Elevated levels of erythropoietin in Hbb th3/+ mice caused increased production of erythroferron, an erythroid-regulating hormone known to suppress hepcidin (Kautz L. et al., Nat. Genet., 2014). Consistent with the reduced plasma erythropoietin levels, erythropoietin mRNA expression was significantly reduced in the spleen of Hbb th3/+ mice treated with Fpn inhibitor compared with mice treated with vehicle alone (Table 10). Erythroferron is produced by the precursors of red blood cells, which proliferate massively in the spleen of Hbb th3/+ mice due to extramedullary erythropoiesis. Thus, the effect of Fpn inhibitors on the expression of erythroferrone in the spleen is through improvement of erythropoiesis.

Increased iron requirement due to ineffective erythropoiesis and chronically low hepcidin levels in thalassemia patients causes iron accumulation in organs and associated pathological manifestations such as hepatocellular carcinoma and heart failure (Rivella S. Haematologica, 2015). In Hbb th3/+ mice, excess iron is absorbed as a consequence of inappropriately low hepcidin levels in conjunction with high iron content in the liver, spleen and kidneys, and increased expression of ferroportin in the duodenum (Gardenghi S., Blood, 2007). Total hepatic iron and ⁵⁸ Fe contents in the organs of Hbb th3/+ mice treated with vehicle or Fpn inhibitors were analyzed by inductively coupled plasma emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICPMS). respectively. ^58Fe concentrations in the liver and spleen of Hbb th3/+ mice treated with Fpn inhibitors were significantly lower than those in mice treated with vehicle alone, demonstrating that Fpn inhibitors prevent iron accumulation in organs (Table 10).

Because Fpn inhibitors are systemically available, they are capable of blocking iron export in all ferroportin-expressing tissues, including the duodenum, spleen, and liver. Accordingly, Fpn inhibitors are expected to prevent iron absorption from the duodenum, but they cannot remove pre-existing iron from the liver and spleen. Indeed, total iron status remained unchanged in mice treated with Fpn inhibitor or vehicle (data not shown). Importantly, Fpn inhibitors significantly reduced ^58Fe concentrations in the spleen and liver of Hbb th3/+ mice, demonstrating the ability of these small molecules to prevent iron accumulation.

- 60 044945

In addition, reactive oxygen species (ROS) were detected in bone marrow cells using a fluorescent indicator, CM-H2DCFDA (Thermo Fisher Scientific, Cat. C6827). Flow cytometric analysis showed that Fpn inhibitors significantly reduced ROS levels in mature erythroid cells compared to control Hbb th3/+ mice treated with vehicle alone (Table 10).

These data demonstrated the ability of orally administered small molecule ferroportin inhibitors to influence disease by improving anemia and ineffective erythropoiesis, as well as mitigating splenomegaly and preventing further iron accumulation in the liver and spleen in a model of β-thalassemia intermedia.

Table 10

Parameter Conn. 1 Conn. 2 Conn. 40 Conn. 127 Reduction of iron in plasma with 49/66% 50/69% 28/58% 68/81% administration of 20/60 mg/kg compound Correction of anemia on days 20-48 at a dose of 20/60 mg/kg 6/20 g/l 3/11 g/l 6 /13 g/l 12/20 g/l Increase in the number of red blood cells 4/8% A 0 / 2/22% 0/36% at a compound dose of 20/60 mg/kg Decrease____________number reticulocytes_____at_____dose compounds 20/60 mg/kg 8/39% 0/11% 19/43% 16/61% Increase in hematocrit at a compound dose of 20/60 mg/kg 0/4% 0/15% 0/1% 3/20% Reduction of RDW at compound dose 20/60 mg/kg 3/16% 0/15% n.d./n.d. 19/25% Decrease in the number of leukocytes at a dose of the compound 20/60 mg/kg 32/44% 29/55% 0/36% 46/66% Reduction of ROS in bone marrow erythrocytes Reducing relative weight 20/45% 13/65% n.d./n.d., n.d./ 75% spleen at a dose of the compound 20/60 mg/kg Decrease in the content of ⁵⁸ Fe in 23/59% 16/47% 23/48% 40/61% spleen at a compound dose of 20/60 mg/kg 14/48% 13/40% 19/51% 43/68% Prevention of ⁵⁸ Fe accumulation in the liver at a compound dose of 20/60 mg/kg 12/40% 14/47% 20/48% 39/59% Reducing________content erythropoietin in blood plasma at a dose of the compound 20/60 mg/kg Decrease in erythropoietin mRNA in 64/78% 4/27% 6/37% 32/33% spleen at a compound dose of 20/60 mg/kg 82/292% 461/639% n.d./n.d. 1012/3031°/ Table 10 - effectiveness of ferroportin inhibitors in a mouse model of thalassemia intermedia (Hbb th3/+ mice). The indicated Fpn inhibitors were administered twice daily within 20 days (Connections 1 and 2), 27 days (Compound 127) or 46 days (Compound 40). The results are expressed as the difference from con- troll group (to which only the vehicle was administered) in the case of hemoglobin, and as % change < regarding

relative to the control group for all other given parameters.

12. Evaluation of activity in the treatment of drepanocytic anemia in a mouse model.

Using the mouse model described by Yulin Zhao et al. in the work MEK1/2 inhibitors reverse acute vascular occlusion in mouse models of sickle cell anemia; The FASEB Journal Vol. 30, No. 3, pp 1171-1186, 2016, determined the activity of the salts of the present invention in the treatment of drepanocytic anemia in the manner described below.

Ferroportin inhibitors prevent acute vascular occlusions and organ damage in a mouse model of sickle cell disease.

Vascular occlusive crisis (VOC) is a leading cause of clinical manifestations and mortality in patients with sickle cell disease (SCD). Hypoxia, dehydration, inflammation or hemolysis all contribute to increased adhesion of sickle red blood cells (SSRBCs), neutrophils and platelets to activated endothelium in small vessels, promoting coagulation, vascular occlusion, painful crises and irreversible damage to various organs. High white blood cell counts, especially activated neutrophils, correlate with early mortality, silent cerebral infarction, hemorrhagic strokes, and acute chest syndrome in patients with SCD (Platt OS, NEJM, 1994). Hemolysis in SCD occurs due to damaged membranes of sickled red blood cells, which causes chronic anemia and the release of Hb into the circulatory system, which provokes inflammation by depleting NO, causing oxidative stress and releasing heme. SSRBC secretes microvesicles that trigger the production of reactive oxygen species (ROS) by endothelial cells, provoke leukocyte adhesion and cause phosphatidylserine-dependent endothelial apoptosis, contributing to the development of vascular occlusive crises in SCD (Camus M, Blood, 2012).

Chronic iron restriction by administration of ferroportin inhibitors reduces ROS formation in the erythrocytes of β-thalassemia mice, as has been shown, for example, for the compounds described in the above unpublished international patent applications PCT/EP2016/075305 and PCT/EP2016/075306 (The Jackson Laboratories, Yang W. et al., PNAS. 1995). Based on these data, it was hypothesized that ferroportin inhibitors may attenuate vascular occlusive crisis (VOC) in SCD by reducing hemolysis and ROS formation in SSRBC, thereby preventing leukocyte adhesion to the endothelium.

To test this hypothesis, vehicle or ferroportin inhibitors were administered orally at 30 or 100 mg/kg twice daily (BID) for 4 weeks in the Townes SCD mouse model (Ryan T., Science, 1990). These mice were genetically engineered to exclusively express human hemoglobin (ha/ha::eS/eS, The Jackson Laboratories). Townes mice have anemia, elevated reticulocyte counts, splenomegaly, vascular inflammation, and are susceptible to developing VOC in response to hypoxia, inflammation, and hemolysis. To study the effect of ferroportin inhibitors on leukocytes and SSRBC adhesion to inflamed endothelium, Townes mice treated with vehicle or ferroportin inhibitor for 25 days were euthanized and a glass chamber was surgically implanted into a dorsal skinfold under sterile conditions as previously described (Kalambur V.S. et al., Am J Hematol. 2004; Zennadi R. et al., Blood, 2007). Three days after surgery, mice were injected with 0.5 μg of TNFa (R&D Systems), causing inflammation leading to VOC. Ninety minutes after TNFa administration, leukocytes and erythrocytes were labeled in vivo by intravenous injection of rhodamine-conjugated Ly6G (Sigma) and phycoerythrin-conjugated anti-TER119 mAb (BioLegend), respectively. Adhesion of leukocytes and erythrocytes to the microvascular endothelium was monitored over the next 90 minutes using fluorescence intravital microscopy, as previously described (Zhao et al., FASEB J, 2016). Briefly, euthanized animals with glass chambers were kept at 37°C, and blood flow parameters and cell adhesion events were recorded using a C2400 digital video camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan) connected to a fluorescence microscope (Axoplan microscope, Carl Zeiss). Twenty to thirty microcapillary segments were examined in each mouse, and cell adhesion was quantified on still frames by measuring the fluorescence intensity of adherent fluorescently labeled cells using ImageJ software. The results were expressed in fluorescence units per million cells.

13. Study of the pharmacokinetics of compound 127 in the form of monosalts with H ₂ SO ₄ or HCl after a single intravenous and oral administration to male Sprague Dawley rats.

To study the pharmacokinetics (PK) of compound 127 as a monosalt with H ₂ SO ₄ (mol. weight 604.6 g/mol) or HCl (mol. weight 444.9 g/mol), a single dose of these salts was administered to male Sprague Dawley rats (n= 3 per route of administration) intravenously (1 mg/kg) or orally (30 mg/kg). These dosages were adjusted for the weight of the compound as free base (molecular weight 408.43 g/mol).

Rats were kept in ventilated cages at temperatures ranging from 22 to 25°C, humidity 40–70% RH, on a 12-hour light/12-hour dark cycle, with standard rodent food and water ad libitum. Before the pharmacokinetic study, rats were fasted overnight and then fed 4 hours after drug administration. This technique has been studied and approved by the Animal Welfare of Contract Research Institutions Committee (GVK Biosciences Pvt. Ltd. Hyderabad, India).

A monosalt of compound 127 with H ₂ SO ₄ or a monosalt of compound 127 with HCl, injected into phosphate-buffered saline (PBS) containing 5% DMSO and 10% solutol, was injected intravenously into the tail vein of rats using a 27-gauge needle in an amount of 5 ml/kg and at a concentration of 0.2 mg/ml.

For oral administration at a dosage of 30 mg/kg, the salts were used at a concentration of 6 mg/ml in a solution of 0.5% methylcellulose containing 5% DMSO and administered orally at a dosage of 5 ml/kg.

Blood samples of 0.20-0.30 ml were taken from the jugular vein of rats into tubes prefilled with lithium heparin at the following time points: 5, 15, 30 minutes, 1, 2, 4, 8 and 24 hours after drug administration. Plasma was separated by centrifugation at 2500xg for 15 min at 4°C. The concentration of compounds in plasma was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS), and standard pharmacokinetic parameters were determined, such as _C0 , _Cmax , _Tmax , Cl, Vd, AUClast, T1/2 , MRT, %F, according to the non-compartmental model using the Phoenix program version 6.4.

The results obtained confirmed a good/improved pharmacokinetic profile.

III. Combination therapy study.

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Regarding the previously mentioned possible options for combination therapy with other active substances for the salts described in the present invention having activity as ferroportin inhibitors, such combination therapy can be studied in a mouse model of beta thalassemia intermedia.

Potential synergistic or additive effects of the salts of the present invention with other therapeutic agents (second agent) are evaluated through combination studies in mouse models of thalassemia major (Hbb ^th3/+ or Hbb ^th1/th1 , Jackson Laboratories) or thalassemia major (C57-FLC ^{th3/ th3} , such as described in the work of Casu S. et al. Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of β-thalassemia intermedia and major; Haematologica, 2017, during which the effect of the salts of the present invention separately is studied ( i.e., administering salt alone) or in combination with additional compound(s) on anemia, hematopoiesis, iron overload, reactive oxygen species (ROS) production, splenomegaly and other biomarkers in thalassemia models.Mice of both sexes aged 12 weeks received the salts of the present invention alone or in combination with one of the following second agents: modified activin receptor type IIA or IIB fusion proteins (such as those described in Suragani RN et al. Modified activin receptor IIB ligand trap mitigates ineffective erythropoiesis and disease complications in murine β-thalassemia. Blood. 2014 Jun 19;123(25):3864-72 and Dussiot M. et al. An activin receptor IIA ligand trap corrects ineffective erythropoiesis in β-thalassemia. Nat Med. 2014 Apr;20(4):398-407), which act as ligand decoys for members of the transforming growth factor beta (TGFβ) superfamily, such as RAP-011 or RAP-536 (murine analogues of ACE-011, sotatercept or ACE-536 , luspatercept (described in patent application WO 2010019261 or claimed in US patent US 8361957), respectively, Acceleron/Celgene) or other antagonists of members of the TGFe superfamily (antibodies, antibody fragments, non-antibody drugs, or ligand decoy producing cells activin receptor).

Inhibitors of JAK1/2 or JAK2, including but not limited to rutoxilinib (Novartis as claimed in US Patents US 7,598,257 and US 8,415,362) or fedratinib (Sanofi), such as those described by Casu C. et al. Short-term administration of JAK2 inhibitors reduces splenomegaly in mouse models of βthalassemia intermedia and major.; Haematologica, 2017.

pan-HDAC inhibitor such as panobinostat (LC Laboratories, USA, and claimed in US patents US 6552065 and US 6833384) or HDAC3 inhibitor RGFP966 (Selleckchem - such as described in Pasricha S.R. et al. Hepcidin is regulated by promoter-associated histone acetylation and HDAC3. Nat Commun. 2017 Sep l;8(1):403).

Matriptase-2 (also known as Tmprss6) antagonists, such as lipid nanoparticle (LNP) Tmprss6 siRNAs or antisense oligonucleotides (ASOs) targeting murine Tmprss6 (such as those described in Guo S. et al Reducing TMPRSS6 ameliorates hemochromatosis and β -thalassemia in mice. J. Clin Invest. 2013 Apr;123(4):1531-41 or in the work of Schmidt P. J. et al. An RNAi therapeutic targeting Tmprss6 decreases iron overload in Hfe(-/-) mice and ameliorates anemia and iron overload in murine β-thalassemia intermedia. Blood. 2013 Feb 14; 121(7): 1200-8).

Exogenous apotransferrin (such as described in Li H. et al. Transferrin therapy ameliorates disease in beta-thalassemic mice. Nat Med. 2010 Feb; 16(2): 177-82).

Hepcidin-inducing steroids (HIS), such as epithiostanol, progesterone and mifepristone, or progesterone receptor membrane component-1 (PGRMC1) antagonists, ref. 7.

Erythroferrone antagonists, such as antibodies or ligand decoys.

Recombinant erythropoietin (EPO). The erythropoietins available for use as therapeutic agents of the present invention are produced by recombinant DNA technology in cell culture and include Epogen/Procrit (epoetin-alpha) and Aranesp (dareroetin-alpha) or Myrcera (epoetin-beta and methoxy polyethylene glycol).

Glycine transporter 1 (GlyT1) inhibitors such as bitopertine (Roche AG).

The salts of the present invention are administered orally to thallasemic mice either as the sole active ingredient twice daily at dosages of 10, 30 and 60 mg/kg, or in combination with one or more of the above compounds (second agents). A control group of mice with thalassemia received only the second drug. Age- and sex-matched thalassemic and wild-type (WT) mice were used as controls. In some experiments, the salts of the present invention could also be dosed in drinking water, facilitating co-administration of other orally administered drugs.

More specifically, the second agent is administered as a separate treatment or administered in conjunction with the salts of the present invention, as described below:

RAP-011 or RAP-536 can be administered subcutaneously twice weekly at 1, 10, or 30 mg/kg for up to 8 weeks.

JAK1/2 inhibitors can be administered orally twice daily, with or without the salts of the present invention, diluted in drinking water.

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Claims (18)

Rutoxilinib (60 or 180 mg/kg) or fedratinib (40 or 120 mg/kg) can be administered orally once daily for 2 weeks, with or without the salts of the present invention, diluted in drinking water. Panobinostat or RGFP966 can be administered once daily at a dosage of 10 or 20 mg/kg, with or without the salts of the present invention, diluted in drinking water. Apotransferrin is administered by injection intraperitoneally at a dosage of 100 or 300 mg/kg per day for 8 weeks. Mifepristone (30 or 100 mg/kg) is administered by injection intraperitoneally once daily for 2 weeks. Erythroferrone-specific antibodies or ligand decoys can be administered twice weekly by subcutaneous injection. Erythropoietin can be injected intraperitoneally at a dosage of 200 IU once daily for 2 weeks. Glycine transporter 1 (GlyT1) inhibitors, such as bitopertine (Roche AG), can also be administered in a suitable manner. Changes in hemoglobin levels in mice were monitored once a week, and at the end of the test, blood and organs were taken for examination. Spleen weight was normalized to body weight and assessed as the effect of administration on extramedullary erythropoiesis. Iron concentrations in the liver, spleen, kidneys and heart were determined by the inductively coupled plasma (ICP) method using an optical emission spectrometer (OES). A complete blood test was performed using an automatic device. Erythropoiesis was assessed in the bone marrow and spleen by labeling cells with antibodies CD71, CD44 and Ter119 and detecting erythroid cells by flow cytometry. The membrane-bound fraction of alpha-globulin on red blood cells was quantified by HPLC. The presence of reactive oxygen species (ROS) in red blood cells was determined by staining with the fluorescent indicator chloromethyl-2',7'dichlorodihydrofluorescein diacetate. Blood iron levels were measured by a colorimetric assay using a Ferene-S reagent (Abbott). The level of erythropoietin in the blood was quantitatively determined by ELISA (R&D, duo set). The level of hepcidin in the blood was measured by ELISA (Intrinsic Lifesciences). Hepcidin levels in the liver and erythroferrone gene expression in bone marrow and spleen were quantified by qRT-PCR. CLAIM 1. Salt of the compound of formula (I) where X ¹ represents N or O; And X ² represents N, S or O; provided that X ¹ and X ² are different; R ¹ represents a hydrogen atom; n is an integer from 1 to 3; A ¹ and A ² are independently methylene or ethane-1,2-diyl; R ² represents a hydrogen atom or a linear or branched C1-C ₄ alkyl; or A ¹ and R ² , together with the nitrogen atom to which they are attached, form an unsubstituted 4-membered ring of the formula R ³ represents H; R ⁴ represents H, wherein said salt is selected from salts of the compound of formula (I), with acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid, characterized in that the ratio of compound (I): acid is from 1:2 to 1:3; and its solvates; and where the following ZNS1 salts are excluded: - 64 044945 and where the following 2HC1-salt is excluded: 2. Salt of the compound of formula (I) according to claim 1, where n = 1; R ² = hydrogen atom; R ³ = hydrogen atom; R ⁴ = hydrogen atom; A ¹ = methylene or ethane-1,2-diyl; A ² = methylene, ethane-1,2-diyl or propan-1,3-diyl; - 65 044945 or A ¹ and R ² together with the nitrogen atom to which they are attached form a 4-membered ring of the formula forming a compound of formula (II) or (III) (ΠΙ), where in formula (II) and (III) m represents the integer 1, 2 or 3, and X ¹ , X ² and R ¹ have the meanings specified in paragraph 1, and its solvates and hydrates. 3. A salt of a compound of formula (I) according to claim 1 or 2, wherein said salts are selected from monosalts, and its solvates and hydrates. 4. A salt of a compound of formula (I) according to any one of claims 1 to 3, wherein the acids are selected from the group consisting of citric acid, hydrochloric acid, maleic acid and sulfuric acid, and solvates and hydrates thereof. 5. A salt of a compound of formula (I) according to any one of claims 1 to 3, wherein the acids are selected from the group consisting of phosphoric acid and sulfuric acid, and their solvates and hydrates. 6. A salt of a compound of formula (I) according to claim 1 or 2, wherein the acid is selected from phosphoric acid, forming a salt having a ratio of compound of formula (I):PO ₄ = 2:1, and its solvates and hydrates. 7. A salt of a compound of formula (I) according to any one of claims 1 to 4, where ZHC1 salts, and their solvates and hydrates are excluded. 8. A salt of a compound of formula (I) according to any one of claims 1 to 7, where the compound of formula (I) is selected from the group - 66 044945 and its solvates and hydrates. 9. A salt of the compound of formula (I) according to claim 8, which is selected from the group consisting of the following compounds: 10. A salt of the compound of formula (I) according to claim 8 or 9, which is selected from the group consisting of the following compounds: F and its solvates and hydrates. - 67 044945 11. A salt of a compound of formula (I) according to any one of claims 1-5, 7-10, which is a 1:1 sulfate salt having the formula 12. A salt of a compound of formula (I) according to any one of claims 1-3, 5, 7-10, which is a 1:1 phosphate salt having the formula 13. A salt of a compound of formula (I) according to any one of claims 1 to 10, which is selected from the group of the following polymorphs of a compound having the structure (i) a citric acid salt polymorph of the said compound, which is characterized by a powder x-ray diffraction pattern (PXRD pattern) containing characteristic crystalline peaks expressed in degrees 2-theta, 24.5, 21.6 and 5.3 ±0.25 degrees , (ii) a maleic acid salt polymorph of said compound, which is characterized by a powder x-ray diffraction pattern (PXRD pattern) containing characteristic crystalline peaks expressed in degrees 2-theta, 9.6, 19.0 and 24.5 ±0.25 degrees, (iii) a phosphoric acid salt polymorph of said compound, which is characterized by a powder X-ray diffraction pattern (PXRD pattern) containing characteristic crystalline peaks expressed in degrees 2-theta, 27.2, 16.8 and 4.6 ±0, 25 degrees, (iv) a phosphoric acid salt polymorph of the said compound, which is characterized by a powder x-ray diffraction pattern (PXRD pattern) containing characteristic crystalline peaks expressed in degrees 2-theta, 26.1, 15.5 and 16.5 ±0 .25 degrees, (v) a sulfuric acid salt polymorph of the said compound, which is characterized by a powder x-ray diffraction pattern (PXRD pattern) containing characteristic crystalline peaks expressed in degrees 2-theta, 25.4, 4.5 and 18.1 ± 0.25 degrees, where Cu-Ka1 radiation is used to determine the PXRD pattern. 14. The use of a salt according to any of the preceding paragraphs and its solvates as a drug acting as a ferroportin inhibitor and/or acting in inhibiting ferroportin-mediated iron transport. 15. The use of a salt according to any one of claims 1 to 13, acting as an inhibitor of ferroportin and/or acting in inhibiting iron transport mediated by ferroportin, and its solvates in the prevention and/or treatment of disorders of iron metabolism leading to elevated iron levels, or increased iron absorption, in the prevention and/or treatment of iron overload and/or in the prevention and/or treatment of diseases associated with or caused by elevated iron levels, increased iron absorption or iron overload. 16. Use according to claim 15, where the diseases associated with or caused by increased iron levels, increased iron absorption or iron overload are selected from hemoglobinopathy, hemoglobin E disease, hemoglobin H disease, hemochromatosis, hemolytic anemia, thalassemia, drepanocytic anemia (sickle cell anemia) and congenital dyserythropoietic anemia. 17. Use according to claim 16, wherein the thalassemia is selected from alpha thalassemia, beta thalassemia and delta thalassemia. 18. Use of a salt according to any one of claims 1 to 13, acting as an inhibitor of ferroportin and/or acting in inhibiting iron transport mediated by ferroportin, and its solvates in the prevention and/or treatment of diseases associated with ineffective erythropoiesis, or as an adjunctive therapy to treatment by limiting the amount of iron available to pathogenic microorganisms in infections caused by said pathogenic microorganisms, or in the prevention and/or treatment of neurodegenerative diseases caused by deposition or increase -