Methods and Compositions for Treating Sickle Cell Disease with the Transferrin Inhibitor (VIT-2763)

Abstract

The present invention relates to a compound of the formula described below and its pharmaceutically acceptable salts, which are used for the treatment of sickle cell disease and for the prevention and treatment of vascular inflammation and vaso-occlusion.

Description

Background Art

[0001] Sickle cell disease (SCD) is a hereditary disorder of hemoglobin synthesis, characterized by lifelong severe hemolytic anemia, recurrent painful crises, chronic organ system damage, and a significantly reduced life expectancy. SCD is caused by a mutation in the hemoglobin (Hb) β gene, which causes an amino acid substitution in β-globin and generates the sickle hemoglobin (HbS) allele βS. In the SCD mouse model (Townes mice), the murine Hb gene has been deleted and replaced with the human sickle Hb gene. Homozygous HbS mice express only human sickle Hb and closely resemble SCD in the human disease process, containing rigid sickle red blood cells (sRBCs) and exhibiting hemolytic anemia, iron overload, splenic red pulp expansion, inflammation, increased adhesion of blood cells to the endothelial vasculature, resulting in vaso-occlusion (VO) and organ damage. Polymerization of deoxy-HbS shortens the lifespan of sickle red blood cells and promotes intravascular and extravascular hemolysis. Intravascular hemolysis releases cell-free Hb from red blood cells (RBCs). Extracellular Hb is readily oxidized from ferrous (Fe 2+ ) Hb to ferric (Fe 3+ ) Hb (metHb), from which heme readily dissociates into the vasculature, causing oxidative stress, inflammation, VO, ischemia, and tissue damage (Umbreit J, Am. J Hematol, 2007).

[0002] Conventional (approved) drugs for treating SCD symptoms associated therewith are hydroxyurea (Droxia, Hydrea, Siklos), L-glutamine oral powder (Endari), crizanlizumab (Adakveo), and voxelotor (Oxbryta).

[0003] Daily administration of hydroxyurea reduces the frequency of painful crises and may reduce the need for blood transfusions and hospitalizations. It may increase the risk of infection.

[0004] L-glutamine oral powder (Endari) helps reduce the frequency of painful crises.

[0005] Crizanlizumab (Adakveo) is an intravenously administered drug that helps reduce the frequency of painful crises. Side effects may include nausea, joint pain, back pain, and fever.

[0006] Voxelotor (Oxbryta) is an orally administered drug that can improve anemia in patients with sickle cell disease. Side effects may include headache, nausea, diarrhea, fatigue, rash, and fever.

[0007] In addition, pain relief medications are regularly administered to SCD patients, which, while helpful in alleviating pain during sickle cell pain crises, do not treat the root cause of the pain.

[0008] Object of the Invention

[0009] The object of the present invention is to provide an improved novel drug or therapy for the treatment of sickle cell disease (SCD). In particular, the novel drug or SCD therapy should improve, alleviate or normalize one or more of the markers, conditions or events associated with SCD and as further defined herein. Another object of the present invention is to provide an SCD drug or therapy with improved safety compared to conventional hydroxyurea treatment. Another object of the present invention is to provide an SCD drug or therapy with at least equivalent or even improved safety compared to conventional voxelotor treatment. The present invention aims to provide an improved SCD therapy, one or more aspects of which will be discussed in more detail below.

[0010] Summary of the Invention and Detailed Description

[0011] Methods for treating sickle cell disease (SCD) are described herein, which include administering a compound of the following formula (Compound 127)

[0012]

[0013] or a pharmaceutically acceptable salt thereof. Among them, suitable salts are: benzoate, hydrochloride (HCl salt), citrate, fumarate, lactate, malate, maleate, mesylate, phosphate, succinate, sulfate, tartrate and tosylate. In various embodiments, the ratio of the compound to the salt is 1:1, 2:1, 1:2 or 1:3. As used herein, unless a specific ratio is specified, the salt of the compound refers to any ratio of the compound to the salt.

[0014] Compound 127 and methods for synthesizing Compound 127 are described in WO2017 / 068089 and WO2017068090A1, which are incorporated herein by reference. Specific salts of Compound 127 and various polymorphs of Compound 127 are described in WO2018 / 192973, which is incorporated herein by reference. The potential use of the specific salts disclosed therein in the treatment of sickle cell disease is generally mentioned in a list of various other indications. Example 13 describes single-dose intravenous and oral pharmacokinetic studies with H2SO4 and the HCl monosalt of Compound 127.

[0015] "First-in-class oral Ferroportin Inhibitor: Mode of Action and Efficacy in a mouse model of Beta-Thalassemia Intermedia" by Vania Manolova (EHA abstract, June 14, 2019) discloses the use of compound 127 in the treatment of beta-thalassemia intermedia, but does not mention any specific salt form thereof. In addition, the potential efficacy of compound 127 in the treatment of sickle cell disease is not mentioned in this disclosure either.

[0016] "Ferroportin inhibition attenuates plasma iron, oxidant stress, and renal injury following red blood cell transfusion in guinea pigs" by J.H. Baek et al. (TRANSFUSION, issue 00, pages 1-11, 2020) reported that intravenous administration of the small molecule ferroportin inhibitor VIT-2653 provided by Vifor (International) immediately after acute red blood cell transfusion in a guinea pig model reduced plasma iron content and NTBI levels and alleviated oxidative stress and cellular injury.

[0017] The ferroportin-hepcidin axis regulates blood iron levels. Compound 127 competes with hepcidin for ferroportin binding and internalization. Compound 127 blocks iron transport into the blood by inhibiting ferroportin.

[0018] In the context of the novel use of the present invention, the term "treatment" includes improving at least one symptom or pathological condition associated with SCD. In the context of the present invention, the term "treatment" also includes prophylaxis. The treatment with compound 127 according to the present invention particularly improves, alleviates or modifies one or more of the following markers, conditions or events, for example, adjusts them to normal levels.

[0019] The therapeutic effect of the present invention

[0020] A specific aspect of the present invention relates to compound 127 as described anywhere herein for treating, preventing or alleviating one or more of the markers, conditions or events described above or below or in particular in the examples.

[0021] In some cases, treating a subject with sickle cell disease (SCD) with Compound 127 reduces the occurrence of hemolysis (e.g., as evaluated by a decrease in cell-free hemoglobin (Hb), a decrease in cell-free heme, a decrease in total plasma bilirubin and indirect plasma bilirubin, or a decrease in serum LDH (lactate dehydrogenase)).

[0022] In some cases, treating a subject with SCD with Compound 127 improves one or more of total serum iron level, serum ferritin level, serum transferrin level, and calculated TSAT (transferrin saturation).

[0023] In some cases, treating a subject with SCD with Compound 127 alleviates reticulocytosis and increases the reticulocyte count and / or reticulocyte percentage.

[0024] In some cases, treating a subject with SCD with Compound 127 decreases one or more of total Hb, RBC count, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (CHCM).

[0025] In some cases, treating a subject with SCD with Compound 127 improves RBC distribution width (RDW) and increases one or more of platelet count and reticulocyte count.

[0026] In some cases, treating a subject with SCD with Compound 127 improves microcytic RBCs (RBC volume-Hb scatterplot).

[0027] In some cases, treating a subject with SCD with Compound 127 alters abnormal RBCs (sickle) and / or improves RBC sickling (peripheral blood smear).

[0028] In some cases, treating a subject with SCD with Compound 127 decreases leukocytosis.

[0029] In some cases, treating a subject with SCD with Compound 127 decreases blood white cell count (e.g., decreases blood neutrophil count and / or blood lymphocyte count).

[0030] In some cases, treating a subject with SCD with Compound 127 reduces the occurrence of extravascular hemolysis and / or intravascular hemolysis.

[0031] In some cases, treating a subject with SCD with Compound 127 improves one or more hemolysis markers (e.g., indirect bilirubin / total bilirubin), blood inflammation markers, such as those measured by hsCRP (high-sensitivity C-reactive protein), IL-1 and IL-6 (interleukins), TNF-α, sVCAM-1, endothelin-1, sP-selectin, sICAM-1, and xanthine oxidase.

[0032] In some cases, treating a subject with SCD with Compound 127 improves one or more RBC indices, including Hb concentration, RBC count, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular Hb (MCH), mean corpuscular Hb concentration (MCHC), mean corpuscular Hb concentration of RBCs (CHCM), RBC distribution width, platelet count, and reticulocyte count, reticulocyte percentage, hypochromic percentage, microcytic RBCs (RBC volume-Hb scatter plot), CHCM (mean corpuscular Hb concentration of RBCs), total serum iron, serum ferritin, serum transferrin, calculated TSAT, hepcidin, EPO (erythropoietin), NTBI (non-transferrin-bound iron), soluble transferrin receptor (sTFR), sTFR-2, and LDH.

[0033] This means that one or more of the parameters mentioned above and below can be determined to evaluate the efficacy of the compounds of the present invention in treating SCD. Compound 127 of the present invention is suitable for improving at least one of these parameters.

[0034] In the context of the present invention, the term "improve" can encompass the modulation or alteration of the corresponding marker or condition in the sense of a therapeutic effect.

[0035] More particularly, the treatment of SCD according to the present invention can result in the following:

[0036] The NTBI level of the patient is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%, which is measured at any time point within a time period of up to 72 hours, up to 60 hours, up to 48 hours, up to 36 hours, up to 24 hours or up to 12 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour and 0.5 hours after administration, and is compared with the NTBI level of the patient measured at any time point within 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours or 48 hours before the start of the treatment of the present invention or at any time point within up to <1 week.

[0037] The total LPI level of the patient is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%, which is measured at any time point within a time period of up to 72 hours, up to 60 hours, up to 48 hours, up to 36 hours, up to 24 hours or up to 12 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour and 0.5 hours after administration, and is compared with the total LPI level of the patient measured at any time point within 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours or 48 hours before the start of the treatment of the present invention or at any time point within up to <1 week.

[0038] At least one of the parameters Hct, MCV, MCH, RDW and reticulocyte count of the patient is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%, which is measured at any time point within a time period of up to one week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 3 months after the first administration, and is compared with the corresponding parameter of the subject measured at any time point within 1 week, 2 weeks, 3 weeks or 4 weeks before the start of the treatment of the present invention.

[0039] The serum ferritin level of the patient is reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%, which is measured at any time point within a time period of up to one week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 3 months after the first administration, and is compared with the serum ferritin level of the patient measured at any time point within 1 week, 2 weeks, 3 weeks or 4 weeks before the start of the treatment of the present invention.

[0040] In some cases, treating a subject with SCD with Compound 127 reduces or prevents further iron deposition in the liver, kidney and / or spleen.

[0041] Thus, on the other hand, the new treatment can cause the hepatic iron concentration, renal iron concentration and / or splenic iron concentration of patients with SCD to be reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or at least 100%, which is measured at any time point within a time period of up to one week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 3 months after the first administration, and is compared with the levels of hepatic iron concentration, renal iron concentration and / or splenic iron concentration of patients with SCD measured at any time point within 1 week, 2 weeks, 3 weeks or 4 weeks before the start of the treatment of the present invention.

[0042] In some cases, treatment with Compound 127 reduces the occurrence of renal iron overload.

[0043] In some cases, treating a subject with SCD with Compound 127 reduces vascular inflammation markers, such as the level of sVCAM-1.

[0044] In some cases, treating a subject with SCD with Compound 127 reduces vascular inflammation.

[0045] In some cases, treating a subject with SCD with Compound 127 reduces the degree of blood cell adhesion in inflamed venules or microvessels and improves blood flow in microvessels.

[0046] In some cases, treating a subject with SCD with Compound 127 reduces the adhesion of blood cells to microvessels, vaso-occlusion (VO) and VO events.

[0047] In some cases, treating a subject with SCD with Compound 127 reduces the frequency of VOC (vaso-occlusive crisis) or painful VOC and / or prevents recurrent painful VOC including ACS (acute chest syndrome).

[0048] In some cases, treating a subject with sickle cell disease (SCD) with Compound 127 one week, two weeks, three weeks, or four weeks, two months, three months, four months, six months, eight months, nine months, twelve months, or twenty-four months before the start of treatment according to the present invention reduces the frequency of vaso-occlusive crises (VOCs) or painful VOCs and / or prevents recurrent painful VOCs including acute chest syndrome (ACS) in the patient; or achieves that SCD patients do not suffer from VOCs or painful VOCs for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks, sixteen weeks, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, twenty-four months, or even longer after such treatment.

[0049] In some cases, treating a subject with SCD with Compound 127 results in a reduction in the average number of painful (VOC) crises within 48 weeks.

[0050] In some cases, treating a subject with SCD with Compound 127 results in a reduction in the average number of painful (VOC) crises in OH uremia-naive patients within 48 weeks.

[0051] Preferred aspects relate to Compound 127 as described anywhere herein for treating, preventing, or reducing vascular inflammation or vaso-occlusive events (VO) and VO events.

[0052] In some cases, treating a subject with SCD with Compound 127 reduces the need for RBC transfusions, e.g., the transfusion burden of the patient is significantly reduced compared to the transfusion burden of the patient one week, two weeks, three weeks, or four weeks, two months, three months, four months, six months, eight months, nine months, twelve months, or twenty-four months before the start of treatment according to the present invention; or achieves that SCD patients do not require red blood cell transfusions for at least two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks, sixteen weeks, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, eighteen months, twenty-four months, or even longer after such treatment until the patient no longer requires red blood cell transfusions at all.

[0053] In another aspect, compared to the quality of life of SCD patients measured one week, two weeks, three weeks, or four weeks before the start of treatment according to the present invention, the new treatment can improve the quality of life of SCD patients. The improvement in the quality of life is measured three months, six months, nine months, twelve months, fifteen months, eighteen months, twenty-one months, or twenty-four months after the start of treatment. The quality of life can be determined by assessing changes in patient-reported outcomes (PRO) using the Adult Sickle Cell Quality of Life Measurement System (ASCQ-ME).

[0054] The determination of the above parameters can be carried out using conventional methods in the art, in particular by those methods described in the unpublished PCT application PCT / EP2020 / 070391, the respective contents of which are incorporated herein by reference.

[0055] Group of patients to be treated

[0056] In principle, the subjects to be treated in the new use according to the invention can be any mammal, such as rodents and primates, and in a preferred aspect, the new medical use relates to the treatment of humans. Subjects suffering from sickle cell disease (SCD) and treated with the new method of the invention are also referred to as "patients".

[0057] The subjects to be treated can be of any age. A preferred aspect of the invention relates to the treatment of children and adolescents. Thus, in a preferred aspect of the invention, the subjects to be treated with the new method described herein are ≤18 years of age. More particularly, the subjects to be treated with the new method described herein are ≤16 years of age, ≤15 years of age, ≤14 years of age, ≤13 years of age, ≤12 years of age, ≤11 years of age, ≤10 years of age, ≤9 years of age, ≤8 years of age, ≤7 years of age, ≤6 years of age or ≤5 years of age. In another aspect of the invention, the subjects to be treated with the new method described herein are 1 to 3 years of age, 3 to 5 years of age, 5 to 7 years of age, 7 to 9 years of age, 9 to 11 years of age, 11 to 13 years of age, 13 to 15 years of age, 15 to 20 years of age, 20 to 25 years of age, 25 to 30 years of age or >30 years of age. Preferably, the pediatric patients to be treated are ≤16 years of age or ≤12 years of age. In the case of treating children, preferably the patients to be treated with the new method described herein are ≥2 years of age, preferably ≥2 years of age and ≤16 years of age or ≥2 years of age and ≤12 years of age.

[0058] In another aspect of treating adults and adolescents, the patient age is ≥12 years of age or ≥16 years of age.

[0059] In the case of treating adults, the subjects to be treated with the new method described herein are preferably 18 to 50 years of age, preferably 18 to 25 years of age, 20 to 25 years of age, 25 to 30 years of age, 30 to 35 years of age, 35 to 40 years of age, 40 to 45 years of age, 45 to 50 years of age. It is also possible to treat the elderly with an age of 50 to 55 years, 55 to 60 years or older than 60 years of age. In the case of treating elderly patients, the subjects to be treated with the new method described herein are 60 to 80 years of age, such as 60 to 65 years of age, 65 to 70 years of age, 70 to 75 years of age, 75 to 80 years of age or older than 80 years of age.

[0060] Treatment of children and adolescents is particularly preferred due to the significant advantages provided by treatment with the compounds of the present invention. The compounds can be administered orally, which is a preferred mode of administration over parenteral administration. In addition, the orally bioavailable compounds of the present invention have been shown to have moderate bioavailability and half-life in vivo, and are thus washed out relatively quickly. This results in fewer side effects and faster drug reversibility, which is particularly important in the treatment of children.

[0061] Compound 127 can be used to treat patients with various forms of SCD, including: HbSS, HbSC, HbSβ0 thalassemia, HbSβ+ thalassemia, HbSD, HbSE, and HbSO. In particular, compound 127 can be used to treat patients with HbSS or HbSβ0 thalassemia.

[0062] Compound 127 can be particularly used to treat patients with SCD as defined anywhere herein, where SCD cannot be adequately controlled in monotherapy (e.g., monotherapy with hydroxyurea).

[0063] As described herein, compound 127 can be used to treat patients with SCD who experience one or more VOCs (vaso-occlusive crises) per year.

[0064] As described herein, compound 127 can be used to treat patients with SCD who experience one or more and no more than 6 VOCs per year.

[0065] As described herein, compound 127 can be used to treat patients with SCD whose absolute reticulocyte count and percentage of reticulocyte count > 1.5 × upper limit of normal (ULN).

[0066] Compound 127 can be used to treat patients with a history of partial or total splenectomy, with a history of any cardiac or pulmonary condition or clinical significant finding, and / or who have received or are receiving regularly or periodically red blood cell (RBC) transfusion therapy (including chronic, defensive, or prophylactic transfusion therapy for SCD).

[0067] Form of administration

[0068] The compounds of the present invention are preferably provided as a medicament or pharmaceutical composition in an oral administration form, including for example pills, tablets (e.g., enteric-coated tablets, film tablets, and layer tablets), sustained-release formulations for oral administration, depot formulations, dragees, granules, emulsions, dispersions, microcapsules, micro-formulations, nano-formulations, liposomal formulations, capsules (e.g., enteric capsules), powders, microcrystalline formulations, dusting powders, drops, ampoules, solutions and suspensions for oral administration.

[0069] In a preferred embodiment of the present invention, the compounds of the present invention are administered in the form of tablets or capsules as defined above. More preferably, capsules filled with the pharmaceutical compound 127. These can be present, for example, as acid-resistant forms or together with pH-dependent coatings.

[0070] The pharmaceutical compound can be filled into the capsule as a pure pharmaceutical substance or in the form of a pharmaceutical composition comprising other pharmaceutically acceptable adjuvants, auxiliaries, solvents, additives, etc.

[0071] Generally, the dosage forms comprising the compounds of the present invention may comprise other pharmaceutically acceptable adjuvants, auxiliaries, fillers, solvents, additives, etc.

[0072] The pharmaceutical composition may comprise, for example, up to 99% by weight or up to 90% by weight or up to 80% by weight or up to 70% by weight of the pharmaceutical compound of the present invention, and the remainder is formed by pharmaceutically acceptable carriers and / or auxiliaries and / or solvents and / or optionally other pharmaceutically active compounds.

[0073] Pharmaceutically acceptable carriers, auxiliary substances or solvents, etc. are common pharmaceutical carriers, auxiliary substances or solvents, including various organic or inorganic carriers and / or auxiliary materials, as they are commonly used for pharmaceutical purposes, especially for solid pharmaceutical preparations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; edible flavors such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers such as citric acid, sodium citrate, acetic acid and polycarboxylic acids from the titriplex series such as diethylenetriaminepentaacetic 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 petrolatum, etc.

[0074] Liquid pharmaceutical preparations, such as solutions, suspensions, and gels, typically contain a liquid carrier, such as water and / or a pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also contain a pH regulator, an emulsifier or dispersant, a buffer, a preservative, a wetting agent, a gelling agent (such as methylcellulose), a dye, and / or a flavoring agent, as defined above. The compositions can be isotonic, i.e., they can have the same osmotic pressure as blood. The isotonicity of the compositions can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid compositions can be adjusted by a pharmaceutically acceptable thickening agent, such as methylcellulose. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickening agent will depend on the reagent selected.

[0075] Pharmaceutically acceptable preservatives can be used to increase the shelf life of the liquid compositions. Benzyl alcohol can be suitable, although a variety of preservatives can also be used, including, for example, parabens, thimerosal, chlorobutanol, and benzalkonium chloride.

[0076] Accordingly, another aspect of the invention relates to the compounds of the invention, including their pharmaceutically acceptable salts, solvates, hydrates, and polymorphs, and the drugs, compositions, and combination preparations containing them, which are used in oral administration form for the treatment of SCD as defined herein.

[0077] Dosage regimen

[0078] The compounds of the invention for the uses of the invention can be administered by one of the following dosage regimens:

[0079] In one aspect, the compounds according to the invention can be administered to a patient in need thereof at a dose of from 0.001 mg to 500 mg, for example, once to four times a day, preferably once or twice a day. However, the dose can be increased or decreased depending on the age, weight, condition, severity of the disease or type of administration of the patient. In another aspect of the invention, the compounds of the invention can be administered at a dose of 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg.

[0080] The preferred dose ranges from 0.5 mg to 500 mg, more preferably from 1 mg to 300 mg or from 3 mg to 300 mg, and even more preferably from 1 mg to 250 mg or from 5 mg to 250 mg.

[0081] The most preferred doses are 5 mg, 15 mg, 30 mg, 60 mg, 120 mg or 240 mg. Particularly preferred doses are 30 mg, 60 mg, 90 mg, 120 mg or 240 mg, and even more particularly preferred doses are 30 mg, 60 mg, 90 mg or 120 mg. The most preferred doses are 30 mg, 60 mg and 120 mg.

[0082] The above doses can be administered as a total daily dose as a single daily dose or as sub-doses divided into two or more daily administrations.

[0083] In another preferred aspect, a daily dose of 30 mg, 60 mg, 90 mg or 120 mg is preferred, which is administered once daily as a single dose. In another aspect, a 60 mg or 120 mg daily dose is administered twice daily as two 30 mg doses or two 60 mg doses, respectively. A 90 mg daily dose can also be administered as three 30 mg doses daily.

[0084] In another aspect, a dose of 0.001 mg / kg to 60 mg / kg body weight, 0.01 mg / kg to 60 mg / kg body weight, 0.1 mg / kg to 60 mg / kg body weight or between 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg up to a maximum of 60 mg / kg body weight can be administered. A dose of 120 mg or up to 240 mg can be administered to patients with a body weight ≥ 50 kg, and a dose of 60 mg can be administered to patients with a body weight ≥ 50 kg, both cases being administered once or twice daily. Preferably, a dose of 60 mg is administered to patients with a body weight ≥ 50 kg, and a dose of 30 mg is administered to patients with a body weight ≤ 50 kg, both cases being administered once or twice daily. Further preferably, the 30 mg and 60 mg doses as defined above are administered to patients with a body weight ≥ 50 kg and ≤ 100 kg.

[0085] Generally, it is possible and in another aspect preferred to adjust the dose according to body weight.

[0086] Also preferably, the compounds of the present invention are administered in an age-appropriate formulation. In particular, for pediatric dosage forms for patients with an age ≥ 2 years, specific administration forms are required, examples including syrups, solutions, drops or formulations for dissolving in liquid beverages.

[0087] In another aspect, one of the doses as defined above can be selected as the initial dose, and the same or different doses as defined above are administered one or more times at a repeat interval of 1 day to 7 days, 1 day to 5 days, preferably 1 day to 3 days or every two days.

[0088] The initial dose and subsequent doses can be selected from the doses defined above and adjusted / changed within the provided range according to the needs of the patient.

[0089] In particular, the amount of the subsequent dose can be appropriately selected according to the individual patient, the course of the disease and the treatment response. One, two, three, four, five, six, seven and more subsequent doses can be administered.

[0090] It is possible that the initial dose is equal to or different from the one or more subsequent doses. Further, it is possible that the subsequent doses are equal or different.

[0091] The repeat interval can be of the same length or can vary according to the individual patient, the course of the disease, and the treatment response.

[0092] Preferably, the amount of the subsequent doses decreases as the number of subsequent administrations increases.

[0093] Preferably, during a treatment period of at least 3 days, at least 5 days, at least 7 days, up to 4 weeks, a dose of 3 mg to 300 mg, more preferably 5 mg to 250 mg, most preferably 5 mg, 15 mg, 30 mg, 60 mg, 90 mg, 120 mg or 240 mg is administered once daily. In a further preferred aspect, a dose of 30 mg, 60 mg or 120 mg is administered once daily. In a further preferred aspect, the total daily dose of 30 mg, 60 mg or 120 mg is achieved by administering a dose of 15 mg, 30 mg or 60 mg twice daily, respectively.

[0094] In another aspect, the total daily dose of 240 mg is achieved by administering a dose of 120 mg twice daily.

[0095] In another aspect, different doses are administered for 1 week, 2 weeks, 3 weeks, 4 weeks or longer starting from an initial dose selected from the doses as defined above, and then increasing doses selected from the doses as defined above are administered for an additional 1 week, 2 weeks, 3 weeks, 4 weeks or longer. Depending on the treatment outcome, it is also possible to start administration at a higher initial dose within the time periods as defined above, and then start subsequent treatment intervals with decreasing doses. Preferably, an initial daily dose of 30 mg or 60 mg is administered for 4 weeks, followed by the same dose for another 4 weeks or followed by increasing doses for another 4 weeks. Such a treatment regimen can include administering 30 mg daily for 4 weeks, followed by administering 60 mg daily for an additional 4 weeks.

[0096] In particular, doses up to a total daily dose of 240 mg have been shown to be safe and well tolerated. The preferred dosing regimens also show rapid oral absorption, with levels detectable as early as 15 minutes to 30 minutes after administration. Even upon repeated dosing, the absorption levels can be maintained stable and no significant deposition has been observed.

[0097] It has further been demonstrated that the preferred dosing regimens effectively reduce the mean serum iron level and the calculated mean transferrin saturation, which indicates their efficiency in treating SCD.

[0098] Combination therapy

[0099] Another object of the present invention relates to a medicament or combination preparation (“combination therapy compound”) comprising a compound of the present invention and at least one other pharmaceutically active compound, which is preferably another active compound for the treatment of sickle cell disease (SCD). The combination therapy compound may be selected from active compounds for the prevention and treatment of iron overload and related symptoms, including iron chelating compounds, or compounds for the prevention and treatment of any condition, disorder or disease associated with or caused by iron overload. Suitable combination therapy compounds may be selected from pharmaceutically active compounds for the prevention and treatment of SCD, thalassemia, haemochromatosis, neurodegenerative diseases (such as Alzheimer's disease or Parkinson's disease) and related symptoms. Preferably, the at least one additional pharmaceutically active combination therapy compound is selected from drugs for the treatment of SCD, such as Hydroxyurea, Voxelotor, (crizanlizumab), L-glutamine oral powder (Endari), fetal hemoglobin (HbF) inducer, PDE9 inhibitor (such as IMR-687) and / or pain relief drugs. The most preferred combination therapy compound from the group of drugs for the treatment of SCD is fetal hemoglobin (HbF) inducer.

[0100] The at least one additional pharmaceutically active combination therapy compound may also be selected from drugs for reducing iron overload (such as Tmprss6-ASO) and iron chelators, especially curcumin, SSP-004184, Deferitrin, deferasirox, deferoxamine and deferiprone, as well as JAK2 inhibitors. The most preferred combination therapy compound from the group of iron chelating compounds is deferasirox.

[0101] Further preferred combination therapy compounds may be selected from drugs for the treatment of β-thalassemia, such as Luspatercept, LentiGlobin BB305 (a gene therapy developed by Bluebird Bio), synthetic human hepcidin (LJPC-401), hepcidin peptide mimetic PTG-300 and antisense oligonucleotides targeting Tmprss6 (IONIS-TMPRSS6-LRX).

[0102] On the other hand, the present invention relates to new uses and medical treatments as defined herein, in which a compound as defined herein is administered to a patient in need thereof in combination therapy with one or more of the combination therapy compounds as defined above, either in fixed doses or free doses, for sequential use. Such combination therapies include co-administering the compound defined by the present invention with the at least one additional pharmaceutically active compound (drug / combination therapy compound).

[0103] The combination therapy in fixed-dose combination therapy includes co-administering the compound defined herein with the at least one additional pharmaceutically active compound in a fixed-dose formulation.

[0104] The combination therapy in free-dose combination therapy includes co-administering the compound defined herein and the at least one additional pharmaceutically active compound in free doses of the respective compounds by simultaneously administering the individual compounds or by sequentially using the individual compounds distributed over a period of time.

[0105] In a particularly preferred embodiment, the combination therapy includes the oral co-administration of Compound 127 and a combination therapy compound from SCD drugs (preferably hydroxyurea and / or pain relief drugs).

[0106] Another embodiment of the present invention relates to the combination therapy described herein, wherein the pharmaceutical compound is selected from those compounds described in WO2020 / 123850A1, particularly one of the specific example compounds described below.

[0107] On the other hand, it relates to providing a novel combination therapy for treating SCD by administering Compound 127 as described herein in combination therapy with hydroxyurea, voxelotor, (crizanlizumab), L-glutamine oral powder (Endari), fetal hemoglobin (HbF) inducer, PDE9 inhibitor (e.g., IMR-687) and / or pain relief drugs. The most preferred combination therapy compound from the group of drugs for co-treatment of SCD with Compound 127 is the fetal hemoglobin (HbF) inducer.

[0108] Other useful compounds

[0109] Other useful compounds for treating sickle cell disease (SCD) are described in WO2017 / 068089, WO2017068090A1 and WO2018 / 192973. Thus, in some embodiments, a patient is treated by administering a compound of formula (I)

[0110]

[0111] wherein,

[0112] X 1 is N or O; and

[0113] X 2 is N, S or O;

[0114] provided that X 1 and X 2 are different;

[0115] R 1 is selected from

[0116] - hydrogen and

[0117] - optionally substituted alkyl;

[0118] n is an integer from 1 to 3;

[0119] A 1 and A 2 are independently selected from alkanediyl;

[0120] R 2 is

[0121] - hydrogen or

[0122] - optionally substituted alkyl;

[0123] or

[0124] A 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted 4- to 6-membered ring;

[0125] R 3 represents one, two or three optional substituents which may be independently selected from:

[0126] - halogen,

[0127] - cyano,

[0128] - optionally substituted alkyl,

[0129] - optionally substituted alkoxy and

[0130] - carboxyl;

[0131] R 4 is selected from

[0132] - hydrogen,

[0133] - halogen,

[0134] - C1-C3 alkyl, and

[0135] - halogen-substituted alkyl.

[0136] In some embodiments:

[0137] n = 1;

[0138] R 2 = hydrogen;

[0139] R 3 = hydrogen;

[0140] R 4 = hydrogen;

[0141] A 1 = methylene or ethane-1,2-diyl;

[0142] A 2 = methylene, ethane-1,2-diyl or propane-1,3-diyl;

[0143] or A 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted 4-membered ring, thereby forming a compound according to formula (II) or (III),

[0144]

[0145] wherein in formula (II) and (III)

[0146] m is an integer 1, 2 or 3, and

[0147] X 1 、X 2 and R 1 have the meanings defined for the compounds according to formula (I).

[0148] In some embodiments, treating a patient with a compound selected from:

[0149]

[0150]

[0151] and its pharmaceutically acceptable salts.

[0152] In a further preferred aspect, the present invention relates to new uses and methods of treatment as defined herein, wherein the compound of formula (I), (II) or (III) is selected from:

[0153]

[0154] and its pharmaceutically acceptable salts.

[0155] In a further preferred aspect, the present invention relates to new uses and methods of treatment as defined herein, wherein the compound of formula (I), (II) or (III) is selected from:

[0156]

[0157] and its pharmaceutically acceptable salts.

[0158] In some embodiments, the method comprises administering a compound selected from

[0159] and its pharmaceutically acceptable salts.

[0160] In Formulas I, II, and III, the substituents are defined as follows:

[0161] Optionally substituted alkyl preferably includes: straight-chain or branched-chain alkyl preferably containing from 1 to 8, more preferably from 1 to 6, particularly preferably from 1 to 4, and even more preferably 1, 2, or 3 carbon atoms, also denoted as C1-C4 alkyl or C1-C3 alkyl.

[0162] Optionally substituted alkyl also includes cycloalkyl preferably containing from 3 to 8, more preferably 5 or 6 carbon atoms.

[0163] Examples of alkyl residues having from 1 to 8 carbon atoms include: methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl 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, 5,5-dimethylhexyl group, 1-propylpentyl group, 2-propylpentyl group, etc. Those groups having from 1 to 4 carbon atoms (C1-C4 alkyl), such as especially methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl are preferred. C1-C3 alkyl, especially methyl, ethyl, propyl and isopropyl are more preferred. Most preferred are C1 and C2 alkyl, such as methyl and ethyl.

[0164] Cycloalkyl residues having from 3 to 8 carbon atoms preferably include: cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl group, cycloheptyl group and cyclooctyl group. Cyclopropyl group, cyclobutyl group, cyclopentyl group and cyclohexyl group are preferred. Cyclopropyl group is particularly preferred.

[0165] The substituents of the optionally substituted alkyl as defined above preferably include 1, 2 or 3 identical or different substituents selected from, for example: halogen as defined below, such as preferably F, cycloalkyl as defined above, such as preferably cyclopropyl, optionally substituted heteroaryl as defined below, such as preferably a benzimidazolyl group, optionally substituted amino as defined below, such as preferably an amino group or a carbobenzoxyamino group, a carboxyl group, an aminocarbonyl group as defined below, and an alkylene group such as especially a methylene group, forming, for example, a methylene-substituted ethyl group (CH3-(C═CH2)- or where * represents the binding site).

[0166] Halogen includes fluorine, chlorine, bromine and iodine, preferably fluorine or chlorine, and most preferably fluorine.

[0167] Examples of straight-chain or branched alkyl residues substituted by halogen and containing 1 to 8 carbon atoms include: fluoromethyl group, difluoromethyl group, trifluoromethyl group, chloromethyl group, dichloromethyl group, trichloromethyl group, bromomethyl group, dibromomethyl group, tribromomethyl group, 1-fluoroethyl group, 1-chloroethyl group, 1-bromoethyl group, 2-fluoroethyl group, 2-chloroethyl group, 2-bromoethyl group, difluoroethyl groups 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, 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, 2,2,3,3,4,4,4-heptafluorobutyl group, perfluorobutyl group, 2-fluoropentyl group, 2-chloropentyl 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, perfluoroheptyl group, etc. Fluoroalkyl, difluoroalkyl and trifluoroalkyl are particularly mentioned, and trifluoromethyl and mono- and difluoroethyl are preferred. Particularly preferred is trifluoromethyl.

[0168] Examples of cycloalkyl-substituted alkyl groups include the above-mentioned alkyl residues containing 1 to 3, preferably 1 cycloalkyl group, such as: cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2-cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, 2-cyclohexylethyl, 2-cyclopropylpropyl or 3-cyclopropylpropyl, 2-cyclobutylpropyl or 3-cyclobutylpropyl, 2-cyclopentylpropyl or 3-cyclohexylpropyl, 2-cyclohexylpropyl or 3-cyclohexylpropyl, etc. Preferred is cyclopropylmethyl.

[0169] Examples of the heteroaryl-substituted alkyl groups include the above-mentioned alkyl residues containing 1 to 3, preferably 1 (optionally substituted) heteroaryl group, such as pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, benzimidazolyl, thienyl or oxazolyl, such as pyridin-2-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-2-yl-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, thien-2-yl-methyl, thien-3-yl-methyl, 3-oxazol-2-yl-methyl.

[0170] Alkyl groups preferably substituted with benzimidazolyl, such as benzimidazol-2-yl-methyl and benzimidazol-2-yl-ethyl.

[0171] Examples of the amino-substituted alkyl residues include the above-mentioned alkyl residues containing 1 to 3, preferably 1 (optionally substituted) amino as defined below, such as aminoalkyl (NH2-alkyl) or mono- or dialkylamino-alkyl, such as aminomethyl, 2-aminoethyl, 2-aminopropyl or 3-aminopropyl, methylaminomethyl, methylaminoethyl, methylaminopropyl, 2-ethylaminomethyl, 3-ethylaminomethyl, 2-ethylaminoethyl, 3-ethylaminoethyl, etc., preferably 3-aminopropyl, or alkyl groups optionally substituted with alkoxycarbonylamino, such as groups according to the following formula

[0172] wherein R defines phenyl, thus forming benzyloxycarbonylaminopropyl.

[0173] Optionally substituted amino preferably includes: amino (-NH2), optionally substituted mono- or dialkylamino (alkyl-NH-, (alkyl)2N-), wherein for "alkyl" reference may be made to the definition of the above-mentioned optionally substituted alkyl. Preferred are mono- or dimethylamino, mono- or diethylamino and monopropylamino. Most preferred are amino (-NH2) and monopropylamino.

[0174] In addition, the carboxyl group represents the group [-(C=O)-OH] and the aminocarbonyl group represents the group [NH2-(C=O)-].

[0175] Optionally substituted alkoxy includes an optionally substituted alkyl - O - group, where reference may be made to the aforementioned definition of the alkyl group. Preferred alkoxy groups are straight - chain or branched - chain alkoxy groups having up to 6 carbon atoms, such as methoxy group, ethoxy group, n - propoxy group, isopropoxy group, n - butoxy group, isobutoxy group, sec - butoxy group, tert - butoxy group, n - pentyloxy group, isopentyloxy group, sec - pentyloxy group, tert - pentyloxy group, 2 - methylbutoxy group, n - hexyloxy group, isohexyloxy group, tert - hexyloxy group, sec - hexyloxy group, 2 - methylpentyloxy group, 3 - methylpentyloxy group, 1 - ethylbutoxy group, 2 - ethylbutoxy group, 1,1 - dimethylbutoxy group, 2,2 - dimethylbutoxy group, 3,3 - dimethylbutoxy group, 1 - ethyl - 1 - methylpropoxy group, and cycloalkoxy groups such as cyclopentyloxy group or cyclohexyloxy group. Methoxy group, ethoxy group, n - propoxy group, and isopropoxy group are preferred. Methoxy and ethoxy groups are more preferred. Particularly preferred is the methoxy group.

[0176] Optionally substituted alkanediyl is preferably a divalent straight - chain or branched - chain alkanediyl having 1 to 6, preferably 1 to 4, more preferably 1, 2, or 3 carbon atoms, which may optionally bear 1 to 3, preferably 1 or 2 substituents selected from halogen, hydroxy (-OH), oxo ((=O; forming a carbonyl or acyl group [-(C=O)-]) and alkyl groups as defined above, such as preferably methyl. The following may be mentioned as preferred examples: methylene, ethane - 1,2 - diyl, ethane - 1,1 - diyl, propane - 1,3 - diyl, propane - 1,1 - diyl, propane - 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. Particularly preferred are methylene, ethane - 1,2 - diyl, ethane - 1,1 - diyl, propane - 1,3 - diyl, propane - 2,2 - diyl, and butane - 2,2 - diyl. Most preferred are methylene, ethane - 1,2 - diyl, and propane - 1,3 - diyl.

[0177] Preferred substituted alkanediyl is a hydroxy - substituted alkanediyl such as hydroxy - substituted ethanediyl, an oxo - substituted alkanediyl such as oxo - substituted methylene or ethanediyl, thereby forming a carbonyl or acyl (acetyl) group, a halogen - substituted alkanediyl group.

[0178] In some cases, A having the meaning of a straight - chain or branched - chain alkanediyl group as defined above 1 and R having the meaning of an optionally substituted alkyl group as defined above 2Together with the nitrogen atom to which they are bonded, form an optionally substituted 4- to 6-membered ring, which ring may be substituted with 1 to 3 substituents as defined above. Thus, A 1 and R 2 may together be from a group according to one of the following formulas

[0179]

[0180] wherein preferably (substituted or unsubstituted) form a 4-membered ring, for example very particularly the group wherein the left binding site represents a direct binding site to the heterocyclic 5-membered ring between positions X 1 and X 2 in formula (I). The right binding site represents a binding site to the group A 2 having the meaning of alkanediyl as defined herein.

[0181] In formula (I) as defined herein, n has the meaning of an integer from 1 to 3, including 1, 2 or 3, and thus represents a methylene group, an ethane-1,2-diyl group or a propane-1,3-diyl group. More preferably n is 1 or 2, and even more preferably n is 1, representing a methylene group.

[0182] In some embodiments:

[0183] A) X 1 is N or O; and

[0184] X 2 is N, S or O;

[0185] provided that X 1 and X 2 are different;

[0186] thereby forming a 5-membered heterocycle according to the following formula,

[0187]

[0188] where * represents the binding site to the aminocarbonyl group and ** represents the binding site to the A 1 group.

[0189] B) n is an integer 1, 2 or 3; preferably n is 1 or 2, more preferably n is 1.

[0190] C) R 1 is selected from

[0191] - hydrogen and

[0192] - optionally substituted alkyl (as defined above);

[0193] Preferably R 1 is hydrogen or methyl, more preferably R1 is hydrogen.

[0194] D) R 2 is selected from

[0195] - hydrogen and

[0196] - optionally substituted alkyl (as defined above);

[0197] Preferably, R 2 is hydrogen or C1-C4 alkyl, more preferably R 2 is hydrogen or methyl, even more preferably R 2 is hydrogen.

[0198] E) R 3 represents one, two or three optional substituents, which may be independently selected from

[0199] - halogen (as defined above),

[0200] - cyano,

[0201] - optionally substituted alkyl (as defined above),

[0202] - optionally substituted alkoxy (as defined above) and

[0203] - carboxyl (as defined above);

[0204] Preferably, R 3 represents one or two optional substituents, which may be independently selected from

[0205] - halogen,

[0206] - cyano,

[0207] - alkyl (as defined above) (which may be substituted by one, two or three halogen atoms (as defined above)), optionally substituted alkoxy (as defined above) and

[0208] carboxyl (as defined above);

[0209] More preferably, R 3 represents one or two optional substituents, which may be independently selected from

[0210] - F and Cl,

[0211] - cyano,

[0212] - trifluoromethyl,

[0213] - methoxy and

[0214] - carboxyl;

[0215] Even more preferably, R3 is hydrogen, representing the unsubstituted terminal benzimidazole ring in formula (I).

[0216] F) R 4 is selected from

[0217] - hydrogen,

[0218] - halogen (as defined above),

[0219] - C1 - C3 alkyl and

[0220] - halogen - substituted alkyl (as defined above);

[0221] Preferably, R 4 is selected from

[0222] - hydrogen,

[0223] - Cl,

[0224] - methyl, ethyl, isopropyl and

[0225] - trifluoromethyl;

[0226] More preferably, R 4 is selected from

[0227] - hydrogen,

[0228] - Cl,

[0229] - methyl and

[0230] - trifluoromethyl;

[0231] Even more preferably, R 4 is selected from

[0232] - hydrogen,

[0233] - Cl and

[0234] - methyl;

[0235] Even more preferably, R 4 is hydrogen.

[0236] G) A 1 is alkanediyl;

[0237] Preferably, A 1 is methylene or ethane - 1,2 - diyl, and more preferably A 1 is ethane - 1,2 - diyl.

[0238] H) A 2 is alkanediyl;

[0239] Preferably, A 2 is methylene, ethane - 1,2 - diyl or propane - 1,3 - diyl;

[0240] More preferably, A 2 is methylene or ethane-1,2-diyl, and even more preferably A 2 is ethane-1,2-diyl.

[0241] I) or A 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted 4- to 6-membered ring as defined above;

[0242] wherein 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;

[0243] wherein 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).

[0244] The substituents of the following compounds (I) may in particular have the following meanings:

[0245] n has any of the meanings according to B) above, and the remaining substituents may have any of the meanings defined in A) and C) to I).

[0246] R 1 has any of the meanings according to C) above, and the remaining substituents may have any of the meanings defined in A) and B) and D) to I).

[0247] R 2 has any of the meanings according to D) above, and the remaining substituents may have any of the meanings defined in A) to C) and E) to H) or I).

[0248] R 3 has any of the meanings according to E) above, and the remaining substituents may have any of the meanings defined in A) to D) and F) to I).

[0249] R 4 has any of the meanings according to F) above, and the remaining substituents may have any of the meanings defined in A) to E) and G) to I).

[0250] A 1 has any of the meanings according to G) above, and the remaining substituents may have any of the meanings defined in A) to F) and H) or I).

[0251] A 2Has any of the meanings according to H) above, and the remaining substituents may have any of the meanings defined in A) to G) and I).

[0252] R 2 and A 1 Has any of the meanings defined in I), and the remaining substituents may have any of the meanings defined in A) to C), E), F) and H).

[0253] In some cases, X 1 is N or O; and X 2 is N, S or O; provided that X 1 and X 2 are different; R 1 is hydrogen; n is 1, 2 or 3; A 1 is methylene or ethane-1,2-diyl; A 2 is methylene, ethane-1,2-diyl or propane-1,3-diyl; R 2 is hydrogen or C1-C4 alkyl;

[0254] or

[0255] 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 one or two optional substituents, which may be independently selected from

[0256] -halogen,

[0257] -cyano,

[0258] -alkyl, which may be substituted by one, two or three halogen atoms,

[0259] -optionally substituted alkoxy and

[0260] -carboxyl;

[0261] R 4 is selected from

[0262] -hydrogen,

[0263] -Cl,

[0264] -methyl, ethyl, isopropyl and

[0265] -trifluoromethyl; or a salt thereof

[0266] In some cases, the salt is selected from salts of the compound of formula (I) with acids selected from 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 the compound (I) to the acid is 1-2:1-3.

[0267] In some cases of the compounds of general formula I: X 1 is N or O; and X 2 is N, S or O; provided that X 1 and X 2 are different; R 1 is hydrogen; n is 1 or 2; A 1 is methylene or ethane-1,2-diyl; A 2 is methylene, ethane-1,2-diyl or propane-1,3-diyl; R 2 is hydrogen or methyl;

[0268] or

[0269] A 1 and R 2 together with the nitrogen atom to which they are attached form an unsubstituted 4-membered ring;

[0270] R 3 represents one or two optional substituents, which can independently be selected from

[0271] -F and Cl,

[0272] -cyano,

[0273] -trifluoromethyl,

[0274] -methoxy and

[0275] -carboxyl;

[0276] R 4 is selected from

[0277] -hydrogen,

[0278] -Cl,

[0279] -methyl and

[0280] -trifluoromethyl;

[0281] In some cases, the salt is selected from the salts of the compound of formula (I) with an acid selected from 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 the compound (I) to the acid is 1 - 2:1 - 3.

[0282] In some embodiments of the compound of formula (I): X 1 is N or O; and X 2 is N, S or O; provided that X 1 and X 2 are different; R 1 is hydrogen; n is 1; A 1is methylene or ethane-1,2-diyl; A 2 is methylene, ethane-1,2-diyl or propane-1,3-diyl; R 2 is hydrogen;

[0283] or

[0284] A 1 and R 2 together with the nitrogen atom to which they are attached form an unsubstituted 4-membered ring; R 3 represents hydrogen, thereby forming an unsubstituted terminal benzimidazolyl ring; R 4 is selected from

[0285] - hydrogen,

[0286] - Cl and

[0287] - methyl or its salt;

[0288] wherein the salt is selected from the salts of the compound of formula (I) and an acid selected from 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 the compound (I) to the acid is 1 - 2:1 - 3.

[0289] In some embodiments of the compound of formula (I): X 1 is N or O; and X 2 is N, S or O; provided that X 1 and X 2 are different; R 1 is hydrogen; n is 1; A 1 is methylene or ethane-1,2-diyl; A 2 is methylene, ethane-1,2-diyl or propane-1,3-diyl; R 2 is hydrogen;

[0290] or

[0291] A 1 and R 2 together with the nitrogen atom to which they are attached form an unsubstituted 4-membered ring; R 3 represents hydrogen, thereby forming an unsubstituted terminal benzimidazolyl ring; and R 4 is hydrogen; or its salt.

[0292] In some embodiments, the salt is selected from the salts of the compounds of formula (I), (II), (III) or the compounds of WO2020 / 123850A1 as defined below and an acid selected from 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 the compound (I) to the acid is 1 - 2:1 - 3; and

[0293] In some embodiments of the compound of formula (I): n = 1; R 3 = hydrogen; R 4 = hydrogen; A 1 = ethane-1,2-diyl; A 2 = methylene, ethane-1,2-diyl or propane-1,3-diyl; R 2 = hydrogen; or A 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted 4-membered ring, thereby forming a compound of formula (II) or (III):

[0294]

[0295] wherein in formulae (II) and (III)

[0296] m is an integer 1, 2 or 3, and

[0297] X 1 、X 2 and R 1 have the meanings defined above in any embodiment comprising the compound of formula (I).

[0298] In particular, in formulae (II) and (III), X 1 and X 2 have the meanings defined in A).

[0299] In formula (II), R 1 and R 2 are preferably hydrogen.

[0300] In formula (III), R 1 is preferably hydrogen and m is preferably 2.

[0301] In another preferred embodiment of the compound of general formula (II): X 1 and X 2 are selected from N and O and are different; R 1 = hydrogen; R 2 = hydrogen; and m = 2.

[0302] The compounds (I), (II) or (III) that form salts or the compounds of WO2020 / 123850A1 as defined below are also referred to as "bases" or "free bases". The compounds of formula (I), (II) or (III) in free base form or the compounds of WO2020 / 123850A1 as defined below have at least one basic group, such as an amino group, to which an acidic group can bind.

[0303] Salts of the compounds of formula (I), (II) or (III) or the compounds of WO2020 / 123850A1 as defined below may be selected from salts having a ratio of base (compound (I), (II) or (III)) to acid of 1 - 2:1 - 3, wherein the acids for salt formation are selected as defined above.

[0304] These compounds may be mixed salts of the base (compound (I), (II) or (III)) with one or more of the above acids, which may have the same or different ratios (base:acid). These acids provide counter anions for the cationic forms of the compounds (I), (II) or (III).

[0305] Particularly preferred is the 3HCl salt of the above - mentioned compound.

[0306] In a particularly preferred embodiment, the method comprises administering the 3HCl salt of compound 127,

[0307]

[0308] In another aspect of the present invention, compound 127 may be administered in the form of one of the following salts:

[0309] 1:1 sulfate having the following formula

[0310]

[0311] 1:1 phosphate having the following formula

[0312]

[0313] 2:1 phosphate (hemiphosphate)

[0314]

[0315] Other compounds that act as ferroportin inhibitors as defined herein and are suitable for the treatment of SCD are those compounds described in WO2020 / 123850A1, which are incorporated herein by reference in their entirety. The specific compounds described in WO2020 / 123850A1 that are suitable for the treatment of SCD as defined herein may be selected from:

[0316]

[0317]

[0318] The compounds described in WO2020 / 123850A1 and selected from the above group can be provided as a novel combination therapy for treating SCD by administering these compounds in combination therapy with a fetal hemoglobin (HbF) inducer. In a preferred embodiment, the compound 2-(2-{[2-(1H-1,3-benzodiazol-2-yl)ethyl]amino}ethyl)-N-[(3-fluoropyridin-2-yl)methyl]-[1,3]oxazolo[4,5-c]pyridin-4-amine is provided in a manner of combination therapy with a fetal hemoglobin (HbF) inducer to treat SCD. BRIEF DESCRIPTION OF THE DRAWINGS

[0319] Figure 1 : Effect of compound 127 on RBC hemolysis in Townes mice. The figure shows plasma levels of cell-free Hb, heme, and lactate dehydrogenase (LDH). Hemolysis markers were measured using commercially available kits (cell-free Hb kit #CSB-E09632h, Cusabio; heme assay kit #MAK316, Sigma Aldrich) and according to the manufacturer's instructions. LDH in plasma was measured using a Hitachi automated clinical chemistry analyzer. Individual values and mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 9 - 10 mice / group.

[0320] Figure 2 : RBC indices in Townes mice treated with compound 127 or vehicle for 6 weeks. Individual values and mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 9 - 10 mice / group.

[0321] Figure 3 : Compound 127 corrected the elevated WBC count in Townes mice. Individual values and mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 5 - 10 mice / group.

[0322] Figure 4: Compound 127 reduced the sizes of the spleen and liver in Townes mice. Individual values and mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 9 - 10 mice / group.

[0323] Figure 5 : Total iron levels and 58 Fe levels in organs of Townes mice treated with compound 127 or vehicle for 6 weeks. Individual values and mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 8 - 10 mice / group.

[0324] Figure 6 : Compound 127 decreased the percentage of mature RBCs containing mitochondria in sickle mice treated with compound 127 for 6 weeks.

[0325] Figure 7 : Compound 127 decreased the plasma level of sVCAM-1 in Townes mice. sVCAM-1 was measured by ELISA. Individual values and mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 6 - 9 mice / group.

[0326] Figure 8 : Plasma iron was measured 3 hours after the last dose of compound 127 was administered on days 43 / 44. Individual values of mean ± SD are shown. Significant differences compared to the HbSS vehicle group are shown: *P < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison test).

[0327] Figure 9 : MCHC (left panel) and CHCM (right panel) of HbSS and HbAA mice were measured using a Siemens Advia 120 automated hematology analyzer on days 43 / 44. Individual values of mean ± SD are shown. Significant differences compared to the HbSS vehicle group are shown: *P < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison test).

[0328] Figure 10: On days 43 / 44, the percentages of hypochromic red blood cells (upper left), microcytic red blood cells (upper right), hyperchromic red blood cells (lower left), and macrocytic red blood cells (lower right) in male and female HbSS and HbAA mice were measured using a Siemens Advia 120 automated hematology analyzer. Individual values are shown as mean ± SD. Significant differences compared to the HbSS vehicle group are shown: *P < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison test).

[0329] Figure 11 : Total bilirubin and indirect bilirubin in the plasma of HbSS and HbAA mice were studied on days 43 / 44. Individual values are shown as mean ± SD. Significant differences compared to the HbSS vehicle group are shown: *P < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison test).

[0330] Figure 12 : Plasma levels of sP-selectin (left panel) and RANTES (right panel) in the plasma of HbSS and HbAA mice at the end of the study are shown. Individual values are shown as mean ± SD. Significant differences compared to the HbSS vehicle group are shown: *P < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison test).

[0331] Figure 13 : Plasma xanthine oxidase (XO) activity and intracellular reactive oxygen species (ROS) in whole blood of HbSS and HbAA mice. Plasma activity of XO and percentage of ROS-positive mature RBCs are shown as individual values of mean ± SD. Significant differences compared to the HbSS vehicle group are shown: *P < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Dunnett's multiple comparison test).

[0332] Figure 14 : Compound 127 reduced intracellular iron ([[]] 56 Fe and [[[]] 58 Fe) in RBCs of Townes mice. Intracellular [[[]] 56 Fe and [[[]] 58 Fe contents in washed RBCs of Townes mice were determined by ICP-MS and normalized to RBC count. Individual values are shown as mean ± SD. Statistical analysis was performed by comparing all treatment groups to the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, n = 6 - 9 mice / group.

[0333] Figure 15: Compound 127 reduced periportal inflammation and chemokine CXCL1 mRNA expression in the livers of Townes mice. The occurrence of periportal inflammation was evaluated on paraffin sections stained with H&E. The mRNA expression of CXCL1 in total liver RNA was evaluated by RT-qPCR. Individual scores or individual delta Ct values and the mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, **p < 0.01, ***p < 0.001, n = 7 - 11 mice / group.

[0334] Figure 16 : Compound 127 reduced biomarkers of liver injury. The activity of alanine transaminase (ALT) in plasma was measured using a Hitachi automatic clinical chemistry analyzer. Individual values of the mean ± SD are shown. Statistical analysis was performed by unpaired two-tailed Student's t-test, where *P < 0.05, **p < 0.01, ***p < 0.001, n = 9 - 10 mice / group.

[0335] Figure 17 : Compound 127 reduced IL-1β mRNA expression in the lungs of Townes mice. The mRNA expression of IL-1β in total lung RNA was evaluated by RT-qPCR. Individual delta Ct values and the mean ± SD are shown. Statistical analysis was performed by comparing all treatment groups with the HbSS vehicle group using one-way ANOVA with Dunnett's multiple comparison test, where *p < 0.05, n = 8 - 12 mice / group.

[0336] In Figures 1 to 17 ,"VIT-2763" refers to compound 127 (which exists in the form of its 3HCl salt).

[0337] DETAILED DESCRIPTION

[0338] Townes mice have been genetically engineered to specifically express human sickle hemoglobin (Ryan et al., Science, Vol. 247: p. 566, 1990). Townes mice have anemia, high reticulocyte counts, splenomegaly, vascular inflammation, and are prone to vaso-occlusion (VO) due to hypoxia, inflammation, and hemolysis.

[0339] The studies described below used homozygous male and female mice with human HbS (HbSS) and control mice (HbAA) expressing wild-type (WT) human hemoglobin HbA.

[0340] The first study ( Figures 1 to 7 ):

[0341] Mice were purchased from The Jackson Laboratory, USA (B6;129Hbbtm2(HBG1,HBB*)TOW / Hbbtm3(HBG1,HBB)Tow Hbatm1(HBA)TOW / J, strain number: 013071; "Townes mice"), 10 to 12 weeks of age, fed a low-iron diet (10 - 13 ppm iron, Granovit) and administered orally twice daily (bid) a dose of 60 mg / kg or 120 mg / kg body weight of compound 127 or vehicle (0.5% methylcellulose / water) for 6 weeks, excluding weekends. Between compound doses, mice had access to drinking water containing the stable iron isotope 58 57Fe (1 mM of 58 Fe(II)SO4, supplemented with 10 mM ascorbic acid as a reducing agent) to replace the iron present in the standard rodent diet (250 ppm iron). Labeled 58 57Fe was used to distinguish iron absorbed during the study period from that absorbed prior to the study.

[0342] The second study ( Figures 8 to 13 ):

[0343] Homozygous Townes mice for HbS (HbSS) (6 weeks old, The Jackson Laboratory, USA, strain #013071) were fed a low-iron diet (LID, Granovit, catalog number 2039, batch 0001906903, iron content 8.6 mg / kg) and administered orally twice daily (bid) a dose of 60 mg / kg of compound 127 or vehicle 0.5% methylcellulose (MC). After the first administration, mice had access to drinking water (DW) supplemented with 1 mM of 58 Fe(II) sulfate and 10 mM ascorbic acid for 6 hours. The concentration of 58 Fe(II) sulfate provided in the DW was adjusted to supplement dietary iron to the standard rodent diet level of 250 mg / kg iron content. Water without iron and ascorbic acid was provided during the remaining 18-hour period. Non-sickle Townes mice (HbAA) expressing normal human hemoglobin (wild type, WT) were administered vehicle twice daily (bid) and these mice were used as the control group. Administration of compound 127 or vehicle and subsequent exposure to water containing 58 57Fe was repeated for 44 days. Administration was suspended during weekends (WE), during which time mice had free access to LID and drank mineral water without 58 57Fe.

[0344] Plasma iron was measured using MULTIGENT Iron Assay (Abbott Diagnostics).

[0345] Hematological parameters were determined in whole blood samples collected on the last day of the study (day 43 / 44) and measured using a Siemens Advia 120 system.

[0346] Reactive oxygen species (ROS) in mature red blood cells (RBCs) were detected using the indicator chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Invitrogen, catalog number C6827) in RBCs labeled with APC-eFluor780-conjugated rat anti-mouse Ter119 and PE-conjugated rat anti-mouse CD71 antibodies (eBioscience, catalog numbers 47-5921-82 and 12-0711).

[0347] The activity of xanthine oxidase in plasma was measured using a xanthine oxidase activity assay kit (Sigma-Aldrich, catalog number MAK078).

[0348] Plasma bilirubin was measured by using an assay kit (Sigma-Aldrich, catalog number MAK126) according to the manufacturer's instructions.

[0349] sP-selectin and RANTES in plasma were measured by ELISA kits (R&D Systems, catalog numbers MVC00 and DY478-05 respectively) according to the manufacturer's instructions.

[0350] The activity of compound 127 in preventing vaso-occlusion (VO) to treat sickle cell anemia (sickle cell disease) can be determined by using the mouse models described in WO2018 / 192973, such as the mouse models described by Yulin Zhao et al. in "MEK1 / 2 inhibitors reverse acute vascular occlusion in mouse models of sickle cell disease"; and the mouse models described in FASEB Journal, Volume 30, Issue 3, pages 1171-1186, 2016. This mouse model can be appropriately adapted to determine the activity of compound 127 or compounds of other embodiments of the present invention in treating VO in sickle cell anemia. Appropriate adjustments can be made to the optimized test conditions, and these adjustments are within the routine work scope of those skilled in the art.

[0351] In the two studies described in Examples 1 to 17, the 3HCl salt of compound 127 was used.

[0352] Example 1: Compound 127 reduced the occurrence of hemolysis in Townes mice

[0353] As demonstrated by the elevated levels of cell-free Hb, heme, and LDH in the HbSS control group treated with solvent, red blood cells (RBCs) in Townes mice are prone to hemolysis( Figure 1 ). Notably, compound 127 significantly reduced the levels of cell-free Hb, heme, and LDH, indicating that the ferroportin inhibitory effect of compound 127 reduced hemolysis in Townes mice( Figure 1 ).

[0354] Example 2: Effect of compound 127 on RBC indices

[0355] The first study ( Figures 1 to 7 ):

[0356] Compared with HbAA mice, HbSS mice are anemic and have hematological parameters indicative of pathologically altered hemolytic anemia, such as reduced RBC count, Hb, and compensatory reticulocytosis, as well as elevated white blood cell count. After 6 weeks of treatment with compound 127, hematological parameters in fresh EDTA-blood were measured on an automated hematology analyzer. Compound 127 was orally administered to HbSS mice for six weeks, and the levels of total Hb, RBC count, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) decreased. A decrease in the concentration of HbS in RBCs of SCD patients is associated with reduced HbS aggregation and clinical benefit (Castro O. Am. J. Hematol., 1994).

[0357] The second study ( Figures 8 to 13 ):

[0358] Similar to the HbAA control group, Townes HbSS mice have plasma iron levels. In a second study, compound 127 was administered to HbSS mice at a dose of 60 mg / kg twice daily for 44 days, and plasma iron was measured 3 hours after the last dose as a marker of acute efficacy. The plasma iron level was significantly reduced in HbSS mice receiving compound 127, demonstrating the efficacy of compound 127 in inhibiting iron transport into the bloodstream( Figure 8 ).

[0359] In addition, the mean corpuscular hemoglobin concentration (MCHC) was significantly reduced in Townes mice treated with compound 127( Figure 9, (left panel). MCHC is calculated by dividing the mean Hb concentration in lysed blood by the hematocrit. In hemolytic diseases (such as SCD), due to the presence of free Hb in hemolyzed blood samples, the MCHC value may be falsely overestimated. To avoid potential artifacts due to excessive hemolysis in Townes mice, the concentration of HbS within RBCs was evaluated based on the mean Hb concentration of RBCs (CHCM) parameter. CHCM is determined by laser scattering and used to back-calculate cellular Hb, which reflects the hemoglobin content in intact RBCs. CHCM is not affected by hemolysis, and this value was significantly reduced in HbSS mice treated with compound 127, further demonstrating that the iron restriction by compound 127 reduces the concentration of HbS in the SCD model ( Figure 9 , (right panel). Additionally, scatter plot analysis of RBC distribution based on mouse volume and Hb concentration showed that in HbSS mice treated with compound 127, the percentage of hypochromic RBCs and microcytes increased significantly, while the percentage of macrocytes decreased ( Figure 10 ). Hematological analysis of blood samples from Townes mice showed that by blocking ferroportin, compound 127 induced iron-restricted erythropoiesis, which in turn reduced the concentration of HbS in RBCs. A reduction in the concentration of HbS in RBCs of SCD patients is associated with a decrease in HbS aggregation and clinical benefit (Castro O. Am. J. Hematol., 1994). Therefore, the reduction of HbS concentration by compound 127 may be a novel treatment for SCD.

[0360] As shown in the first study and demonstrated by the reduction of cell-free Hb, heme, and LDH, compound 127 reduced the occurrence of hemolysis in Townes mice ( Figure 1 ). Additionally, as shown in the second study, compound 127 reduced total bilirubin and indirect bilirubin, which are clinically relevant markers of hemolysis, further demonstrating the efficacy of this compound in reducing the occurrence of hemolysis ( Figure 11 ).

[0361] The interaction of sRBC, activated leukocytes, and free heme with the endothelium causes vascular inflammation and promotes vaso-occlusion and organ damage. Endothelial dysfunction in SCD is associated with elevated levels of soluble adhesion molecules such as sVCAM-1 and sP-selectin.

[0362] Two independent studies showed that compound 127 significantly reduced sVCAM-1 ( Figure 7 , the first study) and sP-selectin ( Figure 12 , left panel, the second independent study), indicating that compound 127 has the potential to reduce the occurrence of vascular inflammation and prevent vaso-occlusion in the Townes model of SCD.

[0363] Heme derived from RBC can serve as a damage-associated molecular pattern, activating the innate immune system and causing oxidant production, inflammation, vaso-occlusion, ischemia, and tissue damage (Belcher JD et al., J. Clin. Invest, 2006). In Townes mice and SCD patients, there is leukocytosis in the blood circulation, producing pro-inflammatory cytokines and chemokines, which attract additional inflammatory cells and activate the endothelium. For example, HbSS mice show elevated plasma levels of the chemokine RANTES (CCL5), which is involved in the recruitment of leukocytes to the site of inflammation. Treatment with compound 127 significantly reduced the RANTES level in HbSS mice ( Figure 12 , right panel), indicating that compound 127 not only reduced the leukocyte count in the blood circulation ( Figure 3 ) but also inhibited their pro-inflammatory activity.

[0364] The plasma activity of xanthine oxidase (XO) is upregulated in SCD patients and is defined as a source of increased production of vascular superoxide and hydrogen peroxide. Increased XO activity has also been reported in the plasma of Townes mice (Osarogiagbon UR et al., Blood, 2000; Aslan M et al., PNAS, 2001), which is considered an important source of ROS production and oxidative tissue damage. In fact, the XO activity in HbSS mice is significantly higher compared to HbAA mice. Compound 127 reduced the XO activity in the plasma of HbSS mice, indicating a reduction in vascular oxidative damage ( Figure 13 , left panel). In addition, flow cytometry analysis of intracellular ROS in blood cells using the fluorescent indicator CM-H2DCFDA showed that most RBCs in HbSS mice had high levels of ROS, while twice-daily (bid) administration with a dose of 60 mg / kg of compound 127 significantly reduced them ( Figure 13 , right panel).

[0365] The reduction of HbS concentration by compound 127 may have a positive effect on sRBC. Importantly, compound 127 significantly reduced the reticulocyte count, which is greatly increased in SCD due to the compensatory response to hemolysis ( Figure 2 ).

[0366] In summary, these data clearly demonstrate that compound 127 has the potential to reduce oxidative stress and vascular inflammation, which may result in reduced adhesion of blood cells to the vascular endothelium and ultimately prevent vaso-occlusive (VO) events in the Townes model of SCD.

[0367] Example 3: Effect of compound 127 on the WBC index

[0368] Leukocytosis in SCD is associated with increased incidence of pain crisis, acute chest syndrome, stroke, and death (Platt, NEJM, 1991). Unexpectedly, blood leukocyte counts were significantly reduced in Townes mice treated with compound 127, particularly neutrophils and lymphocytes( Figure 3 ). This data suggests that compound 127 may have a beneficial effect on inflammation in SCD.

[0369] Example 4: Compound 127 reduced the size of the spleen and liver in Townes mice

[0370] Due to stress erythropoiesis, the spleen in Townes mice is significantly enlarged (7-fold larger compared to WT). Notably, compound 127 reduced the size of the spleen in Townes mice, demonstrating that compound 127 promoted extramedullary erythropoiesis( Figure 4 , left panel). In addition, compound 127 corrected the increased liver weight in Townes mice to near WT levels( Figure 4 , middle panel). The weight of the kidneys in Townes mice was within the range of WT littermates and did not change after treatment with compound 127( Figure 4 , right panel).

[0371] Example 5: Compound 127 prevents organ iron loading and reduces total renal iron in Townes mice

[0372] Due to intravascular and extravascular hemolysis in defective RBCs, Townes mice deposit excessive iron in organs such as the liver, kidney, and spleen. It is estimated that approximately 1 / 3 of SCD hemolysis occurs intravascularly due to mechanical destruction of deformed and inelastic sRBCs, while the other 2 / 3 occurs extravascularly as a result of macrophage removal of abnormal sRBCs (Hebbel RP, Am. J. Hematol., 2011). In addition, anemia leads to upregulation of hypoxia-inducible factor (HIF)-2alpha in the intestine, which causes excessive iron absorption (Das N. et al., J Biol. Chem., 2015).

[0373] Oral administration in rodent PK studies showed that compound 127 is systemically administrable, indicating its ability to block iron export in all tissues expressing membrane ferroportin, including the duodenum (dietary iron absorption), liver (iron storage in hepatocytes and macrophages), and spleen (iron formation in macrophages from senescent red blood cells). To distinguish the effects of compound 127 on pre-existing and newly absorbed iron in organs, mice were provided with drinking water containing stable iron isotopes during the study period 58Drinking water of Fe. The total iron content and 58 Fe content in the organs of HbSS mice treated with the solvent or compound 127 were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Compound 127 did not alter the total hepatic iron concentration and splenic iron content in Townes mice, which is consistent with the inhibition of ferroportin-mediated iron efflux from these tissues ( Figure 5 , upper left and middle panels). Surprisingly, compound 127 significantly decreased the total iron concentration in the kidneys of Townes mice ( Figure 5 , upper right panel). Abnormal renal iron metabolism and cortical iron deposition are characteristics of SCD and are associated with renal complications (Vazquez-Meves G et al., Blood, 2016). Thus, the reduction of renal iron content by compound 127 may have a beneficial effect on renal function in SCD.

[0374] Importantly, compared to solvent-treated mice, the 58 Fe concentrations in the liver, kidneys, and spleen of Townes mice administered compound 127 were significantly reduced, indicating that compound 127 prevented further organ iron deposition ( Figure 5 , second row of figures).

[0375] Example 6: Compound 127 reduced apoptotic markers and increased mitochondrial clearance in mature sRBCs of Townes mice

[0376] Polymerization of HbS triggers the formation of free radicals, dehydration, and membrane damage in sRBCs, which may lead to apoptosis. Apoptotic RBCs expose phosphatidylserine (PS) to the extracellular space, which is a signal for targeted RBC phagocytosis. PS exposure on RBCs was measured by annexin V staining of RBCs and flow cytometry. Compound 127 decreased PS exposure on sRBCs in a dose-dependent manner, indicating that iron restriction improved the cell membrane organization of RBCs and increased the potential survival rate ( Figure 6 , left panel).

[0377] RBC precursors in healthy individuals eliminate their mitochondria through mitophagy during terminal differentiation. Abnormal retention of mitochondria in mature RBCs of SCD patients and SCD mice has been reported (Jagadeeswarn et al., 2017). The retention of mitochondria in RBCs was studied by flow cytometry using Ter119 and CD71 antibodies to distinguish the mature state of RBCs and MitoTracker to detect mitochondria. Notably, RBCs in Townes mice receiving two doses of compound 127 had a lower proportion of mature RBCs containing mitochondria ( Figure 6, (right figure). Mouse models with specific deletions in mitophagy genes showed that due to mitochondrial retention, the survival rate of RBCs decreased (Sandoval et al., 2008; Mortensen et al., 2010). This fully indicates that the reduction of the proportion of RBCs with mitochondrial retention by compound 127 in SCD may have a positive effect on RBC lifespan.

[0378] Example 7: Compound 127 reduced vascular inflammation markers in the Townes model of SCD

[0379] The interaction of sRBCs, activated white blood cells, and free heme with the endothelium causes vascular inflammation and promotes vaso-occlusion (VO) and organ damage. Endothelial dysfunction in SCD is associated with elevated levels of soluble adhesion molecules such as soluble vascular cell adhesion molecule 1 (sVCAM-1). This is consistent with a decrease in hemolysis ( Figure 1 ) and a decrease in white blood cell count ( Figure 3 ). Treatment with compound 127 significantly reduced the level of sVCAM-1 in Townes mice ( Figure 7 ). This data demonstrates the potential of compound 127 to reduce the occurrence of vascular inflammation in the Townes model of SCD, which may prevent VO events.

[0380] Example 8: Determination of the activity of compound 127 in treating VO in sickle cell disease in a mouse model

[0381] The method described in WO2018 / 192973 can be used to determine the activity of the membrane iron transporter inhibitor compounds of the present invention.

[0382] By using the mouse models described by Yulin Zhao et al. in "Yulin Zhao et al. in "MEK1 / 2 inhibitors reverse acute vascular occlusion in mouse models of sickle cell disease"; and the mouse models described in FASEB Journal, Vol. 30, No. 3, pp. 1171-1186, 2016, the activity of compound 127 in treating sickle cell anemia can be determined as follows:

[0383] Vascular occlusion (VO) crisis is a major cause of morbidity and mortality in patients with SCD. Hypoxia, dehydration, inflammation, or hemolysis all contribute to increased adhesion of sickle red blood cells, neutrophils, and platelets to activated endothelium in small blood vessels, thereby promoting coagulation, vascular occlusion, pain crises, and irreversible damage to multiple organs. High white blood cell counts, particularly high counts of activated neutrophils, are associated with early death, silent cerebral infarcts, hemorrhagic stroke, and acute chest syndrome in patients with SCD (Platt OS, NEJM, 1994). Hemolysis in SCD is caused by damaged sickle RBC membranes, resulting in chronic anemia and release of Hb into the blood circulation, which promotes inflammation by consuming nitric oxide, generating oxidative stress, and releasing heme. Sickle RBCs release microvesicles that trigger endothelial cells to produce reactive oxygen species (ROS), promote leukocyte adhesion, and induce endothelial cell apoptosis in a phosphatidylserine-dependent manner, leading to acute VO in SCD (Camus M, Blood, 2012).

[0384] Based on this data, it is hypothesized that the compounds of the present invention may alleviate VO in SCD by reducing hemolysis occurrence in sickle RBCs and continuously preventing leukocyte adhesion to endothelium.

[0385] To test this hypothesis, the vehicle or compound of the present invention was orally administered at a dose of 30 mg / kg or 100 mg / kg twice daily (BID) for 4 weeks in the Townes mouse model of SCD (Ryan T, Science, 1990). These mice are genetically engineered to specifically express human hemoglobin (hα / hα::βS / βS, Jackson Laboratory). Townes mice are characterized by anemia, high reticulocyte counts, splenomegaly, vascular inflammation and are prone to vaso-occlusion (VO) due to hypoxia, inflammation and hemolysis. To study the effect of the ferroportin inhibitor on the adhesion of leukocytes and sickle RBCs to the inflamed endothelium, Townes mice treated with vehicle or ferroportin inhibitor for 25 days were anesthetized as previously described and a chamber was surgically implanted into the dorsal skin fold of the mice under sterile conditions (Kalamur VS et al., Am J Hematol., 2004; Zennadi, R et al., Blood, 2007). Three days after surgery, the mice were injected with 0.5 μg of TNFα (R&D Systems) to induce inflammation leading to the occurrence of VO. Ninety minutes after TNFα administration, leukocytes and red blood cells were labeled in vivo by intravenous injection of rhodamine-conjugated Ly6G (Sigma) and phycoerythrin-conjugated anti-TER119 mAb (BioLegend), respectively. As previously described (Zhao et al., FASEB J, 2016), the adhesion of leukocytes and red blood cells to the microvascular endothelium was monitored by fluorescence intravital microscopy over the next 90 minutes. Briefly, the anesthetized animals with chambers were maintained at a temperature of 37 °C and blood flow and cell adhesion events were recorded using a digital camera C2400 (Hamamatsu Photonics KK, Hamamatsu City, Japan) connected to a fluorescence microscope (Axoplan microscope, Carl Zeiss). Twenty to thirty microcapillary segments were examined per mouse and cell adhesion was quantified on static images by measuring the fluorescence intensity of the fluorescently labeled adhered cells using ImageJ software. Results were expressed as fluorescence units per million cells.

[0386] Conclusion:

[0387] In summary, iron restriction by oral administration of the ferroportin inhibitor compound 127 significantly reduced the occurrence of hemolysis, oxidative stress, vascular inflammation and systemic inflammation and improved the morphology of RBCs, thus alleviating vaso-occlusive events and improving hemodynamics in the Townes model of SCD.

[0388] Ferroportin inhibitors can prevent acute vaso-occlusion and organ damage in a mouse model of sickle cell disease.

[0389] Example 9( Figure 14):RBC iron content

[0390] Sickle RBCs contain several discrete iron compartments, including denatured hemoglobin and free heme, as well as molecular iron associated with membrane phospholipids. Abnormal iron deposition on the sickle RBC membrane is thought to promote oxidative damage to the membrane structure and lead to its dysfunction (Browne P, Shalev O, Hebbel RP., The molecular pathobiology of cellmembrane iron: the sickle red cell as a model., Free Radic Biol Med., April 1998; Vol. 24, No. 6, pp. 1040 - 8). In addition, high concentrations of HbS (MCHC and CHCM) in sickle RBCs are also associated with cumulative membrane abnormalities, including oxidative damage possibly caused by increased membrane iron. Therefore, a decrease in the total intracellular iron content in sickle RBCs may be beneficial for SCD. The intracellular 56 Fe and 58 Fe contents in washed RBCs from Townes mice were measured by ICP - MS and normalized to RBC count. Compared with HbAA mice, vehicle - treated HbSS mice had significantly higher levels of intracellular 56 Fe and 58 Fe, despite having lower hemoglobin levels, indicating abnormal iron deposition. Treatment with compound 127 normalized the intracellular iron content in the RBCs of Townes mice. A decrease in the intracellular iron content in RBCs may be directly related not only to a decrease in Hb content (decreases in MCHC and CHCM were observed) but also to a decrease in membrane iron deposition. This result emphasizes the potential of compound 127 to minimize the harmful toxic effects of free iron on RBCs in SCD.

[0391] Example 10( Figure 15 ):Liver inflammation

[0392] Vascular inflammation caused by the interaction of sRBC, activated white blood cells, and free heme with the endothelium promotes vascular occlusion (VO) and organ damage. Compound 127 significantly reduced vascular inflammation and leukocyte markers in peripheral blood, indicating its potential to reduce inflammation, VO, and subsequent organ damage. To assess organ damage, liver lobes isolated from Townes mice treated with Compound 127 for 6 weeks were fixed in formalin and paraffin sections were stained with hematoxylin and eosin (H&E) for histological examination. The main pathological findings included mononuclear cell inflammatory infiltration in the periportal area, which was subsequently scored by a pathologist. The following histology was used for scoring: no inflammation (0), occasional portal tracts showing mild to moderate inflammation (1), moderate number of portal tracts showing mild to moderate inflammation (2), and large number of portal tracts showing marked inflammation (3). Histopathological evaluation showed a significant reduction in periportal inflammation in Townes mice treated with Compound 127 compared to vehicle-treated Townes mice, further demonstrating the efficacy of the compound in reducing the inflammatory state in SCD.

[0393] Chemokine CXCL1 is a key inflammatory mediator in acute VO crisis in SCD mice (Jang JE., CXCL1 and its receptor, CXCR2, mediate murine sickle cell vaso-occlusion during hemolytic transfusion reactions., J Clin Invest., 2011; Vol. 121, No. 4, pp. 1397-1401). CXCL1 is expressed by activated endothelial cells and is involved in the hepatic recruitment of neutrophils (Hilscher MB., "Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension", Gastroenterology, 2019; Vol. 157, No. 1: pp. 193-209), and it plays an important pathological role in SCD crisis. Compared with vehicle-treated HbAA mice, vehicle-treated Townes mice showed an upward trend in CXCL1 mRNA expression in the liver, which indicated the recruitment of neutrophils in the liver tissue, consistent with the above histopathological analysis. Compound 127 significantly reduced the expression of CXCL1 in the liver of HbSS mice, indicating its protective function against liver inflammation in Townes mice.

[0394] Example 11( Figure 16 ) : ALT

[0395] Liver disease is an important cause of morbidity and mortality in SCD patients. The Townes mouse model of SCD is known to recapitulate hepatocyte injury, which is reflected by an increase in plasma alanine aminotransferase (ALT) levels, a clinically relevant biomarker of liver injury (Aslan M., "Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease", Proc Natl Acad Sci U S A, 2001; Vol. 98, No. 26, pp. 15215-15220). Importantly, compound 127 significantly reduced the plasma ALT levels in Townes mice, highlighting the potential of the compound to reduce VO tissue damage.

[0396] Example 12( Figure 17 ):Pulmonary inflammation

[0397] Acute chest syndrome (ACS) is a pulmonary complication in patients with SCD and significantly overlaps with pneumonia. ACS is the second most common cause of hospitalization in patients and the leading cause of death in patients with SCD. Vaso-occlusive crisis (VOC) usually occurs before ACS and is characterized by RBC sickling, excessive cell adhesion, and hemolysis (Novelli EM., Crises in Sickle Cell Disease. Chest., 2016; Vol. 149, No. 4, pp. 1082-1093). Pro-inflammatory cytokine levels are elevated in the sera of sickle cell patients and are associated with pain crises and VO. In particular, IL-1β is a pro-inflammatory cytokine released by activated monocytes that can induce endothelial cell activation and plays a driving role in the pathophysiology of pulmonary microvascular occlusion in SCD (Pathare A. Cytokines in Sickle Cell Disease., Hematology, 2003; Vol. 8, No. 5, pp. 329-337).

[0398] Compared with vehicle-treated HbAA mice, vehicle-treated Townes mice showed an upward trend in IL-1β mRNA expression in the lungs. Administration of compound 127 decreased the expression of IL-1β in the lungs of HbSS mice. Although the decrease in IL-1β expression in compound-treated mice was not significant, mainly due to the high variability of vehicle-treated HbSS mice, this trend was consistent in independent experiments, indicating that compound 127 has the potential to alleviate pulmonary inflammation and may prevent the occurrence of pulmonary VO and ACS in SCD.

[0399] Other examples

[0400] It should be understood that although the present invention has been described in connection with the detailed description of the present invention, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Use of the 3HCl salt of the compound shown by the following formula for the preparation of a medicament for preventing or treating vascular inflammation and vaso-occlusion in a patient suffering from sickle cell disease, 2. The use according to claim 1, wherein the sickle cell disease is selected from HbSS; HbSC; HbSβ0 thalassemia; HbSβ+ thalassemia, HbSD, HbSE and HbSO.

3. The use according to claim 1 or 2, wherein the compound is in an oral administration form.

4. The use according to any one of claims 1 or 2, wherein the compound is in the form of a filled capsule for oral administration.

5. The use according to claim 1, wherein the medicament is a combination therapy composition, wherein the compound as defined in claim 1 and at least one further pharmaceutically active compound are present in the form of a fixed dose or a free dose combination for co-administration of the compounds in a sequential use manner.

6. The use according to claim 5, wherein the at least one further pharmaceutically active compound is selected from sickle cell disease drugs.

7. The use according to claim 5, wherein the at least one further pharmaceutically active compound is selected from hydroxyurea and / or pain relief drugs.

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