WO2025040685A1

WO2025040685A1 - Quinones for use in the treatment of red blood cell enzymopathies

Info

Publication number: WO2025040685A1
Application number: PCT/EP2024/073371
Authority: WO
Inventors: Magnus Joakim HANSSON; Mats Eskil ELMÉR; Steven James Moss
Original assignee: Abliva Ab
Priority date: 2023-08-21
Filing date: 2024-08-20
Publication date: 2025-02-27
Also published as: WO2025040686A1

Abstract

Class of quinones for use in the treatment restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells. Specific example is use in the treatment of red blood cell enzymopathies, especially in the treatment of defects in red blood cell metabolism such as defects in red blood cell glycolysis. The treatment increases ATP in non-mitochondrial cells. Thus, the present invention relates to a class of quinones for use in the treatment of diseases or conditions associated with or caused by defects in red blood cell glycolysis.

Description

QUINONES FOR USE IN THE TREATMENT OF RED BLOOD CELL ENZYMOPATHIES

FIELD OF THE INVENTION

The present invention relates to the finding that classes of quinones have the ability of restoring, normalizing and/or improving ATP in cells lacking mitochondria such as red blood cells (RBC). Red blood cells depend solely on anaerobic conversion of glucose by the Embden-Meyerhof pathway (glycolysis) for the generation and storage of high-energy phosphates such as ATP, which is necessary for a number of vital functions. Thus, the present invention relates to classes of quinones for use in the treatment of diseases or conditions associated with or caused by defects in red blood cell glycolysis.

BACKGROUND

Human erythrocytes are unique in that they anucleate when mature. Immature erythrocytes have nuclei but during early erythropoiesis prior to becoming circulating reticulocytes they extrude nuclei as well as other organelles such as mitochondria, endoplasmic reticulum, and Golgi apparatus, in order to make room for oxygen-carrying hemoglobin. As a result of lacking mitochondria, mature red blood cells do not utilize any of the oxygen they transport to economically synthesize adenosine phosphate (ATP) as other normal differentiated cells do. Instead, red blood cells depend entirely on anaerobic glycolysis to cycle nicotinamide adenine dinucleotide (NAD+) and to make ATP, an essential energy source largely used to drive ATPase-dependent K⁺/Na⁺ and Ca²⁺ pumps, in order to maintain cell membrane integrity and pliability as they navigate through blood vessels.

During the intravascular lifespan of the red blood cells, they require energy to maintain a number of vital functions including maintenance of glycolysis. Because of the lack of nuclei and mitochondria, mature red blood cells are incapable of generating energy via the oxidative Krebs cycle. Instead, red blood cells depend on anaerobic conversion of glucose by Embden-Meyerhof pathway for the generation and storage of ATP.

Red blood cell enzymopathies may be in any of the steps in the glycolysis of the red blood cell and may lead to various diseases. There is a need to identify substances that can treat red blood cell enzymopathies and diseases caused by or associated with enzymopathies.

The present invention is based on the unique finding that classes of quinones were found to increase the ATP level in red blood cells, and to increase the ATP level in red blood cells wherein the enzyme pyruvate kinase (PK) was inhibited. SUMMARY

In its broadest aspect, the present invention relates to quinone compounds that can be used in restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells. The quinone compounds may be used in conditions/diseases requiring restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells. Specific examples are the use of the quinones in red blood cell enzymopathies and in diseases caused by or associated with enzymopathies. It is contemplated that the quinone compounds also may be effective in hemolytic anemias caused by

• Defective red blood cell metabolism (as in red blood cell glycolysis and pentose phosphate pathway including defects in glucose-6-phosphate dehydrogenase and pyruvate kinase or other enzymes being part of red blood cells metabolism).

Hemolytic anemias are associated with a decreased survival of erythrocytes in circulation where ATP boosting can be argued to increase red blood cell survival by enhanced glycolysis and ATP production. In addition, several of these diseases may also go with ineffective erythropoiesis where enhancing glycolysis and ATP production similarly can be expected to improve erythropoiesis. The latter reason is the primary cause of anemia caused by chronic diseases, myelodysplastic syndrome and sideroblastic anemia.

In specific aspects the invention relates to quinone compounds of Formula (I) or reduced forms thereof, that can be used in a) restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells, and/or b) in treatment of red blood cell enzymopathies, and/or c) in the treatment of defects in red blood cell metabolism such as defects in red blood cell glycolysis, and/or d) in red blood cell enzymopathies that involve a pyruvate kinase (PK) deficiency in red blood cells, and/or e) in red blood cell enzymopathies that involve a pyruvate kinase (PK) deficiency in red blood cell and wherein the PK deficiency is associated with a lack of or decrease in R-type PK (PKR) activity in red blood cells, and/or f) in the treatment of hemolytic anemia caused by defective red blood cell metabolism (as in red blood cell glycolysis and pentose phosphate pathway including defects in glucose-6- phosphate dehydrogenase and pyruvate kinase). More specifically, the invention relates to a compound of formula (I) or a reduced form thereof, a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof

formula (I), wherein,

Xi, is C, or N;

X2, X3 and X4 are independently C, N, O or S, whereby at least two of Xi, X2, X3, and X4 are selected from N, O or S, provided that Xi and X4 are not both N; or

Xi is C, X2 is O, X3 is C(CH3), and X4 is CH2CH2 and the resultant 6-membered heterocycle is not aromatic;

R1, is one or more selected from the group consisting of H, C1-6 alkyl, C1-6 alkyloxy, Ce-io aryl, heteroaryl, halo, nitro, hydroxy, cyano and NRsRs;

R2, R3 and R4 are independently not present if required by the valence of X2, X3 or X4 respectively, or independently selected from the group consisting of H, OH, alkyl, alkyloxy, Ce-io aryl and heterocyclyl, wherein the alkyl may be substituted with Ce-io aryl and the heterocyclyl may be substituted with C(O)Rs;

Re and Re are each independently selected from the group consisting of H, alkyl and C(O)R?, or R5 and Re, are joined with each other to form a heterocyclyl including at least one nitrogen atom in the cycle;

R7 is alkyl;

Rs is alkyloxy; alkyl is any C1-10 linear or branched, or C3-7 cyclic alkyl; alkyloxy is any C1-10 linear or branched alkyloxy, or C3-7 cyclic alkyloxy; heterocyclyl is 3- to 7-membered heterocyclic group having in the cycle at least one hetero atom selected from the group consisting of N, O and S; heteroaryl is 5- to 10 -membered aromatic cyclic group having in the cycle at least one hetero atom selected from N, O and S, and when the aryl or heteroaryl is substituted, its substituent is each at least one selected from the group consisting of halo, alkyl, halo-substituted alkyl and alkyloxy; and — or ^::: is a single bond or a double bond depending on R2, R3, R4, Xi, X2, X3, and X4, for use in the treatment of restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells. A specific example is the use in the treatment of red blood cell enzymopathies, especially in the treatment of defects in red blood cell glycolysis

In the present context, a reduced form of a quinone of formula (I) is either a semiquinone or a hydroquinone thereof, i.e. where either one or both oxygens in the quinone is reduced to -OH.

Specific quinones for use according to the present invention include

or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof,

beta-lapachone, or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof.

Preferably, the present invention relates to the use of KL1333.

As demonstrated in the Examples herein, the quinone compounds have been shown to increase the ATP levels in red blood cells. Moreover, the content of NAD(P)+ in red blood cells increases and reactive oxygen species decreases. Overall, this leads to a more favorable metabolic status and redox status for the red blood cells. DETAILED DESCRIPTION

As mentioned above, the present invention relates to a compound of formula (I) or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof

formula (I), wherein,

Xi, is C, or N;

R5 and Re are each independently selected from the group consisting of H, alkyl and C(O)R?, or R5 and Re, are joined with each other to form a heterocyclyl including at least one nitrogen atom in the cycle;

R7 is alkyl;

Rs is alkyloxy; alkyl is any C1-10 linear or branched, or C3-7 cyclic alkyl; alkyloxy is any C1-10 linear or branched alkyloxy, or C3-7 cyclic alkyloxy; heterocyclyl is 3- to 7-membered heterocyclic group having in the cycle at least one hetero atom selected from the group consisting of N, O and S; heteroaryl is 5- to 10 -membered aromatic cyclic group having in the cycle at least one hetero atom selected from N, O and S, and when the aryl or heteroaryl is substituted, its substituent is each at least one selected from the group consisting of halo, alkyl, halo-substituted alkyl and alkyloxy; and — or ^::: is a single bond or a double bond depending on R2, R3, R4, Xi, X2, X3, and X4, for use in the treatment of restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells.

Specifically, the cells lacking mitochondria are red blood cells.

Specific examples are the use of a quinone of formula I or a reduced form thereof in the treatment of red blood cell enzymopathies. Typically, red blood cell enzymopathies are defects in red blood cell metabolism such as defects in red blood cell glycolysis. In particular, red blood cell enzymopathies involve a pyruvate kinase (PK) deficiency in red blood cells.

Typically, the PK deficiency is associated with a lack of or decrease in R-type PK (PKR) activity in red blood cells.

In embodiments, the quinone compound according to formula (I) is a compound, wherein

Xi, is C, or N;

X2, X3 and X4 are independently C, N, O or S, whereby at least two of Xi, X2, X3, and X4 are selected from N, O or S, provided that Xi and X4 are not both N, and

Ri-Rs are as defined above for formula (I), and/or a compound, wherein

Xi, is C;

X2 is O, X3 is C(CH3), and X4 is CH2CH2 and the resultant 6-membered heterocycle is not aromatic;

R2 is not present; and

R1, Rs-Rs are as defined above for formula (I).

Xi and X3 are each C;

X2 and X4 are each N, and

Ri-Rs are as defined above for formula (I), and/or a compound, wherein

R3 is alkyl, wherein the alkyl may be substituted with Ce-io aryl, and

R1, R2 and R4-R8 are as defined above for formula (I), and/or a compound, wherein

R2, R3 and R4 are independently not present if required by the valence of X2, X3 or X4 respectively, or R2, R3 and R4 are independently selected from the group consisting of H, OH, and alkyl, and

R1, and R4-Rs are as defined above for formula (I), and/or a compound, wherein

R1 is H, and

R3 is a C1-C6 straight or branched alkyl, and

R2, R4, and Rs are as defined above for formula (I), and/or a compound, wherein

R2 is not present or H;

R4 is not present or H; and

R1, R3 and Rs-Rs are as defined above for formula (I), and/or a compound, wherein

Xi and X3 are each C;

X2 and X4 are each N;

R2 is not present or H;

R4 is not present or H; and

Xi and X3 are each C;

X2 and X4 are each N;

R1 is H;

R2 is not present or H;

R3 is branched or straight Ci-Cs alkyl; F is not present or H.

The compound according to formula (I) may be KL1333 having formula (II):

formula (II) or a reduced form thereof, of a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof, or it may be a compound having the following formula (III)

beta-lapachone, formula (III) or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer or a prodrug thereof.

Use of the quinones in restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells.

Specific example is use in the treatment of red blood cell enzymopathies, especially in the treatment of defects in red blood cell metabolism such as defects in red blood cell glycolysis.

As mentioned herein before, the present invention relates to quinone compounds that can be used in restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells. Specific example is use in the treatment of red blood cell enzymopathies, especially in the treatment of defects in red blood cell metabolism such as defects in red blood cell glycolysis. As mentioned herein before, red blood cells depend solely on anaerobic conversion of glucose by the Embden-Meyerhof pathway (glycolysis) for the generation and storage of high-energy phosphates such as ATP, which is necessary for a number of vital functions. The enzymopathies are defects in red blood cell glycolysis. Thus, red blood enzymopathies are enzyme defects in red blood cell glycolysis.

It is contemplated that the quinone compounds also may be effective in hemolytic anemias caused by Defective red blood cell metabolism (as in red blood cell glycolysis and pentose phosphate pathway including defects in glucose-6-phosphate dehydrogenase and pyruvate kinase).

Hemolytic anemias are associated with a decreased survival of erythrocytes in circulation where ATP boosting can be argued to increase red blood cell survival by enhanced glycolysis and ATP production. In addition, several of these diseases may also go with ineffective erythropoiesis where enhancing glycolysis and ATP production similarly can be expected to improve erythropoiesis.

In specific aspects the invention relates to quinone compounds that can be used in a) the treatment of red blood cell enzymopathies, b) the treatment of red blood cell enzymopathies, which involve an enzyme deficiency in red blood cell glycolysis, c) the treatment of red blood cell enzymopathies, which involve a pyruvate kinase (PK) deficiency in red blood cells, d) restoring, normalizing and/or increasing ATP levels in red blood cells, e) the treatment of PK deficiency associated with a lack of or a decrease in R-type PK (PKR) activity in the blood,

Anemia is a blood disorder in which blood has a reduced ability to carry oxygen due to a lower- than-normal number of red blood cells, or a reduction in the amount of hemoglobin. When anemia comes on slowly, the symptoms are often vague such as tiredness, shortness of breath, headaches, and a reduced ability to exercise. When anemia is acute, symptoms may include confusion, feeling like one is going to pass out, loss of consciousness, and increased thirst. Anemia must be significant before a person becomes noticeably pale. Symptoms of anemia depend on how quickly hemoglobin decreases. Additional symptoms may occur depending on the underlying cause.

In the present context the term “hemolytic anemia caused by defective red blood cell metabolism” is used to denote that hemolytic anemia is caused by enzyme defects in red blood cell glycolysis.

In general, hemolytic anemia due to hemolysis may be related to abnormal breakdown of red blood cells, either in the blood vessels (intravascular hemolysis) or elsewhere in the human body (extravascular). This most commonly occurs within the spleen, but also can occur in the reticuloendothelial system or mechanically (prosthetic valve damage). Hemolytic anemia accounts for 5% of all existing anemias. It has numerous possible consequences, ranging from general symptoms to life-threatening systemic effects. The general classification of hemolytic anemia is either intrinsic or extrinsic. Treatment depends on the type and cause of the hemolytic anemia.

Symptoms of hemolytic anemia are similar to other forms of anemia (fatigue and shortness of breath), but in addition, the breakdown of red cells leads to jaundice and increases the risk of particular long-term complications, such as gallstones and pulmonary hypertension.

Symptoms of hemolytic anemia are similar to the general signs of anemia. General signs and symptoms include: fatigue, pallor, shortness of breath, and tachycardia. In small children, failure to thrive may occur in any form of anemia. In addition, symptoms related to hemolysis may be present such as chills, jaundice, dark urine, and an enlarged spleen. Certain aspects of the medical history can suggest a cause for hemolysis, such as drugs, medication side effects, autoimmune disorders, blood transfusion reactions, the presence of prosthetic heart valve, or other medical illness.

Chronic hemolysis leads to an increased excretion of bilirubin into the biliary tract, which in turn may lead to gallstones. The continuous release of free hemoglobin has been linked with the development of pulmonary hypertension (increased pressure over the pulmonary artery); this, in turn, leads to episodes of syncope (fainting), chest pain, and progressive breathlessness. Pulmonary hypertension eventually causes right ventricular heart failure, the symptoms of which are peripheral edema (fluid accumulation in the skin of the legs) and ascites (fluid accumulation in the abdominal cavity).

As mentioned above, the term “hemolytic anemia” is a general term. However, in the present context it is limited to anemia caused by defects in red blood cell metabolism, i.e. red blood cell glycolysis. As discussed herein, erythrocyte metabolism is improved by KL1333 and other quinones as discussed herein, which leads to enhanced glycolysis and ATP production

Moreover, it is contemplated that quinones of the present invention can be used as discussed in the following:

KL1333 and other quinones as discussed herein lead to improvement to ineffective erythropoiesis by enhanced glycolysis and ATP, thus the quinones may increase survival of erythrocytes in circulation, when needed.

KL1333 and other quinones as discussed herein decrease risks of hemolytic crises by enhanced glycolysis and ATP when there is increased stress from infections etc. In the present context, the term “associated with” such as “anemia associated with defective erythrocyte metabolism or enzymopathies” is intended to mean a disorder that is caused by defective erythrocyte metabolism or enzymopathies or where defective erythrocyte metabolism or enzymopathies are observed, and which leads to hemolytic anemia. As mentioned herein before, the defective erythrocyte metabolism or erythrocyte enzymopathies are caused by defects in the enzyme(s) in red blood cell glycolysis.

In the present context the term “decreased compared to normal” or “increased compared to normal” is intended to denote a decrease or an increase that is 5% or more such as 7.5% or more, 10% or more or 5% or more, 7.5% or more, 10% more, 15% or more or 20% or more than the normal values. Thus, the term “decreased survival of erythrocytes in circulation” is intended to mean that the number of erythrocytes in circulation is 20% or lower, such a 15% or lower, 10% or lower, 7.5% or lower or 5% or lower such as about 20%, about 15%, about 10%, about 7.5% or about 5% lower than the normal value.

Red blood cell glycolysis and defects relating thereto

The steps of glycolysis are as follows:

Step 1. Glucose gets phosphorylated by hexokinase, forming glucose-6-phosphate. This step requires one molecule of ATP.

Step 2. Glucose-6-phosphate is isomerized by phosphoglucose isomerase to form fructose-6- phosphate.

Step 3. Fructose-6-phosphate is phosphorylated by phosphofructokinase to form fructose-1 ,6- bisphosphate. This step requires one molecule of ATP.

Step 4. Fructose-1 , 6-bisphosphate is split into two separate sugar molecules, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, by aldolase.

Step 5. The molecule of dihydroxyacetone phosphate is isomerized by triosephosphate isomerase to form a second glyceraldehyde-3-phosphate.

Step 6. Glyceraldehyde-3-phosphate is phosphorylated by glyceraldehyde-3-phosphate dehydrogenase to form 1 ,3-bisphosphoglycerate. This step requires NAD+ as a cofactor.

Step 7. 1 ,3-bisphosphoglycerate is converted to 3-phosphoglycerate (3-PG) by phosphoglycerate kinase. This step involves the transfer of a phosphate molecule to ADP to form 1 molecule of ATP. In mature erythrocytes, 2,3-BPG is also formed via the Luebering-Rapoport pathway in which bisphosphoglycerate mutase catalyzes the transfer of a phosphoryl group from C1 to C2 of 1 ,3- BPG, giving 2,3-BPG. 2,3-bisphosphoglycerate, the most concentrated organophosphate in the erythrocyte, forms 3-PG by the action of bisphosphoglycerate phosphatase.

Step 8. 3-phosphoglycerate rearranges to form 2-phosphoglycerate by the enzyme phosphoglycerate mutase. Step 9. 2-phosphoglycerate is dehydrated to produce phosphoenolpyruvate by the enzyme enolase.

Step 10. Phosphoenolpyruvate is converted to pyruvate by pyruvate kinase. This step involves the transfer of a phosphate molecule to ADP to form 1 molecule of ATP.

Step 11. Pyruvate is converted to lactate by the enzyme lactate dehydrogenase. [ 11 This step involves the oxidation of NADH to NAD+, allowing glycolysis to continue through the glyceraldehyde-3-phosphate dehydrogenase reaction (step 6, see above).

In erythrocytes, the pentose phosphate pathway, a metabolic pathway parallel to glycolysis, is also important as it generates NADPH. Defects in any one of these steps may lead to a disease as the erythrocyte have no other means of generating ATP and NADPH than via these glycolytic pathways. The pentose phosphate pathway involves the use of glucose-6-phosphate dehydrogenase. Defects of this enzyme are also encompassed in the scope of the present invention. Lack of glucose-6-phosphate dehydrogenase is known as favism.

The overall velocity of red blood cell glycolysis is regulated by three rate-limiting enzymes. These are i) hexokinase (HK), ii) phosphofructokinase (PFK), and Hi) pyruvate kinase (PK).

Hexokinase (HK) deficiency

Hexokinase catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P), using ATP as a phosphoryl donor. As the initial step of glycolysis, HK is one of the rate-limiting enzymes of the pathway. The activity of hexokinase is significantly higher in reticulocytes compared with mature red blood cells, in which it is very low. In fact, of all glycolytic enzymes HK has the lowest enzymatic activity in vitro.

Hexokinase deficiency (OMIM 235700) is a rare autosomal, recessively inherited disease with Congenital Non-spherocytic Hemolytic Anemia (CNSHA) as the predominant clinical feature. As with most glycolytic red cell enzyme deficiencies, the severity of hemolysis is variable, ranging from severe neonatal hemolysis and death to a fully compensated chronic hemolytic anemia.

Splenectomy is in general beneficial.

Glucose-6-phosphate isomerase (GPI) deficiency

Glucose 6-phosphate isomerase (GPI) catalyzes the interconversion of G6P into fructose-6- phosphate (F6P) in the second step of the Embden-Meyerhof pathway/glycolysis. As a result of this reversible reaction, products of the hexose-monophosphate shunt can be recycled to G6P. Unlike HK and other age-related enzymes, the GPI activity in reticulocytes is only slightly higher than that of mature erythrocytes. Apart from its role in glycolysis, GPI exerts cytokine properties outside the cell and is involved in several extracellular processes. Because GPI knock-out mice die in the embryologic state, GPI is considered to be a crucial enzyme.

GPI deficiency (OMIM 172 400) is an autosomal recessive disease and is second to PK deficiency in frequency, with respect to glycolytic enzymopathies. Homozygous or compound heterozygous patients have chronic hemolytic anemia of variable severity and display enzymatic activities of less than 25% of normal. Hemolytic crises may be triggered by viral or bacterial infections. Hydrops fetalis appears more common in GPI deficiency than in other enzyme deficiencies.³² In rare cases, GPI deficiency also affects nonerythroid tissues, causing neurologic symptoms and granulocyte dysfunction. Normally, GPI is very stable, but a striking feature of nearly all GPI mutants is their thermolability, whereas kinetic properties are more or less unaffected.

Phosphofructokinase (PFK) deficiency

Phosphofructokinase catalyzes the rate-limiting, ATP-mediated phosphorylation of F6P to fructose- 1 ,6-diphosphate (FBP).

Three different subunits have been identified in humans: PFK-M (muscle), PFK-L (liver), and PFK- P (platelet). The subunits are expressed in a tissue-specific manner and, in erythrocytes, 5 isoenzymes of varying subunit composition (IVU, M3L1, M2L2, ML3, and L4) can be identified.

PFK deficiency (OMIM 171850) is a rare autosomal, recessively inherited disorder. Because red blood cells contain both M and L subunits, mutations affecting either gene will affect enzyme activity. Thus, mutations concerning the L subunit will render red blood cells that contain only M4 and they are partially PFK deficient. In such cases, patients display a mild hemolytic disorder without myopathy. Likewise, a deficiency of the M subunit results in the absence of muscle PFK and, in addition, causes PFK deficiency in erythrocytes. Accordingly, deficiency of the M subunit causes myopathy and a mild hemolytic disorder. Erythrocytes that express only the PFK also show a metabolic block at the PFK step in glycolysis and lowered 2,3-DPG levels.

Aldolase deficiency

Aldolase catalyzes the reversible conversion of FBP to glyceraldehyde-3-phosphate and di hydroxyacetone phosphate (DHAP). Aldolase deficiency (OMIM 103 850) is a very rare disorder. Patients all displayed moderate chronic hemolytic anemia. Severe hemolytic anemia may occur as well as hemolytic anemia myopathy. Enzyme stability may be decreased.

Triosephosphate isomerase (TP!) deficiency

Triosephosphate isomerase (TPI) is the glycolytic enzyme with the highest activity in vitro. TPI catalyzes the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (DHAP).

TPI deficiency (OMIM 190450) is a rare autosomal recessive disorder, characterized by hemolytic anemia at onset, often accompanied by neonatal hyperbilirubinemia requiring exchange transfusion. In addition, patients display progressive neurologic dysfunction, increased susceptibility to infection, and cardiomyopathy. Patients show a 20- to 60-fold increased DHAP concentration in their erythrocytes, consistent with a metabolic block at the TPI step. Most affected individuals die in childhood before the age of 6 years but there are remarkable exceptions.

Glyceraldehyde-3-phosphate dehydrogenase, monophosphoglycerate mutase, enolase, and lactase dehydrogenase

Red blood cell deficiencies of glyceraldehyde-3-phosphate dehydrogenase and enolase have been described in association with hemolytic anemia.

Phosphoglycerate kinase (PGK) deficiency

Phosphoglycerate kinase generates one molecule of ATP by catalyzing the reversible conversion of 1 ,3-bisphosphoglycerate to 3-phosphoglycerate. This reaction can be bypassed by Rapoport- Luebering shunt, thus preventing the formation of the second ATP molecule.

PGK deficiency (OMIM 311800) is characterized by chronic hemolytic anemia, dysfunction of the central nervous system, and myopathy.

Pyruvate kinase (PK) deficiency

In pyruvate kinase deficiency (PKD), two major distinctive metabolic abnormalities are ATP depletion and concomitant increase of 2,3-diphosphoglycerate consistent with accumulation of upper glycolytic intermediates. Moreover, one of the consequences of decreased ATP and pyruvate level is lowered lactate level leading to inability to regenerate NAD+ through lactate dehydrogenase for further use in glycolysis. The lack of ATP disturbs the cation gradient across the red cell membrane, causing the loss of potassium and water, which causes cell dehydration, contraction, and crenation, and leads to premature destruction and diminished lifetime of the red blood cells (RBCs). Such defective RBCs are destroyed in the spleen, and excessive hemolysis rate in the spleen leads to the manifestation of hemolytic anemia. The exact mechanism by which PKD sequesters newly matured RBCs in the spleen to effectively shorten overall half-lives of circulating RBCs is not yet clear, but recent studies suggest that metabolic dysregulation affects not only cell survival but also the maturation process resulting in ineffective erythropoiesis.

Pyruvate kinase catalyzes the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP. The enzyme has an absolute requirement for Mg²⁺ and K⁺ cations to drive catalysis. PK functions as the last critical step in glycolysis because it is an essentially irreversible reaction under physiological conditions. In addition to its role of synthesizing one of the two ATP molecules from the metabolism of glucose to pyruvate, pyruvate kinase is also an important cellular metabolism regulator. It controls the carbon flux in lower-glycolysis to provide key metabolite intermediates to feed biosynthetic processes, such as pentose-phosphate pathway among others, in maintaining healthy cellular metabolism. Because of these critical functions, pyruvate kinase is tightly controlled at both gene expression and enzymatic allosteric levels. In mammals, fully activated pyruvate kinase exists as a tetrameric enzyme. Four different isozymes (M1 , M2, L and R) are expressed from two separate genes. Erythrocyte-specific isozyme PKR is expressed from the PKLR gene ("L gene") located on chromosome 1q21. This same gene also encodes the PKL isozyme, which is predominately expressed in the liver. PKLR consists of 12 exons with exon 1 is erythroid-specific whereas exon 2 is liver-specific. The two other mammalian isozymes PKM1 and PKM2 are produced from the PKM gene ("M gene") by alternative splicing events controlled by hnRNP proteins. The PKM2 isozyme is expressed in fetal tissues and in adult proliferating cells such as cancer cells. Both PKR and PKM2 are in fact expressed in proerythroblasts. However, upon erythroid differentiation and maturation, PKM2 gradually is decreased in expression and progressively replaced by PKR in mature erythrocytes.

Clinically, hereditary PKR deficiency disorder manifests as non-spherocytic hemolytic anemia. The clinical severity of this disorder ranges from no observable symptoms in fully compensated hemolysis to potentially fatal severe anemia requiring chronic transfusions and/or splenectomy at early development or during physiological stress or serious infections. Most affected individuals who are asymptomatic, paradoxically due to enhanced oxygen-transfer capacity, do not require any treatment. The enhanced oxygen-transfer capacity may be due to increases 2,3-DPG levels. However, for some of the most severe cases, while extremely rare population-wise with estimated prevalence of 51 per million, there is no disease-modifying treatment available for these patients other than palliative care. These hereditary non-spherocytic hemolytic anemia (HNSHA) patients present a clear unmet medical need.

The precise mechanisms that lead to a shortened lifespan of the mature PK-deficient erythrocyte are still unknown. The clinical picture varies from severe hemolysis causing neonatal death to a well-compensated hemolytic anemia. Some PK-deficient patients present with hydrops fetalis. Reticulocytosis is almost always observed. Splenectomy often ameliorated the hemolysis, especially in severe cases, and increases the reticulocyte count even further.

Heterogenous genetic mutations in PKR lead to dysregulation of its catalytic activity. There are now nearly 200 different reported mutations associated with this disease reported worldwide. Although these mutations represent wide range genetic lesions that include deletional and transcriptional or translational abnormalities, by far the most common type is missense mutation in the coding region that one way or another affects conserved residues within domains that are structurally important for optimal catalytic function of PKR. The pattern of mutation prevalence seems to be unevenly distributed toward specific ethnic backgrounds.

PK deficiency has been recognized in dogs and mice. In both species, the deficiency causes severe anemia and marked reticulocytosis, closely resembling human PK deficiency.

Pharmaceutical compositions

The quinones for use according to the present invention may be presented in the form of a pharmaceutical composition comprising the quinone together with one or more pharmaceutically acceptable diluents or carriers.

It is contemplated that the quinones or a composition thereof may be administered by any conventional method for example but without limitation it may be administered parenterally, orally, topically (including buccal, sublingual or transdermal), via a medical device (e.g. a stent), by inhalation or via injection (subcutaneous or intramuscular). The treatment may consist of a single dose or a plurality of doses over a period of time.

The treatment may be by administration once daily, twice daily, three times daily, four times daily etc. The treatment may also be by continuous administration such as e.g. administration intravenous by drop.

Whilst it is possible for the quinone to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be “acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Examples of suitable carriers are described in more detail below.

The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the quinone with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the quinone with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The quinones will normally be administered intravenously, orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the quinone, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof, or in the case of solid or semi-solid compositions it may be solid or semi-solid carriers.

For example, the quinones can also be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or control led-release applications.

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

Solutions or suspensions of the quinones suitable for oral administration may also contain excipients e.g. N,N-dimethylacetamide, dispersants e.g. polysorbate 80, surfactants, and solubilisers, e.g. polyethylene glycol, Phosal 50 PG (which consists of phosphatidylcholine, soyafatty acids, ethanol, mono/diglycerides, propylene glycol and ascorbyl palmitate). The formulations according to present invention may also be in the form of emulsions, wherein a compound according to Formula (I) may be present in an aqueous oil emulsion. The oil may be any oil-like substance such as e.g. soy bean oil or safflower oil, medium chain triglyceride (MCT-oil) such as e.g. coconut oil, palm oil etc or combinations thereof. Tablets may contain excipients such as microcrystalline cellulose, lactose (e.g. lactose monohydrate or lactose anhydrous), sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, butylated hydroxytoluene (E321), crospovidone, hypromellose, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium, and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), macrogol 8000, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the quinone in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. povidone, gelatin, hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g. sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the quinone ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

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

Formulations suitable for topical administration in the mouth include lozenges comprising the quinone in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the quinone in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the quinone in a suitable liquid carrier.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, sprays, aerosols or oils, transdermal devices, dusting powders, and the like. These compositions may be prepared via conventional methods containing the quinone. Thus, they may also comprise compatible conventional carriers and additives, such as preservatives, solvents to assist drug penetration, emollient in creams or ointments and ethanol or oleyl alcohol for lotions. Such carriers may be present as from about 1% up to about 98% of the composition. More usually they will form up to about 80% of the composition. As an illustration only, a cream or ointment is prepared by mixing sufficient quantities of hydrophilic material and water, containing from about 5-10% by weight of the compound, in sufficient quantities to produce a cream or ointment having the desired consistency.

Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the quinone may be delivered from the patch by iontophoresis.

For applications to external tissues, for example the mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the quinone may be employed with either a paraffinic or a water-miscible ointment base.

Alternatively, the quinone may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms are prepared utilizing the quinone and a sterile vehicle, for example but without limitation water, alcohols, polyols, glycerine and vegetable oils, water being preferred. The quinone, depending on the vehicle and concentration used, can be either colloidal, suspended or dissolved in the vehicle. In preparing solutions the quinone can be dissolved in water for injection and filter sterilised before filling into a suitable vial or ampoule and sealing.

Advantageously, agents such as preservatives and buffering agents can be dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum. The dry lyophilized powder is then sealed in the vial and an accompanying vial of water for injection may be supplied to reconstitute the liquid prior to use.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability.

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

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents. A person skilled in the art will know how to choose a suitable formulation and how to prepare it (see e.g. Remington’s Pharmaceutical Sciences 18 Ed. or later). A person skilled in the art will also know how to choose a suitable administration route and dosage.

It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound of the invention will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the particular subject being treated, and that a physician will ultimately determine appropriate dosages to be used. This dosage may be repeated as often as appropriate. If side effects develop the amount and/or frequency of the dosage can be altered or reduced, in accordance with normal clinical practice.

Methods for preparing the quinones for use according to the invention

Orthoquinone compounds for use in the treatment of red blood cell enzymopathies are made by standard methods known to one skilled in the art. There are multiple routes to the compounds for use. Typically, the approach would commence from an available bicyclic compound and the third ring (heterocycle) is installed by installing appropriate functionality and then performing ring closure. This includes standard strategies for heterocycle synthesis [e.g. Katritzky et al, Handbook of Heterocyclic Chemistry, Elsevier 2010; Eicher and Hauptmann, The Chemistry of Heterocycles, 2003, Wiley]. Typically, through the synthesis the centre ring is aromatic with a single oxygen substituent (either protected or not) and the quinone introduced via oxidation of the ring to regioselectively introduce a second oxygen as a hydroxyquinone that then is converted to the ortho-quinone.

Suitable methods for synthesizing orthoquinone compounds for the use in treatment of enzymopathies are described in WO2016159577A2, W02022039460 and W02020175851.

In the embodiment whereby the orthoquinone compound for the use in treatment of enzymopathies is beta-lapachone then this may be synthesized as above. Alternatively, it may be extracted from a plant or parts of a plant that naturally produces the compound. Such methods are described in Karthikeyan et al [Karthikeyan R, Sai Koushik O, Kumar PV (2016) Isolation, Characterisation and Antifungal Activity of p-Lapachone from Tecomaria capensis (Thunb.) Spach Leaves, Med. Aromat. Plants 5: 239].

General

It should be understood that any feature and/or aspect discussed above in connections with the compounds according to the invention apply by analogy to the methods described herein. The following figures and examples are provided below to illustrate the present invention. They are intended to be illustrative and are not to be construed as limiting in any way.

Specific embodiments

Specific embodiments include

1. A compound of formula (I) or a pharmaceutically acceptable salt, a hydrate, a solvate, a diastereomer, a tautomer or a prodrug thereof

formula (I), wherein,

Xi, is C, or N;

R1, is one or more selected from the group consisting of H, C1-6 alkyl, C1-6 alkyloxy, Ce-io aryl, heteroaryl, halo, nitro, hydroxy, cyano and NRsRe;

R7 is alkyl; Rs is alkyloxy; alkyl is any C1-10 linear or branched, or C3-7 cyclic alkyl; alkyloxy is any C1-10 linear or branched alkyloxy, or C3-7 cyclic alkyloxy; heterocyclyl is 3- to 7-membered heterocyclic group having in the cycle at least one hetero atom selected from the group consisting of N, O and S; heteroaryl is 5- to 10 -membered aromatic cyclic group having in the cycle at least one hetero atom selected from N, O and S, and when the aryl or heteroaryl is substituted, its substituent is each at least one selected from the group consisting of halo, alkyl, halo-substituted alkyl and alkyloxy; and — or ^::: is a single bond or a double bond depending on R2, R3, R4, Xi, X2, X3, and X4, for use in restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as red blood cells. Specific example is use in the treatment of red blood cell enzymopathies, especially in the treatment of defects in red blood cell metabolism such as defects in red blood cell glycolysis.

2. A compound for use according to item 1 , wherein

Xi, is C, or N;

Ri-Rs are as defined in claim 1 .

3. A compound for use according to item 1 , wherein

Xi, is C;

R2 is not present; and

R1, Rs-Rs are as defined in claim 1.

4. A compound for use according to item 1 or 2, wherein

Xi and X3 are each C;

X2 and X4 are each N, and

Ri-Rs are as defined in claim 1 . 5. A compound for use according to any one of the preceding items, wherein

R3 is alkyl, wherein the alkyl may be substituted with Ce-io aryl, and

R1, R2 and R4-R8 are as defined in claim 1.

6. A compound for use according to any one of items 1-4, wherein

R1, and R4-Rs are as defined in claim 1.

7. A compound for use according to any one of the preceding items, wherein

R1 is H, and

R3 is a C1-C6 straight or branched alkyl, and

R2, R4, and Rs are as defined in claim 1.

8. A compound for use according to any one of the preceding items, wherein

R2 is not present or H;

R4 is not present or H; and

R1, R3 and Rs-Rs are as defined in claim 1.

9. A compound for use according to any one of items 1 , 2, 4-8, wherein

Xi and X3 are each C;

X2 and X4 are each N;

R2 is not present or H;

R4 is not present or H; and

R1, R3 and Rs-Rs are as defined in claim 1.

10. A compound for use according to any one of items 1 , 2, 4-9, wherein Xi and X3 are each C;

X2 and X4 are each N;

R1 is H;

R2 is not present or H;

R3 is branched or straight Ci-Ce alkyl;

R4 is not present or H.

11 . A compound for use according to any one of items 1 , 2, 4-10 having formula (II):

formula (II) or a pharmaceutically acceptable salt, a hydrate, a solvate, a diastereomer, a tautomer or a prodrug thereof.

12. A compound for use according to any one of items 1 , 3, 5-8 having of formula (III)

beta-lapachone, formula (III) or a pharmaceutically acceptable salt, a hydrate, a solvate, an enantiomer, a diasteromer, a tautomer or a prodrug thereof.

13. A compound for use according to any one of the preceding items, wherein the red blood cell enzymopathies involve an enzyme deficiency in red blood glycolysis. 14. A compound for use according to any one of the preceding items, wherein the red blood cell enzymopathies involve a pyruvate kinase (PK) deficiency in red blood cells.

15. A compound for use according to any one of the preceding items, wherein the treatment restores, normalizes and/or increases ATP level in red blood cells.

16. A compound for use according to item 14, wherein the PK deficiency is associated with a lack of or decrease in R-type PK (PKR) activity in red blood cells.

17. A compound for use according to any one of the preceding items, for use in the treatment of hemolytic anemia or ineffective erythropoiesis.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Changes in ATP levels, redox status and cell size of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. Shikonin decreased ATP levels (A, B), increased NAD(P)H autofluorescence (C, D) and increased cell size (E, F) in a dose-dependent manner. Bar graphs depict levels at the final measurement timepoint.

Figure 2. Changes in ATP levels, redox status and cell size of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333 and idebenone all counteracted PK inhibition at the selected concentrations - ATP levels (A, B); NADH (C, D) and FSC (E). Bar graphs depict levels at the final measurement timepoint.

Figure 3. Changes in ATP levels, redox status and cell size of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. KL1333 counteracted PK inhibition in a dose-dependent manner. ATP levels (A, B); NAD(P)H (C, D) and FSC (E). Bar graphs depict levels at the final measurement timepoint.

Figure 4. Changes in ATP levels, redox status and cell size of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. The combination of KL1333 and mitapivat counteracted the effects of PK inhibition to a larger extent than mitapivat only. ATP levels (A, B); NAD(P)H (C, D) and FSC (E). Bar graphs depict levels at the final measurement timepoint.

Figure 5. Changes in ATP levels, redox status and cell size of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333 and idebenone all counteracted the effects of PK inhibition induced by phenyl alanine methyl ester at the selected concentrations. ATP levels (A, B) and FSC (C). Bar graphs depict levels at the final measurement timepoint.

Figure 6. Changes in ATP levels of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. In this experiment the effects of PK inhibition were partially counteracted by beta- lapachone at 1 pM concentration. KL1333 was more effective. No clear effect was seen at 10 pM beta-lapachone. ATP levels (A, B). Bar graphs depict levels at the final measurement timepoint.

Figure 7. Changes in ATP levels of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. In this experiment the effects of PK inhibition were only discretely affected by vatiquinone at 1 pM concentration. KL1333 was more effective. No effect was seen at 10 pM concentration. ATP levels (A, B). Bar graphs depict levels at the final measurement timepoint.

Figure 8. Changes in reactive oxygen species (ROS) levels of non-inhibited, healthy human RBCs following treatment with 1 pM KL1333. Administration of KL1333 clearly decreased the level of intracellular ROS as determined by following the fluorescence of 2-hydroethidium with flow cytometry.

Figure 9. Changes in ATP levels of human RBCs in an ex vivo model of pyruvate kinase (PK) deficiency. In this experiment the effects of PK inhibition were counteracted by napabucasin at 1 pM concentration. KL1333 was more effective. The effect of 1 pM beta-lapachone was similar to that of 1 pM napabucasin.

Description of the results from the Examples presented herein. In the Examples other quinones are included.

Example 1 depicts the established flow cytometry-based ex vivo model of pyruvate kinase (PK) deficiency in human red blood cells (RBCs). The PK-inhibitor Shikonin increased Magnesium Green fluorescence, NAD(P)H autofluorescence, and FSC (forward light scatter), in a dosedependent manner.

Magnesium Green fluorescence is inversely related to intracellular changes in ATP levels. It is a dye that measures Mg2+. As ATP binds to Mg2+ with higher affinity than ADP, the cytosolic magnesium concentration [Mg2+]i will rise upon ATP hydrolysis (and so will the Magnesium Green fluorescence).

NAD(P)H autofluorescence is related to cellular redox status. The higher autofluorescence the higher the intracellular levels of NADPH and NADH.

FSC (forward light scatter) is related to cellular diameter. Much of the ATP produced by RBCs is used to drive membrane ion pumps to maintain cellular integrity. It has been shown that depletion of ATP in RBCs gives a transient increase in size and if prolonged the cells take up calcium, loose potassium and are “dehydrated” i.e., become smaller. During our 2h experiment following PK inhibition the cell size increases.

So, inhibiting pyruvate kinase with shikonin decreases red blood cell ATP levels, increases levels of NAD(P)H and increases cell size. All of this in a dose-dependent manner.

The rest of the examples are using this model of pyruvate kinase (PK) deficiency except for Example 5 which uses another inhibitor of PK - phenyl alanine methyl ester to validate the findings with shikonin. In Example 8 PK is not inhibited.

Example 2 compares the effects of mitapivat, KL1333 and idebenone. The results show that administration of mitapivat, KL1333 or idebenone in shikonin-treated RBC at the selected concentrations counteracts the effects of PK inhibition as indicated by increased ATP levels, decreased NAD(P)H levels and decreased cell size (as compared to shinkon in-treated RBCs with no administration of mitapivat, KL1333 or idebenone).

Example 3 investigates dose-response relationship of KL1333. The results show that KL1333 counteracted PK inhibition in a dose-dependent manner as indicated by dose-dependent increase in ATP levels, decrease in NAD(P)H levels and decrease in cell size in shikonin-treated RBCs.

Example 4 investigates the effect of co-treatment with mitapivat and KL1333. The results show that the combination of KL1333 and mitapivat counteracted the effects of PK inhibition to a larger extent than mitapivat only.

Example 5 investigate the effect of treatment with KL1333, idebenone or mitapivat of human RBCs in an ex vivo model of pyruvate kinase deficiency induced by another PK-inhibitor, phenyl alanine methyl ester. Similar to the findings with the PK-inhibitor shikonin, the results show an increase in ATP and a decrease in cell size following treatment with KL1333, idebenone or mitapivat (as compared to phenyl alanine methyl ester-treated RBCs with no administration of mitapivat, KL1333 or idebenone).

Example 6 investigates the effect of two different concentrations of beta-lapachone (compared to 1 pM KL1333) on pyruvate kinase deficient human RBCs induced by shikonin. The results show a partial increase in ATP level for 1 pM beta-lapachone (and KL1333)..

Example 7 investigates the effect of vatiquinone on pyruvate kinase deficient human RBCs induced by shikonin. The results show a minor increase in ATP levels for 1 pM vatiquinone.

Example 8 investigates the effect of KL1333 on intracellular reactive oxygen species. The results show a decrease in ROS when KL1333 was administered.

Example 9 investigates the effect of 1 pM napabucasin (compared to 1 pM KL1333 and 1 pM beta-lapachone) on pyruvate kinase deficient human RBCs induced by shikonin. The results show clear increases in ATP levels for the three quinones at the compared concentration of 1 pM (KL1333 was the most effective).

Example 10 investigates enzymes and cofactors reducing the quinone compounds KL1333 and idebenone. The results show that in addition to previously described NQO1 activation, KL1333 and Idebenone are also activating NQO2 activity, and KL1333 is activating CYB5R3, which thereby alters the redox state of the nicotinamide adenine dinucleotide-related cofactors NRH and NADH, respectively. Both NQO2 and CYB5R3 being expressed in red blood cells, in contrast to NQO1 which is not.

METHODS AND MATERIALS

Ex vivo assays in human red blood cells

Flow cytometry - measurement of intracellular ATP, cell diameter (cell size) and NAD(P)H autofluorescence

Magnesium Green, NAD(P)H, and forward scatter (FSC)-based flow cytometry was used to detect mitochondrial ATP production, NAD(P)H levels, and cell size, respectively, in human red blood cells (RBCs). RBCs were gated from human blood based on forward and side scattering properties. Magnesium Green fluoresces when bound to free Mg²⁺, which has a higher affinity for ATP than ADP. Magnesium Green fluorescence therefore drops as ATP levels increase (1). NAD(P)H levels were measured using its intrinsic autofluorescence properties, excited by 360 nm UV light and emitting at 470 nm.

RBCs (50 million/ml) were stained with the intracellular, cell-permeant fluorochrome Magnesium Green AM (M3735, Molecular Probes, 1 mM) for 60 min at 37°C in MiR05 buffer with 5 mM glucose (MiR05: 0.5 mM EGTA, 3 mM MgCL, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, 1 g/L free-fatty-acid bovine serum albumin, pH 7.1 ). RBCs were then treated with either the pyruvate kinase (PK) inhibitors phenylalanine methyl ester (1 mM), shikonin (1 , 7, 15, or 30 pM), or DMSO for 20 min before addition of selected concentrations of idebenone, beta-lapachone, napabucasin, vatiquinone, KL1333, mitapivat, or vehicle solution (DMSO). The BD LSR Fortessa and BD LSR Fortessa X20 allow for measurement of fluorescence excitation with the UV laser (355 nm), blue laser (488 nm), and violet laser (405 nm). Magnesium Green fluorescence and NAD(P)H autofluorescence were detected with the 530/30 LP filter and 450/50 filters, respectively.

Flow-cytometry - intracellular radical oxygen species (ROS)

Dihydroethidium (DHE)-based flow cytometry was used to visualize cytoplasmic reactive oxygen species (ROS) levels in human red blood cells (RBCs). RBCs were gated from blood based on forward and side scattering properties. DHE is oxidized by superoxide or hydrogen peroxide present throughout the cell into its fluorescent form 2-hydroethidium. Five billion RBCs/mL were stained with 50 uM DHE for 30 min at 37°C in MiR05 buffer with 5 mM glucose. RBCs were then treated with 1 uM of KL1333, or DMSO and followed for two hours. DHE fluorescence was read every fifteen minutes following addition of treatment. The BD LSR Fortessa allows for measurement of fluorescence excitation using the Blue laser (488 nm). DHE was detected with the 610/20 bandpass filter (PI channel).

Enzyme kinetics

To a 96 well plate was added 140 pL of a pH 7.5 buffer (50 mM tris-hydrochloride (Apollo Scientific, BIT1513, CAS#1185-53-1), 0.14% BSA (Sigma-Aldrich, A9418-5G, CAS#9048-46-8)). 20 pL of the substrate solution (KL1333 (ISC01-878-s8) or Idebenone (BLD, BD134310, CAS#58186-27-9) was then added, followed by 20 pL of enzyme solution. Finally, 20 pL of cofactor solution was added, the resulting solution was mixed, the bubbles removed, and the plate reader immediately started. Plate reader equipments were BioTek Epoch (Agilent), BioTek Synergy H1 (Agilent), 96 well microplate, round wells, flat bottom (Starlab, E2996-1600). Mean velocity was recorded (a.u./min) using read data between 2-12 minutes, measuring absorbance at 340 nm at room temperature. The enzymes used were recombinant Human CYB5R3 (antibodies.com, A59277), Recombinant Human NQO2 (Abeam, AB93933-1001 and antibodies.com, enz-515), Recombinant Human NQO1 (Sigma-Aldrich, D1315), and cofactors NADH (Glentham Life Sciences, GK2960-1G, CAS# 606-68-8), NADPH (Apollo Scientific, BIB3014, CAS#2646-71-1 ), NRH (BLD, BD221061 , CAS#19132-12-8).

Data analysis

Mean fluorescence intensity (MFI) values were compared using FlowJo software v. 10 (BD Biosciences). Changes in Magnesium Green fluorescence are inverted and presented as changes in ATP levels. Statistical analysis (one-way ANOVA with Dunnett’s multiple comparison post hoc test) was performed using Graph Pad PRISM version 9 (La Jolla, California, USA). Data are presented as means ± SEM.

Materials

All reagents used were obtained from commercial sources. EXAMPLES

Example 1 - Simulation of pyruvate kinase (PK) deficiency in human red blood cells (RBCs) using shikonin (a pyruvate kinase inhibitor).

Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP (a decrease in cellular ATP leads to an increase in Magnesium Green fluorescence). Cells were then administered vehicle or the pyruvate kinase inhibitor shikonin at escalating concentrations to inhibit PK. Changes in ATP-levels, NAD(P)H autofluorescence (redox status) and FSC (forward scatter, cell diameter/size) were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results are shown in Figure 1.

The rest of the examples are using this model of pyruvate kinase (PK) deficiency except for Example 5 which uses another inhibitor of PK - phenyl alanine methyl ester to validate the findings with shikonin. In Example 8 PK is not inhibited. Example 2 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by shikonin) - effect of mitapivat, KL1333 and idebenone.

Example 2 compares the effects of mitapivat, KL1333 and idebenone.

Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were subsequently administered vehicle or shikonin 7.5 pM to inhibit PK. This was followed (after 20 min) by administration of vehicle, 5 pM mitapivat, 1 pM KL1333 and 10 pM idebenone. ATP-levels, NAD(P)H autofluorescence (redox status) and FSC (forward scatter, cell diameter/size) were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results show that administration of mitapivat, KL1333 or idebenone in shikonin-treated RBC at the selected concentrations counteracts the effects of PK inhibition as indicated by increased ATP levels, decreased NAD(P)H levels and decreased cell size (as compared to shinkonin-treated RBCs with no administration of mitapivat, KL1333 or idebenone).

The results are shown in Figure 2.

Example 3 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by shikonin). KL1333 - assessment of dose-response relationship.

Example 3 investigates dose-response relationship of KL1333.

Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were then administered vehicle or shikonin 7.5 pM to inhibit PK followed (after 20 min) by vehicle or KL1333 at escalating concentrations. ATP levels, NAD(P)H autofluorescence (redox status) and FSC (forward scatter, cell diameter/size) were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results show that KL1333 counteracted PK inhibition in a dose-dependent manner as indicated by dose-dependent increase in ATP levels, decrease in NAD(P)H levels and decrease in cell size in shikonin-treated RBCs.

The results are shown in Figure 3.

Example 4 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by shikonin) - effect of co-treatment with mitapivat and KL1333.

Example 4 investigates the effect of co-treatment with mitapivat and KL1333. Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were then administered vehicle or shikonin 7.5 pM to inhibit PK followed (after 20 min) by vehicle, 500 nM mitapivat, and 500 nM mitapivat + 500 nM KL1333. ATP levels, NAD(P)H autofluorescence (redox status) and FSC (forward scatter, cell diameter/size) were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results show that the combination of KL1333 and mitapivat counteracted the effects of PK inhibition to a larger extent than mitapivat only.

The results are shown in Figure 4.

Example 5 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by phenyl alanine methyl ester) - effect of mitapivat, KL1333 and idebenone.

Example 5 investigates the effect of treatment with KL1333, idebenone or mitapivat of human RBCs in an ex vivo model of pyruvate kinase deficiency induced by another PK-inhibitor, phenyl alanine methyl ester.

Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were then administered vehicle or 1 mM phenyl alanine methyl ester to inhibit PK followed (after 20 min) by vehicle, 5 pM mitapivat or 1 pM KL1333. ATP levels and FSC (forward scatter, cell diameter/size) were then assessed repeatedly using flow cytometry for the duration of the experiment.

Similar to the findings with the PK-inhibitor shikonin, the results show an increase in ATP and a decrease in cell size following treatment with KL1333, idebenone or mitapivat (as compared to phenyl alanine methyl ester-treated RBCs with no administration of mitapivat, KL1333 or idebenone).

The results are shown in Figure 5.

Example 6 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by shikonin) - effect of beta-lapachone.

Example 6 investigates the effect of two different concentrations of beta-lapachone (compared to 1 pM KL1333) on pyruvate kinase deficient human RBCs induced by shikonin.

Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were subsequently administered vehicle or shikonin 7.5 pM to inhibit PK. This was followed (after 20 min) by administration of vehicle, 1 pM betalapachone, 10 pM betalapachone, and 1 pM KL1333. ATP-levels were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results show a partial increase in ATP level for 1 pM beta-lapachone (and KL1333).

The results are shown in Figure 6.

Example 7 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by shikonin) - limited effect of vatiquinone.

Example 7 investigates the effect of vatiquinone on pyruvate kinase deficient human RBCs induced by shikonin.

Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were subsequently administered vehicle or shikonin 7.5 pM to inhibit PK. This was followed (after 20 min) by administration of vehicle, 1 pM vatiquinone, 10 pM vatiquinone, and 1 pM KL1333. ATP-levels were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results show minor increases in ATP levels for 1 pM vatiquinone.

The results are shown in Figure 7.

Example 8 - The effect of KL1333 on intracellular reactive oxygen species as determined using RBCs stained with dihydroethidium (DHE).

Example 8 investigates the effect of KL1333 on intracellular reactive oxygen species.

Human RBCs were firstly stained with DHE to make it possible to follow changes in intracellular reactive oxygen species (ROS). RBCs were then treated with 1 uM of KL1333, or DMSO and followed with flow cytometry for two hours. DHE fluorescence was read every fifteen minutes following addition of treatment.

The results show a decrease in ROS when KL1333 was administered.

The results are shown in Figure 8.

Example 9 - Treatment of an ex vivo human model of pyruvate kinase (PK) deficiency (induced by shikonin) - effect of napabucasin as compared to beta-lapachone and KL1333.

Example 9 investigates the effect of 1 pM napabucasin (compared to 1 pM KL1333 and 1 pM betalapachone) on pyruvate kinase deficient human RBCs induced by shikonin. Human RBCs were firstly stained with Magnesium Green AM (cell permeant) to make it possible to follow changes in intracellular ATP. Cells were subsequently administered vehicle or shikonin 7.5 pM to inhibit PK. This was followed (after 20 min) by administration of vehicle, 1 pM napabucasin, 1 pM beta-lapachone, or 1 pM KL1333. ATP-levels were then assessed repeatedly using flow cytometry for the duration of the experiment.

The results show clear increases in ATP levels for the three quinones at the compared concentration of 1 pM (KL1333 was the most effective).

The results are shown in Figure 9.

Example 10 - Reductive metabolism of compounds KL1333 and idebenone and activation of enzymes NQO1 , NQO2 and CYB5R3.

Example 10 investigates enzymes and cofactors reducing the quinone compounds KL1333 and idebenone.

Enzyme kinetics were established using a 96 well plate reader of absorbance rate at 340 nm of the substrate, enzyme and cofactor solutions.

The results are shown in Table 1.

Table 1. Enzyme kinetics of KL1333 and Idebenone using human recombinant enzymes NQO1 , NQO2 and CYB5R3 with cofactors.

The results show that in addition to previously described NQO1 activation, KL1333 and Idebenone are also activating NQO2 activity, and KL1333 is activating CYB5R3, which thereby alters the redox state of the nicotinamide adenine dinucleotide-related cofactors NRH and NADH, respectively. Both NQ02 and CYB5R3 being expressed in red blood cells, in contrast to NQ01 which is not.

Claims

1. A compound of formula (I) or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer of formula (I) or a reduced form thereof

formula (I), wherein,

Xi, is C, or N;

R7 is alkyl;

Rs is alkyloxy; alkyl is any C1-10 linear or branched, or C3-7 cyclic alkyl; alkyloxy is any C1-10 linear or branched alkyloxy, or C3-7 cyclic alkyloxy; heterocyclyl is 3- to 7-membered heterocyclic group having in the cycle at least one hetero atom selected from the group consisting of N, O and S; heteroaryl is 5- to 10 -membered aromatic cyclic group having in the cycle at least one hetero atom selected from N, O and S, and when the aryl or heteroaryl is substituted, its substituent is each at least one selected from the group consisting of halo, alkyl, halo-substituted alkyl and alkyloxy; and — or ^::: is a single bond or a double bond depending on R2, R3, R4, Xi, X2, X3, and X4, for use in restoring, normalizing and/or improving ATP in cells lacking mitochondria, such as in red blood cells.

2. The compound for use according to claim 1 , wherein the cells lacking mitochondria are red blood cells.

3. The compound for use according to claim 1 or 2, wherein the use is in treatment of red blood cell enzymopathies.

4. The compound for use according to any one of the preceding claims, wherein the use is in the treatment of defects in red blood cell metabolism such as defects in red blood cell glycolysis.

5. The compound for use according to claim 3 or 4, wherein the red blood cell enzymopathies involve a pyruvate kinase (PK) deficiency in red blood cells.

6. The compound for use according to claim 5, wherein the PK deficiency is associated with a lack of or decrease in R-type PK (PKR) activity in red blood cells.

7. The compound for use according to any one of the preceding claims, wherein

Xi, is C, or N;

Ri-Rs are as defined in claim 1 .

8. The compound for use according to any one of claims 1-6, wherein

Xi, is C; X2 is O, X3 is C(CH3), and X4 is CH2CH2 and the resultant 6-membered heterocycle is not aromatic;

R2 is not present; and

R1, Rs-Rs are as defined in claim 1.

9. The compound for use according to any one of claimsl-7, wherein

Xi and X3 are each C;

X2 and X4 are each N, and

Ri-Rs are as defined in claim 1 .

10. The compound for use according to any one of the preceding claims, wherein

R3 is alkyl, wherein the alkyl may be substituted with Ce-io aryl, and

R1, R2 and R4-R8 are as defined in claim 1.

11 . The compound for use according to any one of claims 1-9, wherein

R1, and R4-Rs are as defined in claim 1.

12. The compound for use according to any one of the preceding claims, wherein

R1 is H, and

R3 is a C1-C6 straight or branched alkyl, and

R2, R4, and Rs are as defined in claim 1.

13. The compound for use according to any one of the preceding claims, wherein

R2 is not present or H;

R4 is not present or H; and

Ri, R3 and Rs-Rs are as defined in claim 1.

14. The compound for use according to any one of claims 1-7, 9-13, wherein

Xi and X3 are each C;

X2 and X4 are each N;

R2 is not present or H;

R4 is not present or H; and

R1, R3 and Rs-Rs are as defined in claim 1.

15. The compound for use according to any one of claims 1-7, 9-14, wherein

Xi and X3 are each C;

X2 and X4 are each N;

R1 is H;

R2 is not present or H;

R3 is branched or straight Ci-Ce alkyl;

R4 is not present or H.

16. The compound for use according to any one of claims 1-7, 9-15 having formula (II):

formula (II) or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof.

17. The compound for use according to any one of claims 1-6, 8, 10-13 having of formula (III)

beta-lapachone, formula (III) or a reduced form thereof, or a pharmaceutically acceptable salt, a hydrate, a solvate, or a tautomer thereof.

18. The compound for use according to any one of the preceding claims, wherein the red blood cell enzymopathies involve an enzyme deficiency in red blood glycolysis.