2',3'-diacetyluridine with acetoacetyl substituted at the 5' position.

Abstract

5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine is useful in the treatment or prevention of disorders characterized by cerebral metabolic energy insufficiency or reduced mitochondrial energy storage capacity.

Description

Background Art

[0001] Oral delivery of uridine for therapeutic purposes is limited due to low bioavailability, which is about 7% in both humans and mice. Although ester prodrugs of uridine have been found to improve its bioavailability, only one, 2’,3’,5,-tri-O-acetyluridine (or uridine triacetate), has been found to be suitable for delivering sufficient uridine for clinical purposes. The bioavailability of oral uridine triacetate has been measured to be about 50% (Ashour 1996). Therefore, there is room for improvement in the efficiency of uridine delivery, which is important in that relatively large doses of uridine are required for therapeutic applications.

Summary of the Invention

Means for Solving the Problems

[0002] The present invention provides the compound 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine. It provides a method for treating or preventing a disorder characterized by metabolic energy deficiency of the brain or reduced mitochondrial energy storage capacity in a mammalian subject, comprising the step of administering to the subject an amount of the compound of the present invention effective in treating the disorder. The present invention also provides the compound of the present invention for use in the treatment or prevention of a disorder characterized by metabolic energy deficiency of the brain or reduced mitochondrial energy storage capacity in a mammalian subject, or for the manufacture of a pharmaceutical for treatment or prevention. Furthermore, it provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier. The present invention also provides a method for producing 5'-O-acetoacetyl-2',3'-di-O-acetyluridine, comprising the steps of (a) mixing 2',3'-O-isopropylideneuridine and 2,2,6-trimethyl-4H-1,3-dioxin-4-one in dimethylformamide under conditions for producing 5'-O-acetoacetyl-2',3'-O-isopropylideneuridine; and (b) the 5'-O-acetoacetyl-2',3'-O-isopropylideneuridine from step (a). The present invention provides a method comprising the steps of: (a) mixing with an aqueous acetic acid solution under conditions that produce crude 5'-O-acetoacetyluridine; (b) dissolving the crude 5'-O-acetoacetyluridine from step (b) in a mixture of dichloromethane and pyridine, then adding acetic anhydride and stirring for several days; and (d) neutralizing the mixture obtained from step (c) with saturated NaHCO3 by switching the solvent to ethyl acetate to obtain crude 5'-O-acetoacetyl-2',3'-di-O-acetyluridine. The present invention also provides 5'-O-acetoacetyluridine as a crude intermediate and isolated compound.

[0003] Acetate substituents improve absorption from the gastrointestinal tract into circulation and are rapidly removed by nonspecific esterase activity. Acetate is a benign metabolite, but is pharmacologically and therapeutically neutral. The discovery that introducing an acetoacetyl substituent at the 5' position provides beneficial pharmacological activity in disorders requiring uridine delivery, in addition to significantly improving uridine bioavailability for therapeutic purposes, thus achieving dual therapeutic activity with a single molecule, represents a significant advance. [Brief explanation of the drawing]

[0004] [Figure 1] This figure shows the plasma [uridine + uracil] of mice after oral administration of a uridine prodrug. UTA = uridine triacetate (2',3',5'-tri-O-acetyluridine) Tri-AcAcU = 2',3',5'-tri-O-(acetoacetyl)uridine AcAcDAU = 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine DA-HU = 2',3'-di-O-acetyl-5'-O-heptanoyluridine [Figure 2] This figure shows the plasma uridine levels of mice after oral administration of uridine prodrugs. Tri-AcAcU = 2',3',5'-tri-O-(acetoacetyl)uridine AcAcDAU = 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine DA-HU = 2',3'-di-O-acetyl-5'-O-heptanoyluridine [Figure 3] This figure shows the plasma uridine levels of mice after oral administration of a uridine prodrug. UTA = Uridine triacetate (2',3',5'-tri-O-acetyluridine) DAU = 2',3'-di-O-acetyluridine AcAcU = 5'-O-(acetoacetyl)uridine [Figure 4] This figure shows the plasma uridine levels of mice after oral administration of AcAcDAU. AcAcDAU = 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine [Figure 5]This figure shows the plasma uracil levels of mice after oral administration of AcAcDAU. AcAcDAU = 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine [Figure 6] This figure shows the plasma uridine + uracil levels of mice after oral administration of AcAcDAU. AcAcDAU = 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine [Figure 7] This figure shows the plasma beta-hydroxybutyrate levels of mice after oral administration of AcAcDAU. BHB = beta-hydroxybutyrate [Figure 8] This figure shows the survival rate of mice treated with 3-nitropropionic acid 60 mg / kg / day and oral uridine triacetate (UTA) 1000 mg / kg twice daily or 5'-O-acetoacetyl-2',3'-di-O-acetyluridine (AcAcDAU) 1113.5 mg / kg twice daily. [Modes for carrying out the invention]

[0005] As used herein, the transitional term “including” is open-ended. Claims using this term may include elements in addition to those described in such claims. Thus, for example, a claim may encompass a therapeutic regimen that includes other therapeutic or therapeutic viral doses not specifically listed, insofar as the listed elements or their equivalents exist.

[0006] Abbreviation Certain compounds are referred to herein by their chemical names, abbreviations, or structural formulas as shown below. Compound AcAcDAU is within the scope of the present invention. UTA = Uridine triacetate (2',3',5'-tri-O-acetyluridine) Tri-AcAcU=2',3',5'-tri-O-(acetoacetyl)uridine, MW496.42

[0007] [ka]

[0008] AcAcDAU = 5’-O-(Acetoacetyl)-2’,3’-di-O-acetyluridine, MW 412.35

[0009]

Chem.

[0010] DA-HU = 2’,3’-di-O-acetyl-5’-O-heptanoyluridine, MW 440.44

[0011]

Chem.

[0012] DAU = 2’,3’-di-O-acetyluridine AcAcU = 5’-O-(Acetoacetyl)uridine (also called 5’-O-acetoacetyluridine)

[0013]

Chem.

[0014] BHB = beta-Hydroxybutyrate (also called 3-hydroxybutyrate) DCM = Dichloromethane (also called methylene chloride, chemical formula CH2Cl2) DMF = Dimethylformamide, chemical formula (CH3)2NC(O)H

[0015] According to the method, compound for use, use, and pharmaceutical composition of the present invention, any conventional disorder characterized by impaired cerebral metabolic energy or reduced mitochondrial energy storage capacity in a mammalian subject can be treated or prevented. In one embodiment, the disorder is a neurological disorder, such as a hereditary neurodegenerative disease, an age-related neurodegenerative disorder, and a traumatic or hypoxic cerebrovascular injury. Examples of such hereditary neurodegenerative diseases include, but are not limited to, Down syndrome dementia, Huntington's disease, and amyotrophic lateral sclerosis. Examples of such age-related neurodegenerative disorders include, but are not limited to, Parkinson's disease and senile dementia. The category of senile dementia includes, for example, Alzheimer's disease and vascular dementia. Examples of such traumatic or hypoxic cerebrovascular injuries include, but are not limited to, secondary injury after traumatic brain injury, secondary injury after hypoxic-ischemic encephalopathy, secondary injury after neonatal asphyxia, secondary injury after ischemic stroke, secondary injury after hemorrhagic stroke, secondary injury after cardiac arrest, and secondary injury after drowning.

[0016] In another embodiment, the disorder is a neuromuscular disorder. Examples of such neuromuscular disorders include, but are not limited to, age-related sarcopenia, disuse atrophy of muscle, muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, and Friedreich's ataxia.

[0017] In another embodiment, the disorder is a heart failure. Examples of such heart failure include, but are not limited to, dilated cardiomyopathy, right ventricular failure (including right ventricular failure secondary to pulmonary hypertension), acute heart failure, and chronic heart failure.

[0018] In another embodiment, the disorder is a primary hereditary mitochondrial disease. Examples of such primary hereditary mitochondrial diseases include Mitochondrial Encephalomyopathy with Lactic Acidemia and Stroke-like episodes (MELAS), Myoclonus, epilepsy, and myopathy with ragged red fibers (MERRF), Neurogenic muscular weakness, ataxia, and retinitis pigmentosa (NARP), and Neurogenic muscular weakness, ataxia, and retinitis pigmentosa / maternally inherited Leigh syndrome (NARP / MILS).Leigh syndrome (also known as retinitis pigmentosa or Maternally inherited Leigh syndrome), Leber's hereditary optic neuropathy (LHON), Kearns-Sayre syndrome (KSS), Pearson Marrow-Pancreas syndrome (PMPS), Progressive external ophthalmoplegia (PEO), Chronic progressive external ophthalmoplegia (CPEO), Leigh syndrome, Mitochondrial neurogastrointestinal encephalopathy (MNGIE) This includes, but is not limited to, mitochondrial complex syndromes, Alper syndrome, multiple mtDNA deletion syndrome, MtDNA depletion syndrome, mitochondrial complex I deficiency, mitochondrial complex II (SDH) deficiency, mitochondrial complex III deficiency, mitochondrial complex IV (cytochrome c oxidase) deficiency, mitochondrial complex V deficiency, adenine nucleotide translocator (ANT) deficiency, pyruvate dehydrogenase (PDH) deficiency, multiple mitochondrial DNA deletion syndrome, Barth syndrome, mitochondrial myopathy, mitochondrial epilepsy, and mitochondrial tubular acidosis.

[0019] Further examples of disorders that can be treated or prevented according to the present invention include ethylmalonic aciduria with lactic acid bacteremia, 3-methylglutaconic aciduria with acidemia, refractory epilepsy with reduced remission during infection, Asperger syndrome with reduced remission during infection, autism with reduced remission during infection, cerebral palsy with reduced remission during infection, dyslexia with reduced remission during infection, maternal thrombocytopenia and leukemia syndrome, MARIAHS syndrome (mitochondrial ataxia, recurrent infections, aphasia, hypouricemia / myelin sheathing, seizures, and dicarboxylic aciduria), ND6 dystonia, cyclic vomiting syndrome with reduced remission during infection; 3-hydroxyisobutyrateuria with lactic acid bacteremia, diabetes mellitus with lactic acid bacteremia, uridine-responsive neurologic syndrome (URNS), and familial bilateral striatal necrosis (FBSN). This includes necrosis, aminoglycoside-related hearing loss, splenic lymphoma, Wolfram syndrome, tubular acidosis / diabetes / ataxia syndrome, lactic bacteremia, encephalomyopathy, 1+ proteinuria, aminoaciduria, hydroxyprolylinuria, cardiolipin deficiency, neuromuscular degenerative diseases, developmental delay in cognitive, motor, language, executive, or social abilities, epilepsy, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic bowel dysfunction, sensorineural hearing loss, neurogenic bladder dysfunction, migraine, ataxia, tubular acidosis, dilated cardiomyopathy, fatty liver disease, hepatic failure, and lactic bacteremia. Examples of developmental delays in cognitive, motor, language, executive function, or social abilities include, but are not limited to, pervasive developmental delay, pervasive developmental delay not otherwise specified (PDD-NOS), attention-deficit hyperactivity disorder (ADHD), Rett syndrome, and several forms of autism.

[0020] According to the present invention, the compound can be administered to any mammalian subject. In one embodiment, the mammalian subject is a human subject. According to the present invention, any conventional route of administration can be used. Preferably, the compound is administered orally. A skilled physician can adjust and optimize the dosage for a specific patient. Typically, the compound is administered to a body surface area of ​​1 m². 2 It is administered orally to human patients in doses of 1-3 g per dose. Typically, the dose is administered 2 or 3 times per day.

[0021] It has been found that when the acetacetate substituent at the 5' position of the ribose moiety is combined with acetate substituents at the 2' and 3' positions, novel compounds are obtained that deliver uridine to the circulation at a rate equivalent to or better than uridine triacetate. Prodrug substituents are further selected to provide additional or complementary therapeutic effects beyond simply facilitating uridine delivery.

[0022] In contrast, compounds such as uridine triacetate (2',3',5'-tri-O-acetoacetyluridine; acetoacetate is a ketone body, an oxidized form of beta-hydroxybutyrate) or 5'-O-heptanoyl-2',3'-di-O-acetyluridine resulted in relatively little systemic uridine delivery after oral administration. Similarly, 5'-O-acetoacetyluridine delivered very little uridine into circulation, and 2',3'-di-O-acetyluridine delivered less than 30% of the uridine into circulation compared to an equimolar dose of uridine triacetate. In contrast, a hybrid combining structural parts of these four relatively inactive molecules, 5'-O-acetoacetyl-2',3'-di-O-acetyluridine, delivered more uridine and uridine catabolites into circulation than an equimolar dose of uridine triacetate.

[0023] Acetoacetate, a ketone body with neuroprotective properties, is also delivered in parallel with uridine when incorporated into 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine. Both acetoacetate and beta-hydroxybutyrate, delivered by oral administration of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine, are problematic for oral delivery, partly due to their unpleasant taste. Therefore, ester prodrugs of these ketone bodies, particularly enantiomerically concentrated 3-hydroxybutyl 3-hydroxybutyrate with respect to the (3R,3R') enantiomer (whereas in mammalian biochemistry the R[or D] enantiomer is the natural form, while the chemical synthesis of BHB initially produces a racemic mixture of enantiomers), have been proposed. 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine overcomes the palatability issues of free keto acids by first delivering acetoacetate cleaved from the uridine skeleton, which is converted to R-BHB in vivo without requiring enantiomer enrichment during chemical synthesis and purification. A single agent with dual complementary pharmacological activity is also advantageous in terms of regulatory issues, including the complexity of clinical trials of combination drugs, and in reducing the patient burden that may limit compliance.

[0024] The conversion of acetacetate to BHB by the enzyme beta-hydroxybutyrate dehydrogenase uses NADH as an electron donor and triggers the regeneration of NAD+. In several disease states associated with mitochondrial respiratory chain dysfunction, the respiratory chain's ability to receive electrons from NADH is limited, and a high NADH / NAD+ ratio leads to significant secondary pathologies, as the NADH / NAD+ ratio affects the activity of numerous cytoplasmic enzymes. Maintaining an excessively high NADH / NAD+ ratio is called "reductive stress" and is recognized as a contributing factor to tissue dysfunction and adverse effects on lifespan in disorders involving mitochondrial electron transport chain deficiencies. Administration of exogenous AcAcDAU induces net oxidation of NADH, producing NAD+ and generating BHB, which itself has important pharmacological and physiological benefits as an alternative fuel for the brain and other tissues, and as a signaling molecule with known anti-inflammatory, anti-epileptic, and other effects. Therefore, AcAcDAU is useful for mitigating reductive stress in disorders characterized by NADH accumulation secondary to electron transport chain dysfunction by complementing the improvement of mitochondrial bioenergy efficiency mediated by uredin moiety through acetacetate-mediated NADH oxidation. Furthermore, BHB is known to have multifaceted potential benefits, including anti-inflammatory and anti-seizure activity, although not necessarily mediated by the reduction of reductive stress at concentrations achieved after oral administration of AcAcDAU.

[0025] In one embodiment of the method for producing the above 5'-O-acetoacetyl-2',3'-di-O-acetyluridine, step (a) is carried out at a temperature of 90°C to 110°C, for example, at about 110°C. In another embodiment, step (b) is carried out at a temperature of room temperature to 75°C, for example, at about 65°C. In yet another embodiment, step (c) is carried out at approximately room temperature, which is typically about 25°C. Preferably, the method for producing the above 5'-O-acetoacetyl-2',3'-di-O-acetyluridine further comprises, as step (e), purifying the crude 5'-O-acetoacetyl-2',3'-di-O-acetyluridine from step (d) to obtain purified 5'-O-acetoacetyl-2',3'-di-O-acetyluridine.

[0026] The present invention will be better understood by referring to the following examples, which illustrate but do not limit the present invention as described herein. [Examples]

[0027] Example 1: Synthesis of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine, 5'-O-(acetoacetyl)-2'-O-acetyl-3'-O-formyluridine, and 5'-O-(acetoacetyl)-3'-O-acetyl-2'-O-formyluridine

[0028] [ka]

[0029] A mixture of 2',3'-O-isopropylideneuridine (5.21 g, 18.3 mmol) and 25 mL of N-methylpyrrolidinone was heated to 110°C, and then 2,2,6-trimethyl-4H-1,3-dioxin-4-one (3.00 mL, 22.6 mmol) was added. After 2 hours, the mixture was cooled and partitioned between ethyl acetate (3 × 100 mL) and water (2 × 100 mL). The organic phase was washed with brine (100 mL), dried over anhydrous MgSO4, and concentrated in vacuum to obtain a dark brown oil. Rf 0.50 (10% MeOH / DCM). The crude mixture was heated in a mixture of 20 mL of formic acid and 20 mL of water at 65°C for 4 hours. Then, the volatile components were evaporated in vacuum. Rf 0.29 (10% MeOH / DCM).

[0030] The crude reaction product and a mixture of 6 mL of pyridine and 36 mL of DCM were cooled in an ice bath. Acetyl chloride (3.00 mL, 42.0 mmol) was then slowly added. After 2 hours, 5 mL of water was added, and the mixture was concentrated. The residue was dissolved in ethyl acetate (100 mL) and washed sequentially with water, saturated sodium bicarbonate, water, 1 M HCl, and 100 mL of water. The aqueous phase was extracted with ethyl acetate (2 × 100 mL). The organic phase was washed with brine (100 mL), dried over anhydrous MgSO4, and concentrated under vacuum. Purification of the main product by flash chromatography using step gradients of 1%, 2%, and 3% MeOH / DCM yielded 1.6 g of a pale yellow substance. Rf 0.24 (5% MeOH / DCM). Analysis by LC / MS revealed that the substance is a 9.4:1 mixture of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine and 5'-O-(acetoacetyl)-2'-O-acetyl-3'-O-formyluridine and 5'-O-(acetoacetyl)-3'-O-acetyl-2'-O-formyluridine.

[0031] [ka]

[0032] Example 2: Improvement of 5'-O-acetoacetyl-2',3'-di-O-acetyluridine synthesis

[0033] [ka]

[0034] 5'-O-acetoacetyl-2',3'-O-isopropylideneuridine A mixture of 2',3'-O-isopropylideneuridine (40 g, 141 mmol) and 2,2,6-trimethyl-4H-1,3-dioxin-4-one (18.7 mL, 141 mmol) dissolved in 60 mL of DMF was heated at 110°C for 2 hours. The mixture was then cooled to evaporate the volatile components. The residue was partitioned between ethyl acetate and water, the organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated by evaporation. Purification by flash chromatography (3% MeOH / DCM) yielded a product contaminated with colored substances. Partial decolorization with 2 g of activated carbon dissolved in ethyl acetate yielded 35 g of the product as a light brown syrup. Rf 0.71 (10% MeOH / DCM).

[0035] 5'-O-acetoacetyluridine A mixture of 5'-O-acetoacetyl-2',3'-O-isopropylideneuridine (35 g, 95 mmol), 100 mL of acetic acid, and 100 mL of water was heated at 65°C for 24 hours. At this point, partial TLC of the mixture indicated that the starting material had been almost completely consumed. The volatile components were evaporated under vacuum. The crude product was obtained. Rf 0.32 (10% MeOH / DCM).

[0036] 5'-O-acetoacetyl-2',3'-di-O-acetyluridine The crude product obtained above was dissolved in 100 mL of DCM and 30 mL of pyridine, and the solvent was then evaporated under vacuum. The residue was dissolved in 250 mL of DCM and 19 mL of pyridine. Once the mixture was homogenized, 22 mL of acetic anhydride was added. The mixture was stirred for 3 days. Next, the solvent was replaced with 500 mL of ethyl acetate and neutralized with saturated NaHCO3. Volatile components were evaporated to remove excess pyridine. The residue was partitioned between ethyl acetate and water, washed with 1 M HCl, water, and brine, the organic phase was dried over anhydrous MgSO4, and concentrated under vacuum. Purification by flash chromatography (3% MeOH / DCM) yielded 25.7 g of the product as a white foamy solid. 1 H NMR(400MHz,CDCl3)δ9.04(br s,1H), 7.47(d,1H,J=8Hz), 6.07(d,1H,J=6Hz), 5.84(d,1H,J=8Hz), 5.38~5.35 (m,1H), 5.31~5.28(m,1H), 4.90(ABX,1H,J=2.5,12.5Hz), 4.39(ABX,1H,J=3.7, 12.4Hz), 4.35~4.32 (m,1H), 3.61 (AB,1H,J=16Hz), 3.56 (AB,1H,J=16Hz), 2.97 (s,3H), 2.14 (s,3H), 2.10 (s,3H); LC / MS 99.4% purity (260nm); MW: calculated value 412, measured value 412; Rf 0.24 (5% MeOH / DCM).

[0037] [ka]

[0038] Example 3: Improvement of 5'-O-acetoacetyluridine synthesis

[0039] [ka]

[0040] 5'-O-acetoacetyluridine A mixture of 5'-O-acetoacetyl-2',3'-O-isopropylideneuridine (11.4 g, 31.0 mmol), 75 mL of acetic acid, and 75 mL of water was heated at 65°C for 24 hours. At this point, partial TLC of the mixture indicated that the starting material had been almost completely consumed. The volatile components were evaporated under vacuum. The residue was purified by flash chromatography (5% and 7% MeOH / DCM step gradients) to obtain 6.10 g of the product as a white foamy solid. 1 H NMR(400MHz,DMSO-d6)δ11.36(s,1H), 7.59(d,1H,J=8Hz), 5.76(d,1H,J=5Hz), 5.64(d,1H,J=8Hz), 5.46(d,1H,J=6Hz), 5.29(d,1H,J=6Hz), 4.31(1H,ABX,J=3,12Hz), 4.22(1H,ABX,J=5,12Hz), 4.07(q,1H,J=5Hz), 4.02~3.98(m,1H), 3.95(q,1H,J=5Hz), 3.68(2H,AB), 2.19(s,3H); 13 C NMR(100MHz,DMSO-d6)δ201.63, 167.07, 163.01, 150.62, 140.75, 101.99, 88.48, 81.04, 72.63, 69.68, 64.26, 49.46, 30.10;Rf 0.32 (10%MeOH / DCM).

[0041] Example 4: Plasma uridine and [uridine + uracil] of mice after oral administration of uridine prodrug. Chemical substances: (Hydroxypyrrolyl)methylcellulose (HPMC, (Hydroxypropyl)methyl cellulose) (SIGMA-Aldrich: Catalog No. H3785, CAS 9004-65-3); Uridine triacetate (2',3',5'-tri-O-acetyluridine): UTA, Product No. D000156, Lot No. Q000001095, manufactured by Almac Sciences; 2',3',5'-tri-O-(acetoacetyl)uridine: Tri-AcAcU; 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine: AcAcDAU; 2',3'-di-O-acetyl-5'-O-heptanoyluridine: DA-HU. (Note: The AcAcDAU used in this experiment was a mixture of 9.4 parts of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine from Example 1 above, and 1 part of a combination of 5'-O-(acetoacetyl)-2'-O-acetyl-3'-O-formyluridine and 5'-O-(acetoacetyl)-3'-O-acetyl-2'-O-formyluridine.)

[0042] Vessel: Water-soluble HPMC was used as the vehicle for oral administration of uridine derivatives.

[0043] Dosage formulation: UTA and other uridine derivatives were prepared in 0.75% HPMC. UTA and other uridine derivatives were added to 0.75% HPMC and homogenized to remove clumps. The suspension was prepared to the desired volume and concentration and sonicated to break down any remaining small clumps into fine particles. The suspension was stored at 4°C until use. The suspension was used within 24 hours of preparation.

[0044] Administration: Mice were orally administered UTA at a dose of 600 mg / kg at a rate of 0.02 ml per gram of body weight. Other uridine derivatives were administered in the same manner at concentrations containing an equimolar amount of uridine compared to UTA.

[0045] Animal: Female CD-1 mouse.

[0046] [Table 1]

[0047] The general initial plan for the experiment involved force-administering a uridine derivative orally to a group of six mice, followed by blood sampling at several time points (three mice were sampled at two time points (15 and 60 minutes), and the other three mice at two other time points (30 and 120 minutes)). Each experiment included HPMC (vegetative medium only) time points using three mice to establish a baseline of blood uridine.

[0048] [Table 2]

[0049] Blood samples were collected in plasma separation tubes, centrifuged immediately after blood collection, and a portion of the plasma was frozen for further processing. The plasma was later deproteinized, and uridine and uracil were quantified by liquid chromatography using UV absorbance detection and mass spectrometry.

[0050] The delivery of uridine into the bloodstream was assessed by monitoring plasma uridine and the total of uridine and uracil [uridine + uracil], since uracil is the first product in the enzymatic degradation of uridine. Mice convert administered uridine to uracil more rapidly and on a larger scale than humans.

[0051] Of the compounds tested, oral administration of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine (AcAcDAU) caused systemic delivery of uridine and [uridine + uracil] to a greater extent than oral administration of uridine triacetate.

[0052] Figures 1 and 2 show plasma [uridine + uracil] concentrations and plasma uridine levels after administration of a series of uridine prodrugs. Figure 3 shows plasma uridine levels in mice after oral administration of a different series of uridine prodrugs.

[0053] Example 5: Plasma uridine and beta-hydroxybutyrate of mice after oral administration of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine A key feature of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine (AcAcDAU) is that it delivers both uridine and acetacetate simultaneously or in parallel via a single drug molecule. Acetoacetate in plasma is in equilibrium with beta-hydroxybutyrate (BHB), which reflects the free NADH / NAD+ ratio in hepatic mitochondria, and this equilibrium is strongly supported because BHB is more common than the other ketone body under normal physiological conditions. Furthermore, BHB remains stable after blood collection and processing, while acetacetate is unstable. Therefore, plasma BHB was measured together with plasma uridine in mice orally administered AcAcDAU.

[0054] Chemicals: HPMC (hydroxypropyl) methylcellulose (SIGMA-Aldrich: catalog number H3785, CAS 9004-65-3), 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine (AcAcDAU; lot number 432168B). This experiment used the AcAcDAU from Example 2.

[0055] Vessel: Water-soluble HPMC was used as the vehicle for oral administration of uridine derivatives.

[0056] Dosage formulation: UTA and other uridine derivatives were prepared in 0.75% HPMC. AcAcDAU was added to 0.75% HPMC and homogenized to remove clumps. The suspension was prepared to the desired volume and concentration and sonicated to break down any remaining small clumps into fine particles. The suspension was stored at 4°C until use. The suspension was used within 24 hours of preparation.

[0057] Administration: Mice were orally administered AcAcDAU 2350 mg / kg at a dose of 0.02 ml per gram of body weight. Control mice were administered only with 0.75% HPMC media.

[0058] Animal: Female CD-1 mouse.

[0059] [Table 3]

[0060] General experimental design: The general experimental plan involved force-administering AcAcDAU to mice and then collecting blood samples at several time points (see the table below for details). The experiment included two groups of mice that were force-administered HPMC alone to determine baseline plasma uridine levels, with blood samples collected at 60 and 120 minutes.

[0061] [Table 4]

[0062] Mice were administered a suspension of 117 mg / ml AcAcDAU (0.75% aqueous solution of hydroxypropyl methylcellulose) at a dose of 2,350 mg per kg of body weight, at a rate of 0.02 ml per g of body weight. At each time point (15, 30, 60, and 120 minutes after administration), blood samples (approximately 100 microliters) from four mice were collected in plasma separation tubes. The samples were rapidly centrifuged, and the resulting plasma was frozen with dry ice. Subsequently, the plasma samples were deproteinized, and reverse-phase HPLC analysis was performed to measure plasma uridine and its initial metabolite, uracil. A portion of the same plasma samples was also used to measure BHB concentration using a commercially available enzyme assay kit.

[0063] As shown in Figures 4, 5, and 6, plasma uridine and its initial metabolite, uracil, increased after oral administration of AcAcDAU. As shown in Figure 7, plasma beta-hydroxybutyrate also increased after administration of AcAcDAU, with an area under the curve (AUC) of 24,212 nmol / ml × min.

[0064] Example 6: Protection from 3-nitropropionic acid-induced death by 5'-acetoacetyl-2',3'-di-O-acetyluridine and 2'3'5'-tri-O-acetyluridine 5'-O-acetoacetyl-2',3'-di-O-acetyluridine (AcAcDAU) delivers both uridine and acetacetate (and acetacetate-derived beta-hydroxybutyrate [BHB]) into circulation after oral administration. The study was designed to compare the relative efficacy of UTA versus AcAcDAU in a model of progressive lethal mitochondrial energy deficiency. Mitochondrial dysfunction was induced by daily intraperitoneal injection of 3-nitropropionic acid (3-NP, 60 mg / kg / day). 3-NP is an irreversible inhibitor of succinate dehydrogenase (complex II of the mitochondrial electron transport chain). Daily administration of 3-NP progressively reduces the ability of mitochondria to maintain ATP production, ultimately leading to death from both neurodegeneration and cardiomyopathy.

[0065] Female CD-1 mice, approximately 16 weeks old, were divided into groups of 10 mice each, matched in weight. All mice received daily intraperitoneal injections of 3-NP (60 mg / kg) in a volume of 0.01 ml per gram of body weight.

[0066] In addition to daily injections of 3-NP, the three groups of mice were administered either 1) the medium, 2) UTA, or 3) AcAcDAU. This experiment used AcAcDAU as in Example 2. These treatments were administered orally via enteral nutrition at a volume of 0.02 ml per gram of body weight.

[0067] group: 1. Controlled medium (0.75% HPMC; hydroxypropyl methylcellulose) 2. Uridine triacetate 1000 mg / kg twice daily (suspended in 0.75% HPMC) 3. AcAcDAU 1113.5 mg / kg (equimolar to UTA 1000 mg / kg) twice daily (dissolved in 0.75% HPMC)

[0068] The treatment plan was as follows:

[0069] [Table 5]

[0070] During repeated treatment with 3-NP, the mice lost body weight, so their body weight was recorded daily to adjust the drug dose.

[0071] The protective effects of the test drugs were quantified and compared using survival time (median survival time, when 50% of the animals in the group died) and survival rate at the end of the 11-day study.

[0072] result: The final survival rate and median survival time at the end of the study are shown in Table 1 and Figure 8 below.

[0073] [Table 6]

[0074] Equimolar doses of both UTA and AcAcDAU improved survival in mice with progressive mitochondrial dysfunction compared to the medium alone. AcAcDAU treatment resulted in a longer median survival time than UTA administration in mice with progressive mitochondrial energy deficiency.

[0075] Example 7: Protection against death caused by 3-nitropropionic acid by 5'-acetoacetyl-2',3'-di-O-acetyluridine and 2'3'5'-tri-O-acetyluridine Example 6 demonstrated that AcAcDAU treatment, when administered orally at a dose of 1113.5 mg / kg, resulted in better survival in a model of lethal progressive mitochondrial failure compared to equimolar UTA (1000 mg / kg / dose). In this example, the effects of high-dose UTA (2000 mg / kg / dose) versus equimolar AcAcDAU (2227 mg / kg / dose) were compared in the same model system.

[0076] group: 1. Controlled medium (0.75% HPMC; hydroxypropyl methylcellulose) 2. Uridine triacetate 2000 mg / kg twice daily (suspended in 0.75% HPMC)

[0077] AcAcDAU 2227 mg / kg (equimolar to UTA 2000 mg / kg) twice daily (dissolved in 0.75% HPMC). This experiment used the AcAcDAU from Example 2. The treatment plan was as follows:

[0078] [Table 7]

[0079] During repeated treatment with 3-NP, the mice lost body weight, so their body weight was recorded daily to adjust the drug dose.

[0080] The protective effect of the test drug was quantified and compared using the survival rate of 10 mice in each group at the end of the 10-day study.

[0081] result: The final survival rates at the end of the trial are shown in Table 2 below.

[0082] [Table 8]

[0083] Both UTA and AcAcDAU improved survival in mice with progressive mitochondrial failure compared to the medium alone. AcAcDAU treatment resulted in better survival than administration of an equimolar dose of oral UTA at day 10 in mice with progressive mitochondrial energy deficiency.

Claims

1. The compound is 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine.

2. A compound according to claim 1 for use in the treatment of a disorder characterized by metabolic energy deficiency of the brain or reduced mitochondrial energy storage capacity in a mammalian subject, wherein the use comprises the step of administering to the subject an amount of the compound according to claim 1 that is effective in treating the disorder.

3. The compound according to claim 1, for use in the manufacture of a pharmaceutical product for the treatment or prevention of a disorder characterized by metabolic energy deficiency of the brain or reduced mitochondrial energy storage capacity in mammalian subjects.

4. The compound for use according to claim 2 or 3, wherein the disorder is a neurological disorder.

5. The compound for use according to claim 4, wherein the neurological disorder is selected from the group consisting of hereditary neurodegenerative diseases, age-related neurodegenerative disorders, and traumatic or ischemic brain injury.

6. The compound for use according to claim 5, wherein the neurological disorder is a hereditary neurodegenerative disease.

7. Hereditary neurodegenerative diseases, Down syndrome dementia, Huntington's disease, and Amyotrophic lateral sclerosis (ALS) A compound for use according to claim 6, selected from the group consisting of the following.

8. The compound for use according to claim 5, wherein the neurological disorder is age-related neurodegenerative disorder.

9. Age-related neurodegenerative disorders Parkinson's disease, and senile dementia A compound for use according to claim 8, selected from the group consisting of the following.

10. The compound for use according to claim 9, wherein the senile dementia is selected from the group consisting of Alzheimer's disease and vascular dementia.

11. The compound for use according to claim 5, wherein the neurological disorder is traumatic or ischemic brain injury.

12. Traumatic or ischemic brain injury, Secondary injuries following traumatic brain injury, Secondary injury following hypoxic-ischemic encephalopathy, Secondary injuries after birth asphyxia, Secondary injury after ischemic stroke, Secondary injury after hemorrhagic stroke, Secondary injuries after cardiac arrest, and Secondary injuries after drowning A compound for use according to claim 11, selected from the group consisting of the following.

13. The compound for use according to claim 2 or 3, wherein the disorder is a neuromuscular disorder.

14. Neuromuscular disorders, Age-related sarcopenia, Disuse atrophy of muscles, Muscular dystrophy, Myotonic dystrophy, Chronic fatigue syndrome, and Friedreich ataxia A compound for use according to claim 13, selected from the group consisting of the following.

15. The compound for use according to claim 2 or 3, wherein the disorder is heart failure.

16. Heart failure, Dilated cardiomyopathy, right ventricular failure, Acute heart failure, and chronic heart failure A compound for use according to claim 15, selected from the group consisting of the following.

17. The compound for use according to claim 2 or 3, wherein the disorder is a primary hereditary mitochondrial disease.

18. Primary hereditary mitochondrial disease, Mitochondrial encephalomyopathy (MELA) with lactic acid bacteremia and stroke-like symptoms, Myoclonus, epilepsy, and myopathy with red rag fibers (MERRF), Neuropathic muscle weakness, ataxia, retinitis pigmentosa (NARP), Neuropathic muscle weakness, ataxia, retinitis pigmentosa / maternally inherited Leigh syndrome (NARP / MILS), Leber hereditary optic neuropathy (LHON), "mitochondrial blindness", Kearns-Sayre syndrome (KSS), Pearson myelopancreatic syndrome (PMPS), progressive external ophthalmoplegia (PEO), chronic progressive external ophthalmoplegia (CPEO), Leigh syndrome, Mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE), Alper syndrome, Multiple mtDNA deletion syndrome, MtDNA depletion syndrome, Mitochondrial complex I deficiency, Mitochondrial complex II (SDH) deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV (cytochrome c oxidase) deficiency, Mitochondrial complex V deficiency, Adenine nucleotide translocator (ANT) deficiency, Pyruvate dehydrogenase (PDH) deficiency, Multiple mitochondrial DNA deletion syndrome, Birth syndrome, Mitochondrial myopathy, Mitochondrial epilepsy, and Mitochondrial tubular acidosis A compound for use according to claim 17, selected from the group consisting of the following.

19. The compound for use according to claim 2 or 3, wherein the mammalian target is a human target.

20. The compound for use according to claim 2 or 3, wherein the compound is administered orally.

21. The compound is present in 1-3 g / m² ２ The compound for use according to claim 20, administered in the dose of [amount].

22. The compound for use according to claim 21, wherein the dose is administered two or three times per day.

23. A pharmaceutical composition comprising an amount effective for treating or preventing a disorder characterized by metabolic energy deficiency of the brain or reduced mitochondrial energy storage capacity in mammalian subjects, and a pharmaceutically acceptable carrier.

24. A method for producing 5'-O-acetoacetyl-2',3'-di-O-acetyluridine, (a) Mixing 2',3'-O-isopropylidenuridine and 2,2,6-trimethyl-4H-1,3-dioxin-4-one in dimethylformamide under conditions that produce 5'-O-acetoacetyl-2',3'-O-isopropylidenuridine; (b) Mixing the 5'-O-acetoacetyl-2',3'-O-isopropylideneuridine with an aqueous acetic acid solution under conditions that produce crude 5'-O-acetoacetyluridine; (c) Dissolve the crude 5'-O-acetoacetyluridine in a mixture of dichloromethane and pyridine, then add acetic anhydride and stir for several days; and (d) Switch the solvent in the mixture to ethyl acetate and saturated NaHCO ３ Neutralize with to obtain crude 5'-O-acetoacetyl-2',3'-di-O-acetyluridine; The method, including the method described above.

25. The method according to claim 24, wherein (a) is carried out at a temperature of 90°C to 110°C.

26. The method according to claim 24, wherein (b) is carried out at a temperature of room temperature to 75°C.

27. ​​The method according to claim 26, wherein (b) is carried out at a temperature of 65°C.

28. The method of claim 24, wherein (c) is carried out at room temperature.

29. The method according to claim 24, further comprising purifying the crude 5'-O-acetoacetyl-2',3'-di-O-acetyluridine of (d) to obtain purified 5'-O-acetoacetyl-2',3'-di-O-acetyluridine.

30. The compound, 5'-O-acetoacetyluridine.

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