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

Abstract

5'-O-(acetylacetyl)-2',3'-di-O-acetylxanthosine can be used in the treatment or prevention of a disorder characterized by a failure of brain metabolic energy or a decrease in mitochondrial energy reserve capacity.

Description

[0001] Oral delivery of uridine for therapeutic purposes is limited by its poor bioavailability, which is approximately 7% in both humans and mice. Ester prodrugs of uridine have been found to improve bioavailability; however, only one prodrug capable of delivering sufficient uridine for clinical purposes has been identified: 2',3',5'-tri-O-acetyluridine (or uridine triacetate). The oral bioavailability of uridine triacetate has been measured at approximately 50% (Ashour, 1996). Therefore, there is room for improvement in the delivery efficiency of uridine, which is important for therapeutic applications requiring relatively high doses of uridine. Summary of the Invention

[0002] This invention provides a compound, 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine. It provides a method for treating or preventing a condition in a mammalian subject characterized by brain metabolic energy depletion or decreased mitochondrial energy reserves, comprising administering to the subject an amount of the compound of the invention capable of effectively treating the condition. This invention also provides a compound of the invention for treating or preventing a condition in a mammalian subject characterized by brain metabolic energy depletion or decreased mitochondrial energy reserves, or for preparing a medicament for treating or preventing the aforementioned condition. Furthermore, it provides a pharmaceutical composition comprising the compound of the invention and a pharmaceutically acceptable carrier. The present invention also provides a method for producing 5'-O-acetylacetyl-2',3'-di-O-acetyluridine, comprising the following steps: (a) mixing 2',3'-O-isopropyluridine with 2,2,6-trimethyl-4H-1,3,-dioxane-4-one in dimethylformamide under conditions for producing 5'-O-(acetylacetyl)-2',3'-O-isopropyluridine; (b) in the production Under the conditions for crude 5'-O-acetylacetyluridine, 5'-O-(acetylacetyl)-2',3'-O-isopropylideneuridine from step (a) is mixed with an aqueous solution of acetic acid; (c) the crude 5'-O-acetylacetyluridine from step (b) is dissolved in a mixture of dichloromethane and pyridine, then acetic anhydride is added and the mixture is stirred for several days; (d) the mixture obtained in step (c) is subjected to a solvent of ethyl acetate and neutralized with saturated NaHCO3 to produce crude 5'-O-acetylacetyl-2',3'-di-O-acetyluridine. The present invention also provides 5'-O-acetylacetyluridine as a crude intermediate and as a separated compound.

[0003] Acetate substituents improve absorption from the gastrointestinal tract to the circulatory system and are rapidly cleared by nonspecific esterase activity. Besides being pharmacologically and therapeutically neutral, acetate is also a mild metabolite. The discovery that introducing an acetoacetyl substituent at the 5' position not only provides beneficial pharmacological activity for the delivery of uridine but also improves the bioavailability of uridine to make it suitable for therapeutic purposes, producing dual therapeutic activity in a single molecule, represents a significant advance. Attached Figure Description

[0004] Figure 1 : Mouse plasma [uridine + uracil] after oral administration of uridine prodrug

[0005] UTA = uridine triacetate (2',3',5'-tri-O-acetyluridine)

[0006] Tri-AcAcU=2',3',5'-Tri-O-(acetylacetyl)uridine

[0007] AcAcDAU = 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine

[0008] DA-HU = 2',3'-Di-O-acetyl-5'-O-heptanyluridine

[0009] Figure 2 : Plasma uridine in mice after oral administration of uridine prodrug

[0010] Tri-AcAcU=2',3',5'-Tri-O-(acetylacetyl)uridine

[0011] AcAcDAU = 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine

[0012] DA-HU = 2',3'-Di-O-acetyl-5'-O-heptanyluridine

[0013] Figure 3 : Plasma uridine in mice after oral administration of uridine prodrug

[0014] UTA = uridine triacetate (2',3',5'-tri-O-acetyluridine)

[0015] DAU = 2'3'-di-O-acetyluridine

[0016] AcAcU=5'-O-(acetylacetyl)uridine

[0017] Figure 4 : Uric acid in mouse plasma after oral administration of AcAcDAU

[0018] AcAcDAU = 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine

[0019] Figure 5 : Uracil in mouse plasma after oral administration of AcAcDAU

[0020] AcAcDAU = 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine

[0021] Figure 6 : Urine and uracil in mouse plasma after oral administration of AcAcDAU

[0022] AcAcDAU = 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine

[0023] Figure 7 β-hydroxybutyrate in mouse plasma after oral administration of AcAcDAU

[0024] BHB = β-hydroxybutyric acid

[0025] Figure 8 Survival rate of mice treated with 60 mg / kg / day of 3-nitropropionic acid followed by oral administration of 1000 mg / kg bid uridine triacetate (UTA) or oral administration of 1113.5 mg / kg bid 5'-O-acetylacetyl-2',3'-di-O-acetyluridine (AcAcDAU). Detailed Implementation

[0026] As used in this invention, the transitional term "comprising" is open-ended. A claim using this term may include elements other than those referenced in the claim. Thus, for example, a claim may be interpreted as a treatment regimen that also includes other therapeutic agents or doses of a virus not specifically listed herein, provided that the listed elements or their equivalents are present.

[0027] abbreviation

[0028] Some compounds are indicated herein by their chemical names or the abbreviations or structural formulas shown below. The compound AcAcDAU is within the scope of this invention.

[0029] UTA = uridine triacetate (2',3',5'-tri-O-acetyluridine)

[0030] Tri-AcAcU = 2',3',5'-tri-O-(acetylacetyl)uridine, molecular weight 496.42

[0031]

[0032] AcAcDAU = 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine, molecular weight 412.35

[0033]

[0034] DA-HU = 2',3'-di-O-acetyl-5'-O-heptayluridine, molecular weight 440.44

[0035]

[0036] DAU = 2'3'-di-O-acetyluridine

[0037] AcAcU = 5'-O-(acetoacetyl)uridine (also known as 5'-O-acetoacetyluridine)

[0038]

[0039] BHB = β-hydroxybutyric acid (also known as 3-hydroxybutyric acid)

[0040] DCM = dichloromethane (also known as methylene chloride, chemical formula CH2Cl2)

[0041] DMF = dimethylformamide, chemical formula (CH3)2NC(O)H

[0042] According to the methods, compounds, applications, and pharmaceutical compositions of the present invention, any existing condition in mammalian subjects characterized by brain metabolic energy depletion or decreased mitochondrial energy reserves can be treated or prevented. In one embodiment, the aforementioned condition is a neurological condition, such as a hereditary neurodegenerative disease, an age-related neurodegenerative disease, and a traumatic or ischemic brain injury. Examples of the aforementioned hereditary neurodegenerative diseases include, but are not limited to, Down syndrome dementia, Huntington's disease, and amyotrophic lateral sclerosis (ALS). Examples of the aforementioned age-related neurodegenerative diseases include, but are not limited to, Parkinson's disease and Alzheimer's disease. Categories of Alzheimer's disease include, for example, Alzheimer's disease and vascular dementia. Examples of the aforementioned traumatic or ischemic brain injuries include, but are not limited to, secondary injuries following traumatic brain injury, secondary injuries following hypoxic-ischemic encephalopathy, secondary injuries following birth asphyxia, secondary injuries following ischemic stroke, secondary injuries following hemorrhagic stroke, secondary injuries following cardiac arrest, and secondary injuries following drowning.

[0043] In another embodiment, the aforementioned condition is a neuromuscular disorder. Examples of such neuromuscular disorders include, but are not limited to, age-related sarcopenia, muscle disuse atrophy, muscular dystrophy, myotonic dystrophy, chronic fatigue syndrome, and Friedreich ataxia.

[0044] In another implementation, the aforementioned condition is heart failure. Examples of 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.

[0045] In another embodiment, the aforementioned condition is a primary hereditary mitochondrial disease. Examples of such primary hereditary mitochondrial diseases include, but are not limited to, MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like seizures), MERRF (myoclonic epileptic myopathy with broken red fibers), NARP, NARP / MILS (neurogenic myasthenia-ataxia-retinitis pigmentosa / maternally inherited Leigh syndrome), LHON (Lebers optic neuropathy) "mitochondrial blindness," KSS (Karns-Sell syndrome), PMPS (Pearson's myelopancreatic syndrome), PEO (progressive extraocular muscle palsy), CPEO (chronic progressive extraocular muscle palsy), L... Eigh syndrome, MNGIE (mitochondrial neurogastrointestinal encephalopathy syndrome), Alpert 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 transporter (ANT) deficiency, pyruvate dehydrogenase (PDH) deficiency, multiple mitochondrial DNA deletion syndrome, Barth syndrome, mitochondrial myopathy, mitochondrial epilepsy, and mitochondrial renal tubular acidosis.

[0046] Additional examples of conditions that can be treated or prevented according to the present invention include, but are not limited to: ethylmalonic aciduria with lactic acidemia, 3-methylglutaric aciduria with acidemia, refractory epilepsy with decreased urination during infection, Asperger's syndrome with decreased urination during infection, autism with decreased urination during infection, cerebral palsy with decreased urination during infection, maternally inherited thrombocytopenia and leukemia syndrome, MARIAHS syndrome (mitochondrial ataxia-recurrent infection-aphasia-hypouricemia / demyelination-seizures and dicarboxylic aciduria), ND6 dystonia, periodic vomiting syndrome with decreased urination during infection, 3-hydroxyisobutyric aciduria with lactic acidemia, and diabetes mellitus with lactic acidemia. The following conditions are considered as developmental delays: Urea-responsive neurosis (URNS), familial bilateral striatal necrosis (FBSN), aminoglycoside-related hearing loss, splenic lymphoma, Wolfram syndrome, renal tubular acidosis / diabetes / ataxia syndrome; lactic acidosis, encephalomyopathy, proteinuria + 1, aminoaciduria, hydroxyprolineuria, cardiolipin deficiency, neuromuscular degenerative diseases, cognitive-motor-linguistic-executive or social skill developmental delays; epilepsy, peripheral neuropathy, optic neuropathy, autonomic neuropathy, neurogenic bowel dysfunction, sensorineural hearing loss, neurogenic bladder dysfunction, migraine, ataxia, renal tubular acidosis, dilated cardiomyopathy, steatohepatitis, liver failure, and lactic acidosis. Examples of cognitive-motor-linguistic-executive or social skill developmental delays include, but are not limited to: pervasive developmental delay, pervasive developmental disorder-unspecified (PDD-NOS), attention deficit hyperactivity disorder (ADHD), Rett syndrome, and certain forms of autism.

[0047] According to the present invention, the above-described compound can be administered to any mammalian subject. In one embodiment, the mammalian subject can be a human subject. According to the present invention, any conventional route of administration can be used. Preferably, the above-described compound can be administered orally. A technician can titrate the drug dosage to optimize the dosage for a specific patient. Typically, the above-described compound is administered at a dose of 1 to 3 g / m². 2 The dosage based on body surface area is administered orally to human patients. The above dosage is typically administered 2 or 3 times daily.

[0048] A novel compound has been found to be produced by combining an acetoacetate substituent at the 5' position of the riboside group with acetate substituents at the 2' and 3' positions, which delivers uridine into the circulatory system as well as or better than uridine triacetate. The aforementioned prodrug substituents are further selected to provide additional or complementary therapeutic benefits beyond aiding uridine delivery.

[0049] Conversely, compounds such as uridine triacetyl acetate (2',3',5'-tri-O-acetoacetyluridine; acetoacetyl is a ketone body and an oxidized form of β-hydroxybutyric acid) or 5'-O-heptanyl-2',3'-di-O-acetyluridine provide relatively poor systemic uridine delivery after oral administration. Similarly, 5'-O-acetoacetyluridine delivers very little uridine into the circulation, while 2',3'-di-O-acetyluridine delivers less than 30% of the uridine delivered by an equimolar dose of uridine triacetyl. Conversely, the hybrid of these four relatively inactive molecular moieties, namely 5'-O-acetoacetyl-2',3'-di-O-acetyluridine, delivers more uridine and uridine metabolites into the circulation than an equimolar dose of uridine triacetyl.

[0050] When conjugated to 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine, acetoacetate (a ketone body with neuroprotective properties) is delivered simultaneously with uridine. Oral administration of both acetoacetate and β-hydroxybutyrate (both deliverable via oral administration of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine) is difficult, partly due to unpleasant taste; therefore, ester prodrugs of these ketone bodies have been proposed. Notably, 3-hydroxybutyrate-3-hydroxybutyl ester is enriched in the (3R, 3R') enantiomer (the R [or D] enantiomer is the naturally occurring form in mammalian biochemistry, while the initial chemical synthesis of BHB yielded a racemic mixture of enantiomers). 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine overcomes the palatability problem of free keto acids and delivers the acetoacetate cleaved from the uridine backbone, which is converted to R-BHB in vivo without enantiomeric enrichment during chemical synthesis and purification. This single agent with dual complementary pharmacological activities is advantageous from the perspective of regulatory issues related to the complexity of combination therapy clinical trials and also beneficial for reducing the burden on patients who may have limited adherence.

[0051] AcAcDAU utilizes NADH as an electron donor in the conversion to BHB via β-hydroxybutyrate dehydrogenase, leading to NAD+ regeneration. In some disease states associated with mitochondrial respiratory chain dysfunction, high NADH / NAD+ ratios, resulting from the limited ability of the respiratory chain to accept electrons from NADH, can cause significant secondary pathologies because the NADH / NAD+ ratio affects the activity of enzymes in a large number of cytoplasm. Maintaining an excessive NADH / NAD+ ratio is known as "reductive stress" and is a recognized factor in tissue dysfunction and adverse lifespan effects in conditions with impaired mitochondrial electron transport chain defects. Exogenous AcAcDAU administration induces net oxidation of NADH to produce NAD+ and generates BHB, which has important pharmacological and physiological benefits. It can serve as an alternative fuel for the brain and other tissues, as well as a signaling molecule, and has known anti-inflammatory and anti-epileptic effects. Therefore, AcAcDAU can be used to alleviate reductive stress in conditions characterized by NADH accumulation secondary to electron transport chain dysfunction, and acetoacetate-induced NADH oxidation compensates for the improvement in mitochondrial bioenergy efficiency mediated by uridine groups. Furthermore, BHB is known to have the potential pleiotropic benefits at concentrations reached after oral administration of AcAcDAU, which are not necessarily mediated by the relief of decompression stress, including but not limited to anti-inflammatory and antiepileptic activities.

[0052] In one embodiment of a method for producing the above-described 5'-O-acetylacetyl-2',3'-di-O-acetyluridine, step (a) is carried out at a temperature of 90°C to 110°C, for example, about 110°C. In another embodiment, step (b) is carried out at a temperature of room temperature to 75°C, for example, about 65°C. In yet another embodiment, step (c) is carried out at room temperature. Room temperature is typically 25°C. Preferably, the method for producing the above-described 5'-O-acetylacetyl-2',3'-di-O-acetyluridine further comprises purifying the crude 5'-O-acetylacetyl-2',3'-di-O-acetyluridine in step (d) as described in step (e) to obtain purified 5'-O-acetylacetyl-2',3'-di-O-acetyluridine.

[0053] The invention will be better understood by referring to the following embodiments, which illustrate but do not limit the invention.

[0054] Example

[0055] Example 1: Synthesis of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine and 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

[0056]

[0057] A mixture of 2',3'-O-isopropylideneuridine (5.21 g, 18.3 mmol) and 25 mL of N-methylpyrrolidone was heated to 110 °C, followed by the addition of 2,2,6-trimethyl-4H-1,3-dioxane-4-one (3.00 mL, 22.6 mmol). 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 under vacuum to give a dark brown oil. Rf 0.50 (10% MeOH / DCM). The crude mixture was heated at 65 °C for 4 hours in a mixture of 20 mL formic acid and 20 mL water. The volatile components were then evaporated under vacuum. Rf 0.29 (10% MeOH / DCM).

[0058] The crude reaction product was cooled in an ice bath with a mixture of 6 mL pyridine and 36 mL DCM. 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 absorbed in ethyl acetate (100 mL) and washed successively with 100 mL of water, saturated sodium bicarbonate, water, 1 M HCl, and 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. The major product was purified by rapid chromatography using a step gradient of 1%, 2%, and 3% MeOH / DCM to give 1.6 g of a pale yellow substance. Rf 0.24 (5% MeOH / DCM). LC / MS analysis showed that the substance was a 9.4:1 mixture of 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine, 5'-O-(acetylacetyl)-2'-O-acetyl-3'-O-formyluridine, and 5'-O-(acetylacetyl)-3'-O-acetyl-2'-O-formyluridine.

[0059] Option 1

[0060]

[0061] Example 2: Improved Synthesis of 5'-O-acetylacetyl-2',3'-di-O-acetyluridine

[0062]

[0063] 5'-O-acetylacetyl-2',3'-O-isopropyluridine

[0064] A mixture of 2',3'-O-isopropylideneuridine (40 g, 141 mmol) and 2,2,6-trimethyl-4H-1,3-dioxane-4-one (18.7 mL, 141 mmol) was heated in 60 mL of DMF at 110 °C for 2 hours. The mixture was then cooled and the volatile components were evaporated. The residue was partitioned between ethyl acetate and water, and the organic phase was washed with brine, dried over anhydrous MgSO4, and concentrated by evaporation. The major product was purified by rapid chromatography (3% MeOH / DCM) to obtain a product contaminated with colored substances. Partial decolorization with 2 g of activated carbon in ethyl acetate yielded 35 g of a light brown syrupy product. Rf 0.71 (10% MeOH / DCM).

[0065] 5'-O-acetylacetyluridine

[0066] A mixture of 5'-O-acetylacetyl-2',3'-O-isopropylidene uridine (35 g, 95 mmol), 100 mL acetic acid, and 100 mL water was heated at 65 °C for 24 h, during which TLC of aliquots of the mixture showed that the starting material was almost completely consumed. The volatile components were evaporated under vacuum. The crude product was used for further reactions. Rf 0.32 (10% MeOH / DCM).

[0067] 5'-O-acetylacetyl-2',3'-di-O-acetyluridine

[0068] The crude product obtained above was absorbed in 100 mL of DCM and 30 mL of pyridine, and then the solvent was evaporated under vacuum. The residue was absorbed 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. Then, the solvent was replaced with 500 mL of ethyl acetate and neutralized with saturated NaHCO3. The volatile components were evaporated to remove excess pyridine. The residue was partitioned between ethyl acetate and water, 1 M HCl, water, and brine. The organic phase was dried over anhydrous MgSO4 and concentrated under vacuum. Purification by rapid chromatography (3% MeOH / DCM) yielded 25.7 g of a white, foamy solid product. 1HNMR(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); Molecular weight: calculated 412, found 412; Rf 0.24 (5% MeOH / DCM).

[0069] Option 2

[0070]

[0071] Example 3: Improved Synthesis of 5'-O-acetylacetyluridine

[0072] 5'-O-acetylacetyluridine

[0073] A mixture of 11.4 g (31.0 mmol) of 5'-O-acetylacetyl-2',3'-O-isopropylideneuridine, 75 mL of acetic acid, and 75 mL of water was heated at 65 °C for 24 h, during which TLC of aliquots of the mixture showed that the starting material was almost completely consumed. The volatile components were evaporated under vacuum. The residue was purified by rapid chromatography (step gradient of 5% and 7% MeOH / DCM) to give 6.10 g of a white, foamy solid product. 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); 13C 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).

[0074] Example 4: Plasma uridine and [uridine + uracil] levels in mice after oral administration of uridine prodrug

[0075] Chemicals: HPMC (hydroxypropyl) methylcellulose (SIGMA-Aldrich: cat#H3785, CAS 9004-65-3); uridine triacetate (2',3',5'-tri-O-acetyluridine): UTA, drug code D000156, Lot#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-heptanyluridine: DA-HU. (Note: The AcAcDAU used in this experiment is a mixture of 9.4 parts of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine and 1 part of the combination of 5'-O-(acetoacetyl)-2'-O-acetyl-3'-O-formyluridine and 5'-O-(acetoacetyl)-3'-O-acetyl-2'-O-formyluridine from Example 1 above.)

[0076] Carrier: Aqueous HPMC is used as a carrier for the oral administration of uridine derivatives.

[0077] Dosage Formulation: UTA and other uridine derivatives are prepared in 0.75% HPMC. UTA and other uridine derivatives are added to 0.75% HPMC and homogenized to eliminate clumps. The suspension is prepared to the desired volume and concentration and sonicated to break down any remaining small clumps into fine particles. The suspension is stored at 4°C until use. Use the suspension within 24 hours of preparation.

[0078] Administration: Mice were administered 600 mg / kg of UTA by gavage at a concentration of 0.02 ml / g body weight. Other uridine derivatives were administered in the same manner at a concentration containing an equimolar amount of uridine as that of UTA.

[0079] Experimental animals: female CD-1 mice.

[0080]

[0081] The overall initial setup for the above experiments involved gavage of 6 mice in each group with uridine derivatives and subsequent blood sampling at several time points (blood was collected from 3 mice at 2 time points (15 and 60 minutes), and from another 3 mice at two additional time points (30 and 120 minutes)). Each experiment included a time point using 3 mice with HPMC (vector only) to establish a baseline for blood uridine levels.

[0082]

[0083] Blood samples were collected into plasma separation tubes and centrifuged immediately after collection. Aliquots of the frozen plasma were used for subsequent processing. The plasma was then deproteinized, and uridine and uracil were quantified using liquid chromatography with UV absorption detection and mass spectrometry.

[0084] Since uracil is the first product of the enzymatic degradation of uridine, the delivery of uridine into the bloodstream is assessed by monitoring plasma uridine and the sum of uridine and uracil [uridine + uracil]. Compared to humans, mice convert administered uridine to uracil more rapidly and extensively.

[0085] Among the compounds tested, oral administration of 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine resulted in similar or greater systemic uridine and [uridine + uracil] delivery compared to uridine triacetate.

[0086] After administration of a group of uridine prodrugs, the plasma [uridine + uracil] and plasma uridine concentrations were... Figure 1 and Figure 2 It is displayed in the middle. Figure 3 This indicates plasma uridine levels in mice after oral administration of different groups of uridine prodrugs.

[0087] Example 5: Plasma in mice after oral administration of 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine Uridine and β-hydroxybutyrate

[0088] A key characteristic of 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine (AcAcDAU) is its simultaneous or concurrent delivery of uridine and acetoacetate via a single drug molecule. Plasma acetoacetate and β-hydroxybutyrate are in equilibrium, reflecting the free NADH / NAD+ ratio in liver mitochondria, and under normal physiological conditions, BHB is more strongly favored as the more dominant of the two ketone bodies. Furthermore, BHB is stable after blood sampling and processing, while acetoacetate is unstable. Therefore, plasma BHB was measured along with plasma uridine in mice receiving oral AcAcDAU.

[0089] Chemicals: HPMC (hydroxypropyl) methylcellulose (SIGMA-Aldrich: cat#H3785, CAS9004-65-3), 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine (AcAcDAU; lot#432168B). This experiment used AcAcDAU from Example 2.

[0090] Carrier: HPMC aqueous solution was used as a carrier for oral administration of uridine derivatives.

[0091] Dosage Formulation: UTA and other uridine derivatives are prepared in 0.75% HPMC. AcAcDAU and other uridine derivatives are added to 0.75% HPMC and homogenized to eliminate clumps. The suspension is prepared to the desired volume and concentration and sonicated to break down any remaining small clumps into fine particles. The suspension is stored at 4°C until use. Use the suspension within 24 hours of preparation.

[0092] Administration: Mice were administered AcAcDAU at a dose of 2350 mg / kg via gavage at a concentration of 0.02 ml / g body weight. Control mice received only 0.75% HPMC carrier.

[0093] Experimental animals: female CD-1 mice.

[0094]

[0095] Overall experimental design: The overall arrangement of the experiment involved gavage administration of AcAcDAU to mice and blood sampling at several subsequent time points (details are shown in the table below). The experiment also included a group of two mice gavaged with HPMC alone, with blood samples collected at 60 and 120 minutes to determine baseline plasma uridine levels.

[0096] AcAcDAU A1, A2, A3, A4 15 and 60 minutes AcAcDAU A5, A6, A7, A8 30 and 120 minutes HPMC C1, C2 60 and 120 minutes

[0097] Mice were administered a suspension of AcAcDAU (0.75% hydroxypropyl methylcellulose aqueous solution) at a concentration of 117 mg / mL (0.02 ml / g body weight) to achieve a dose of 2350 mg / kg body weight. At each time point (15, 30, 60, and 120 minutes after administration), blood samples (~100 μL) were collected from four mice into plasma separation tubes. The samples were rapidly centrifuged, and the resulting plasma was frozen on dry ice. The plasma samples were then deproteinized and subjected to reversed-phase HPLC analysis to measure plasma uridine and its initial metabolite uracil. Aliquots of the same plasma samples were also used to measure BHB concentration using a commercially available enzyme assay kit.

[0098] like Figure 4 , Figure 5 and Figure 6As shown, plasma uridine and its initial metabolite uracil increased after oral administration of AcAcDAU. Figure 7 As shown, plasma β-hydroxybutyrate also increased due to the administration of AcAcDAU, with an area under the curve (AUC) of 24,212 nmol / ml × min.

[0099] Example 6: 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine and 2',3',5'-tri-O-acetyl The protective effect of uridine against death induced by 3-nitropropionic acid.

[0100] 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine (AcAcDAU) delivers uridine and acetoacetate (and its derivative β-hydroxybutyrate [BHB]) into circulation simultaneously after oral administration. A study was designed to compare the relative efficacy of UTA vs AcAcDAU in a model of progressive lethal mitochondrial energy failure. Mitochondrial dysfunction was induced by daily intraperitoneal administration of 3-nitropropionic acid (3-NP; 60 mg / kg / day). 3-NP is an irreversible inhibitor of succinate dehydrogenase (mitochondrial electron transport chain complex II). Daily administration of 3-NP led to a progressive decrease in the mitochondrial capacity to maintain ATP production, ultimately resulting in death due to a combination of neurodegeneration and cardiomyopathy.

[0101] Female CD-1 mice, approximately 16 weeks old, were divided into weight-matched groups of 10 mice each. All mice received daily intraperitoneal injections of 0.01 mL of 3-NP per gram of body weight (60 mg / kg / day).

[0102] In addition to daily injections of 3-NP, the three groups of mice also received 1) a vector; 2) UTA; and 3) AcAcDAU. AcAcDAU from Example 2 was used in this experiment. These were administered orally via gavage at a volume of 0.02 mL per gram of body weight.

[0103] Grouping:

[0104] 1. Carrier control (0.75% HPMC; aqueous solution of hydroxypropyl methylcellulose)

[0105] 2. Uridine triacetate 1000 mg / kg BID (suspended in 0.75% HPMC)

[0106] 3. AcAcDAU 1113.5 mg / kg (equimolar equivalent to 1000 mg / kg UTA) BID (dissolved in 0.75% HPMC)

[0107] The processing plan is as follows:

[0108] 1 HPMC HPMC 3-NP (60mg / Kg) 2 UTA UTA 3-NP (60mg / Kg) 3 AcAcDAU AcAcDAU 3-NP (60mg / Kg)

[0109] Because the mice lost weight when treated with repeated 3-NP therapy, their weight was recorded daily to adjust the dosage.

[0110] Survival time (median survival; the time point at which 50% of the animals in a group die) and the percentage of survival at the end of the 11-day study were used to quantify and compare the protective effects of the test drug.

[0111] result:

[0112] The final survival percentage and median survival at the end of the study are shown in Table 1 below. Figure 8 As shown.

[0113] Table 1: Survival of mice treated with 60 mg / kg / day of 3-nitropropionic acid and oral administration of 1000 mg / kg UTA bid or 1113.5 mg / kg AcAcDAU bid

[0114] Vector control 10％(1 / 10) 5.5 days UTA 30％(5 / 10) 7 days AcAcDAU 50％(2 / 10) >11 days

[0115] Equimolar doses of both UTA and AcAcDAU improved survival in patients with progressive mitochondrial failure compared to the carrier alone. Mice undergoing progressive mitochondrial energy failure treated with AcAcDAU showed a longer median survival than those treated with UTA.

[0116] Example 7: 5'-O-(acetylacetyl)-2',3'-di-O-acetyluridine and 2',3',5'-tri-O-acetyl The protective effect of uridine against death induced by 3-nitropropionic acid.

[0117] Example 6 demonstrates that, in a lethal, progressive mitochondrial failure model, AcAcDAU treatment at an oral dose of 1113.5 mg / kg resulted in higher survival rates compared to an equimolar amount of UTA (1000 mg / kg / dose). In this example, a higher dose of UTA (2000 mg / kg / dose) was compared with an equimolar amount of AcAcDAU (2227 mg / kg / dose) in the same model system.

[0118] Grouping:

[0119] 1. Carrier control (0.75% HPMC; aqueous solution of hydroxypropyl methylcellulose)

[0120] 2. Uridine triacetate 2000 mg / kg BID (suspended in 0.75% HPMC)

[0121] AcAcDAU 2227mg / kg BID (dissolved in 0.75% HPMC)

[0122] This experiment uses AcAcDAU from Example 2. The processing plan is as follows.

[0123] 1 HPMC HPMC 3-NP (60mg / Kg) 2 UTA UTA 3-NP (60mg / Kg) 3 AcAcDAU AcAcDAU 3-NP (60mg / Kg)

[0124] Because the mice lost weight when treated with repeated 3-NP therapy, their weight was recorded daily to adjust the dosage.

[0125] At the end of the 10-day study, the survival percentage of 10 mice in each group was used to quantify and compare the protective effect of the drug.

[0126] result:

[0127] The final survival percentage at the end of the study is shown in Table 2 below.

[0128] Table 2: Survival of mice treated with 60 mg / kg / day of 3-nitropropionic acid and oral administration of 2000 mg / kg UTA bid or 2227 mg / kg 5'-O-acetylacetyl-2',3'-di-O-acetyluridine (AcAcDAU) bid

[0129] Vector control 30％(3 / 10) UTA 70％(7 / 10) AcAcDAU 100％(10 / 10)

[0130] Both UTA and AcAcDAU improved survival in progressive mitochondrial failure compared to the carrier alone. In mice undergoing progressive mitochondrial energy failure, treatment with AcAcDAU at the 10-day time point resulted in better survival compared to oral administration of an equimolar dose of UTA.

Claims

1. A compound: 5'-O-(acetoacetyl)-2',3'-di-O-acetyluridine.

2. The compound of claim 1, wherein the compound is capable of increasing the levels of uridine, NAD+, acetoacetic acid and / or BHB in mammalian subjects in need.

3. The compound of claim 2, wherein, The mammalian subjects mentioned are human subjects.

4. The compound of claim 2, wherein, The compound is administered orally.

5. The compound of claim 4, wherein, The compound was prepared at 1 g / m 2 Up to 3 g / m 2 Dosage administration.

6. The compound of claim 5, wherein, The dosage is applied two or three times daily.

7. A pharmaceutical composition comprising: an amount of the compound of claim 1, effective in treating or preventing a condition in a mammalian subject characterized by brain metabolic energy depletion or decreased mitochondrial energy reserves, and a pharmaceutically acceptable carrier.

8. A method for producing 5'-O-acetylacetyl-2',3'-di-O-acetyluridine, comprising: (a) Under conditions for the production of 5'-O-acetylacetyl-2',3'-O-isopropyluridine, 2',3'-O-isopropyluridine is mixed with 2,2,6-trimethyl-4H-1,3,-dioxane-4-one in dimethylformamide. (b) The 5'-O-acetylacetyluridine is mixed with an aqueous solution of acetic acid under conditions for producing crude 5'-O-acetylacetyluridine. (c) The crude 5'-O-acetylacetyluridine was dissolved in a mixture of dichloromethane and pyridine, and then acetic anhydride was added and stirred for several days; (d) The solvent in the mixture is replaced with ethyl acetate and neutralized with saturated NaHCO3 to produce crude 5'-O-acetylacetyl-2',3'-di-O-acetyluridine.

9. The method of claim 8, wherein, (a) The process is carried out at a temperature of 90°C to 110°C.

10. The method of claim 8, wherein, (b) The procedure shall be carried out at a temperature between room temperature and 75°C.

11. The method of claim 10, wherein, (b) The procedure shall be carried out at a temperature of 65°C.

12. The method of claim 8, wherein, (c) Performed at room temperature.

13. The method of claim 8, further comprising purifying the crude 5'-O-acetylacetyl-2',3'-di-O-acetyluridine to obtain purified 5'-O-acetylacetyl-2',3'-di-O-acetyluridine.

14. A compound: 5'-O-acetylacetyluridine.

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