5'-o-benzoyl uridine and therapeutic uses thereof

Abstract

Disclosed are 5'-O-benzylacetyluridine and therapeutic uses thereof. 5'-O-benzylacetyluridine effectively delivers phenyl acetate and uridine to a subject. It can be used to treat hepatic encephalopathy and genetic disorders of the liver nitrogen cycle.

Description

[0001] This application is a divisional application. The original application's international application number is PCT / US2021 / 061053, its Chinese national application number is 202180080169.0, its application date is November 30, 2021, and its invention title is "5'-O-phenylacetyluridine and its therapeutic use". Technical Field

[0002] This invention relates to 5'-O-phenylacetyluridine and its therapeutic uses. Background Technology

[0003] Phenyl acetate and its precursor phenyl butyrate have important therapeutic applications. The activity of phenyl acetate is utilized as an ammonia scavenger to eliminate excess ammonia in conditions such as hepatic encephalopathy and genetic disorders of the nitrogen cycle. Furthermore, phenyl acetate has been used as a molecular chaperone to alleviate protein misfolding in the endoplasmic reticulum and as an inhibitor of histone deacetylation, yielding therapeutic benefits in various other diseases and disease models.

[0004] In conditions such as hepatic encephalopathy, excessive ammonia is not the only factor in the pathogenesis and progression of the disease, as neuroinflammation and reduced mitochondrial function are also involved. The pyrimidine nucleoside uridine has broad systemic and brain anti-inflammatory activity and supports defective mitochondrial electron transport and oxidative phosphorylation in cells, including neurons. Oral administration of 2′,3′,5′-tri-O-acetyluridine increases plasma uridine more effectively than oral administration of uridine itself, partly due to the dose-limiting side effects of oral uridine. Summary of the Invention

[0005] This invention provides the compound 5'-O-phenylacetyluridine. This invention provides a method for treating or preventing a condition in a subject selected from the group consisting of hepatic encephalopathy and hepatic nitrogen cycle genetic disorders, comprising administering to the subject an amount of the compound of the invention effective in treating the condition. This invention also provides the compound of the invention for treating or preventing a condition selected from the group consisting of hepatic encephalopathy and hepatic nitrogen cycle genetic disorders, or for preparing a medicament for treating or preventing a condition selected from the group consisting of hepatic encephalopathy and hepatic nitrogen cycle genetic disorders. And it provides pharmaceutical compositions comprising the compound of the invention and a pharmaceutically acceptable carrier.

[0006] It has been found that when administered orally, the compound 5′-O-phenylacetyluridine (PAU) increases plasma uridine more effectively than uridine itself. Furthermore, phenylbutyrate is more advantageous as a drug prodrug than phenyl acetate due to its unpleasant odor, and it has been approved by the FDA for use as an ammonia scavenger.

[0007] In one aspect, the present invention provides a compound, 5'-O-phenylacetyluridine.

[0008] In another aspect, the present invention provides a method for treating or preventing a condition in a mammalian subject, wherein the condition is selected from the group consisting of hepatic encephalopathy and genetic diseases of the liver nitrogen cycle, the method comprising administering an effective amount of the compound described in item 1 to the subject, thereby treating or preventing the condition.

[0009] In one implementation, the administration is oral.

[0010] In one implementation, the mammalian subject is a human subject.

[0011] In one implementation, the administration is oral.

[0012] In one embodiment, the effective amount is 1-5 grams per square meter of body surface area, applied once, twice, or three times daily.

[0013] In another aspect, the present invention provides the above-described compound for use in treating or preventing conditions in mammalian subjects, wherein the conditions are selected from the group consisting of hepatic encephalopathy and genetic disorders of the liver nitrogen cycle.

[0014] In one embodiment, the compound is administered orally to the mammalian subject.

[0015] In one implementation, the mammalian subject is a human subject.

[0016] In one embodiment, the compound is administered orally to the human subject.

[0017] In one embodiment, the compound is administered at a dose of 1 to 5 grams per square meter of body surface area, once, twice, or three times daily.

[0018] In another aspect, the present invention provides the above-mentioned compound for use in the manufacture of a medicament for the treatment or prevention of a condition in a mammalian subject, wherein the condition is selected from the group consisting of hepatic encephalopathy and genetic disorders of the liver nitrogen cycle.

[0019] In one embodiment, the drug is formulated for oral administration to the mammalian subject.

[0020] In one implementation, the mammalian subject is a human subject.

[0021] In one embodiment, the drug is formulated for oral administration to the human subject.

[0022] In one embodiment, the drug is formulated for application of the compound at a dose of 1 to 5 grams per square meter of body surface area, once, twice, or three times daily.

[0023] In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an amount of the compound of item 1 that is effective in treating or preventing a condition in a mammalian subject, wherein the condition is selected from the group consisting of hepatic encephalopathy and genetic diseases of the liver nitrogen cycle.

[0024] In one embodiment, the pharmaceutical composition is formulated for oral administration to the mammalian subject.

[0025] In one implementation, the mammalian subject is a human subject.

[0026] In one embodiment, the pharmaceutical composition is formulated for oral administration to the human subject.

[0027] In one embodiment, it is formulated for application of the compound at a dose of 1 to 5 grams per square meter of body surface area, once, twice, or three times daily. Attached Figure Description

[0028] Figure 1 Plasma uridine in mice after oral administration of 5'-phenylacetyluridine

[0029] Figure 2 Plasma uracil in mice after oral administration of 5'-phenylacetyluridine

[0030] Figure 3 Plasma uridine + uracil in mice after oral administration of 5'-phenylacetyluridine Detailed Implementation

[0031] As used herein, the transitional term "comprising" is open-ended. Claims using this term may include elements other than those set forth in the claim. Thus, for example, a treatment regimen described in a claim may also include other therapeutic agents or doses of the virus not specifically listed herein, provided that the listed elements or their equivalents are present.

[0032] abbreviation: PAU or 5'-PAU: 5'-O-phenylacetylurea, also known as 5'-phenylacetyluridine PA: Phenyl acetate PB: Phenyl butyrate NaPB: Sodium phthalate DMF: Dimethylformamide TLC: Thin-layer chromatography DMSO: Dimethyl sulfoxide HPMC: Hydroxypropyl Methylcellulose LPS: Lipopolysaccharide TNF: Necrosis Factor

[0033] In addition to providing therapeutic benefits derived from the simultaneous delivery of uridine and phenyl acetate, an important safety factor is achieved by using the combination of uridine and phenyl acetate, as uridine can alleviate the metabolic imbalance caused by phenyl acetate or its precursor phenyl butyrate.

[0034] According to the methods, compounds used, applications, and pharmaceutical compositions of the present invention, any conventional disease in mammalian subjects characterized by the potential benefits conferred by phenyl acetate and uridine can be treated or prevented. Such diseases are selected based on any of several known activities and mechanisms of action of phenyl acetate or its precursor phenyl butyrate, which also demonstrate broad tissue-neuroprotective and tissue-protective activities conferred by therapeutic doses of uridine. In one embodiment, hepatic encephalopathy is treated with 5'-phenylacetyluridine, wherein phenyl acetate partially scavenges excess circulating ammonia and histamine associated with the disease, and uridine provides direct neuroprotective activity against neuroinflammation and mitochondrial dysfunction, which also characterize hepatic encephalopathy. Hepatic encephalopathy of all severity stages, from acute crisis to mild clinical symptoms, is treated with 5'-phenylacetyluridine. For severe hepatic encephalopathy, 1 to 9 g / m² body surface area of ​​phenylacetyluridine is administered once to three times daily, with the dosage selected according to the severity of ammonia overload in the specific patient.

[0035] Right ventricular failure and pulmonary artery remodeling in pulmonary hypertension involve pathological metabolic remodeling affecting the heart and pulmonary arteries, accompanied by excessive aerobic glycolysis, similar to the Warburg effect in many cancers. Phenyl acetate inhibits pyruvate carboxylase, partially alleviating pathological aerobic glycolysis. Furthermore, uridine improves the contractility of pressure-overloaded myocardium. Therefore, 5'-phenylacetyluridine, either as a single agent or in combination with triacetyluridine to regulate the uridine / phenyl acetate ratio, provides more complete protection of the right ventricle in pulmonary hypertension than either agent alone; right ventricular failure is a leading cause of death in patients with primary pulmonary hypertension. For the treatment of pulmonary hypertension, the dosage of 5'-phenylacetyluridine is 1–5 g / m² body surface area, administered 1–3 times daily.

[0036] According to the invention, the compound can be administered to any mammalian subject. In one embodiment, the mammalian subject is a human subject. According to the invention, any conventional route of administration can be used. The compound is preferably administered orally. A skilled physician can titrate to optimize the dosage for a particular patient. Typically, the compound is administered orally to human patients at a dose of 1 to 5 grams per square meter of body surface area. This dose is typically administered 1 to 3 times daily.

[0037] PB is typically administered to patients as a sodium salt (Buphenyl®) at doses of 5-25 g / day. Since sodium contains 12% sodium by weight, a 10 g / day NaPB treatment will result in a sodium intake of 1.2 g. The recommended daily sodium intake is 2.3 g, and excessive sodium intake is associated with an increased incidence of hypertension, myocardial infarction, and stroke (Strazzullo, D'Elia et al. 2009; Frieden and Briss 2010). One object of this invention is to provide a bioactive derivative of PB in a form that does not increase excessive sodium intake. This is achieved through an ester bond with uridine.

[0038] Glutamine depletion induced by PB represents additional metabolic impairment in mammals without urea cycle disorders or excessive ammonia levels. Glutamine depletion and the resulting ammonia loss can lead to the consumption of pyrimidines (uridine and cytidine), since pyrimidine synthesis requires glutamine. Pyrimidine synthesis can be altered by the level of glutamine or dietary protein (Monks, Chisena et al. 1985; Nelson, Qureshi et al. 1993; Zaharevitz, Grubb et al. 1993). Ureidine is supplied using uridine ester-linked PB-related derivatives, thus preventing uridine consumption.

[0039] The invention will be better understood by referring to the following embodiments, which illustrate but do not limit the invention described herein.

[0040] Example

[0041] Example 1: Preparation of 5'-O-phenylacetyluridine

[0042] 1. Preparation of 2',3'-O-cyclohexyluridine

[0043] Uric acid (50 g, 205 mmol) was dissolved in anhydrous DMF (250 mL), and a catalytic amount of p-toluenesulfonic acid (1.8 g) was added while stirring at room temperature under argon. After 20 minutes, 31 mL or 2.0 equivalents of 1,1-dimethoxycyclohexane was added. The reaction mixture was stirred overnight at room temperature under argon. The reaction was checked for completeness by TLC (EtOAc, Rf = 0.2), 250 mL of methanol was added, and the mixture was evaporated twice. The crude reaction mixture was purified by rapid chromatography using silica gel and ethyl acetate to give 33 g of the desired product (50% yield). 1 H NMR (400 MHz, DMSO-d6) 1.34 – 1.69 (m, 10H), 3.55 (width s, 2H) 4.03 – 4.05 (m, 1H), 4.73 (d, 1H, J = 3.6 Hz), 4.86 – 4.88 (m, 1H), 5.06 (s, OH), 5.61 (d, 1H, J = 8.1 Hz), 5.82 (dd, 1H, J = 2.6, 7.0 Hz), 7.76– 7.80 (m, 1H), 11.35 (s, NH)

[0044] 2. Preparation of 2',3'-O-cyclohexyl-5'-O-phenylacetyluridine

[0045] Under argon atmosphere, 2',3'-O-cyclohexyluridine (26.0 g, 84.4 mmol) was dissolved in 250 mL of anhydrous CH2Cl2, 125 mL of pyridine was added, and the mixture was cooled to 0°C. Then, phenylacetyl chloride (16 mL, 1.2 eq) was added dropwise over 30 minutes. The reaction mixture was stirred at 0°C for 4 hours, and then stirred again at room temperature for 4 hours. The completion of the reaction was confirmed by TLC (EtOAc, Rf = 0.6). The reaction was quenched with methanol and evaporated to dryness. The residue was dissolved in EtOAc (500 mL), washed with 0.1 N HCl, 0.05 N HCl, water, saturated NaHCO3, and finally washed with water. The crude product was purified by rapid chromatography using silica gel and 50% EtOAc / hexane to give 30 g of the desired product (85% yield). 1 H NMR (400 MHz, DMSO-d6) 1.33 – 1.69 (m, 10H), 3.69 (s, 2H), 4.19 – 4.24 (m, 3 H), 4.73 – 4.74 (m,1H), 4.88 (dd, 1H, J = 2.2, 6.6 Hz), 5.61 (d, 1H, J = 8.1 Hz), 5.77 (d, 1H, J= 1.9 Hz), 7.24 – 7.32 (m, 5H), 7.60 (d, 1H, J= 8.1 Hz), 11.42 (s, NH).

[0046] 3. Preparation of 5'-O-phenylacetyluridine

[0047] 2′,3′-O-cyclohexyl-5′-O-phenylacetyluridine (32.80 g, 77.1 mmol) was dissolved in 300 mL of a 1:1 mixture of formic acid and water. The reaction mixture was heated at 65–70 °C for 4 hours. The consumption of the starting material was confirmed by TLC (ethyl acetate) different from the reaction curve. Water and formic acid were removed by vacuum evaporation. The crude product was dissolved in water (250 mL) and evaporated to dryness twice. Purification was achieved by rapid chromatography using silica gel and ethyl acetate to give 15.1 g (56%) of the desired product. Attempts to recrystallize the product from solvents such as ethyl acetate / hexane, diethyl ether / hexane, methanol / hexane, ethanol / hexane, ethanol / diethyl ether, acetone / hexane, ethanol / diethyl ether, and methanol / diethyl ether were unsuccessful. LC-MS analysis showed that... 230 92% of the total absorbance of light corresponds to the product. 1 H NMR (400 MHz, DMSO-d6) 3.71 (ABq, 2H), 3.85 – 4.05 (m,3H), 4.15 – 4.30 (m, 2H), 5.28 (d, OH, J = 5.1 Hz), 5.43 (d, OH, J = 6.5 Hz), 5.61 (d, 1H, J = 8.1 Hz), 5.72 (d, 1H, J = 5.1 Hz), 7.24 – 7.31 (m, 5H), 7.46 (d, 1H, J = 8.1 Hz), 11.35 (s, NH). Melting point 55-57°C

[0048] Example 2: Plasma uridine and uracil pharmacokinetics after oral administration of 5'-O-phenylacetyluridine (PAU)

[0049] Chemicals: HPMC (hydroxypropyl methylcellulose; SIGMA-Aldrich: cat #H3785, CAS 9004-65-3), 5'-O-phenylacetyluridine (PAU, lot 432-132)

[0050] Carrier: Aqueous HPMC (0.75%) is used as a suspension carrier for oral administration.

[0051] Dosage formulation: Add PAU to 0.75% HPMC and homogenize to eliminate clumps. Make up to the desired volume and concentration with the suspension and sonicate to deagglomerate any remaining small clumps into fine particles. Store the suspension at 4°C until use. Use the suspension within 24 hours of preparation.

[0052] Administration: Mice were given a dose of 587 mg / kg PAU (molar equivalent of 400 mg / kg uridine) by gavage at a dose of 0.02 ml / g body weight.

[0053] Animal: Female CD-1 mouse.

[0054] (The rest of the page is intentionally left blank.)

[0055] The general initial setup of the experiment involved groups of 6 mice gavaged with PAU, followed by blood sampling at several time points (3 mice bled at 2 time points (15 and 60 minutes), and another 3 mice bled at another 2 time points (30 and 120 minutes). Each experiment included HPMC (carrier-only) time points for 3 mice to establish a baseline for blood uridine levels.

[0056]

[0057] Blood samples were collected into plasma separation tubes, centrifuged immediately after collection, and aliquots of plasma were frozen for subsequent processing. The plasma was then deproteinized, and uridine and uracil were quantified by liquid chromatography using UV absorbance detection and mass spectrometry.

[0058] The delivery of uridine into the bloodstream is assessed by monitoring plasma uridine and the sum of uridine and uracil [uridine + uracil], as uracil is the first product in the enzymatic degradation of uridine. Compared to humans, mice convert administered uridine to uracil more rapidly and extensively, making uridine + uracil a better dose and pharmacokinetic conversion index for humans than measuring uridine alone.

[0059] Plasma concentrations of uridine, uracil, and [uridine + uracil] after PAU administration are shown in the figures below. Figure 1 , 2 and 3.

[0060] The delivery of phenyl acetate into the bloodstream after oral administration of PAU was also evaluated. Sodium chlorate is used in clinical practice as an ammonia scavenger in hepatic encephalopathy or hereditary nitrogen cycle disorders, or as an inhibitor of chaperone proteins or histone deacetylases. It is primarily metabolized to phenyl acetate via β-oxidation in the liver, and phenyl acetate mediates the therapeutic benefits of phenyl butyrate. PAU was administered orally at a dose equimolar to 200 mg / kg sodium chlorate. Female BALB / c mice (n=3–5 groups) were orally administered the test compound, and plasma was obtained by retro-orbital bloodletting 30 minutes later. A table summarizing the comparative bioavailability of PAU and sodium chlorate derivatives indicates the maximum concentration (C0.05) obtained in serum after oral administration of these compounds at an equimolar bioavailability index. MaxAn equimolar dose of uridine was used as a comparative measure of the efficacy of delivering uridine into circulation via oral administration of PAU.

[0061] Table 1. Plasma concentrations of uridine, PB, and corresponding metabolites after oral administration of sodium chlorbutate or PAU

[0062] Plasma concentration is measured in micromoles per liter (μM).

[0063] NaPB = Sodium phthalate

[0064] PB = phenyl butyrate

[0065] PA = phenyl acetate

[0066] PAU = 5'-O-phenylacetyluridine

[0067] Oral PAU is substantially more effective than an equimolar dose of oral sodium chlorate in increasing plasma phenylacetate concentrations, producing >3 times higher circulating phenylacetate concentrations within 30 minutes of administration. Furthermore, PAU is superior to oral equimolar doses of uridine in increasing plasma uridine and uracil levels.

[0068] Example 3: Anti-inflammatory effects of 5'-O-phenylacetyluridine (PAU)

[0069] Injection of bacterial cell wall component lipopolysaccharide (LPS) has been used as an inflammation model. When unexamined, the inflammatory cascade can lead to cellular and organ damage. Tissue necrosis factor-α (TNFα) is strongly induced during inflammation, and attenuating this response can be protective in many diseases, including rheumatoid arthritis, psoriasis, vasculitis, and Alzheimer's disease.

[0070] Forty-one-week-old female BALB / c mice (n=7-8 / group) were treated with an oral carrier (0.75% HPMC) or PAU, followed by administration of LPS (2.5 mg / kg intraperitoneally) 30 minutes later. One hour after LPS administration, the mice were sacrificed and blood was collected for plasma TNFα measurement.

[0071] Table 2: Effects of PAU on LPS-induced changes in plasma TNFα

[0072] + indicates p < 0.05 compared to the vector control.

[0073] PAU provides significant anti-inflammatory effects in LPS-induced increases in plasma TNFα.

Claims

1. A compound, 5'-O-phenylacetyluridine.

2. A method for treating or preventing a condition in a mammalian subject, wherein, The condition is selected from the group consisting of hepatic encephalopathy and genetic diseases of the liver nitrogen cycle, and the method comprises administering an effective amount of the compound of claim 1 to the subject to treat or prevent the condition.

3. The method according to claim 2, wherein, The administration is by oral administration.

4. The method according to claim 3, wherein, The mammalian subjects mentioned are human subjects.

5. The method according to claim 4, wherein, The administration is by oral administration.

6. The method according to claim 5, wherein, The effective dosage is 1-5 grams per square meter of body surface area, applied 1, 2 or 3 times daily.

7. The compound of claim 1, used to treat or prevent a condition in a mammalian subject, wherein the condition is selected from the group consisting of hepatic encephalopathy and genetic disorders of the liver nitrogen cycle.

8. The compound according to claim 7, wherein, The compound was administered orally to the mammalian subject.

9. The compound according to claim 7, wherein, The mammalian subjects mentioned are human subjects.

10. The compound according to claim 9, wherein, The compound was administered orally to the human subjects.

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