A bioactive nucleotide compound for ameliorating LCAT activity and in-situ process for preparation thereof

Abstract

The invention disclosed herein relates to a bioactive nucleotide compound for regulating Lecithin-cholesterol acyltransferase (LCAT) activity in human and in situ process for preparation thereof. Particularly, the process comprising steps of in-situ esterification followed by phosphorylation and deprotection. Furthermore, the present bioactive nucleotide compound(s) is useful in the treatment of disorders related to dysregulation of Lecithin–cholesterol acyltransferase (LCAT) activity such as kidney disease, cardiovascular disease, liver diseases and ocular disease.

Description

[0001] TITLE OF THE INVENTION:

[0002] A bioactive nucleotide compound for ameliorating LCAT activity and in- situ process for preparation thereof.

[0003] FIELD OF THE INVENTION:

[0004] The present invention relates to a bioactive nucleotide compound(s) aimed at treating disorders associated with the dysregulation of Lecithin-cholesterol acyltransferase (LCAT) activity. Specifically, this invention concerns a nucleotide compound(s) that regulates cholesterol metabolism and improves lipoprotein function.

[0005] Additionally, the invention pertains to Optimized in-situ process for the preparation of bioactive nucleotide compound(s), with a focus on achieving high purity and high yield.

[0006] The present bioactive nucleotide compound(s) is particularly beneficial for treating disorders related to LCAT dysregulation, such as kidney disease, cardiovascular conditions, liver diseases, and ocular disorders.

[0007] BACKGROUND OF THE INVENTION:

[0008] Lecithin-cholesterol acyltransferase (LCAT) is a crucial enzyme in the process of reverse cholesterol transport, which helps maintain cholesterol homeostasis in the body. LCAT facilitates the conversion of free cholesterol into cholesteryl esters, which are then transported to the liver for further metabolism. Dysregulation of LCAT activity is associated with several metabolic and cardiovascular disorders, including kidney disease, atherosclerosis, ocular and liver dysfunction. The key functions of LCAT are cholesterol esterification, HDL formation and maturation and regulation of plasma lipid levels. In cholesterol esterification, LCAT catalyzes the conversion of free cholesterol into cholesteryl esters. This is achieved by transferring a fatty acid from the phospholipid lecithin (phosphatidylcholine) to the free cholesterol, forming cholesteryl esters and lysolecithin. In HDL formation and maturation, LCAT promotes the conversion of nascent discoidal HDL particles into mature spherical HDL by esterifying cholesterol on the surface of these particles. In regulation of plasma lipid levels, LCAT helps maintain cholesterol homeostasis in the bloodstream, promoting the transport of cholesterol in a more hydrophobic form (cholesteryl esters) that can be stored in HDL particles and eventually delivered to the liver.

[0009] Proper function of LCAT is associated with cardiovascular health, as it supports the removal of excess cholesterol from peripheral tissues, reducing the risk of atherosclerosis. Mutations or deficiencies in LCAT can lead to disorders like familial LCAT deficiency (FLD) characterized by abnormal cholesterol metabolism, corneal opacities and low levels of HDL. Despite the critical role of LCAT, existing therapies for treating LCAT-related disorders remain limited. Current therapeutic approaches are largely palliative and focus on managing cholesterol levels through statins, HDL-raising agents, or other lipid-lowering therapies but they do not directly address LCAT dysfunction. However there are two major therapies which are currently used in managing LCAT deficiency, are called as Enzyme Replacement Therapy (ERT) and gene therapy. In ERT, recombinant LCAT is administered to patients and gene therapy introduces a functional LCAT gene into patient cells.

[0010] US5049488 relates to the expression of lecithin cholesterol acyltransferase in recombinant host cell culture which comprises steps (a) constructing a vector comprising nucleic acid encoding lecithin cholesterol acyltransferase; (b) transforming a host cell culture with the vector of step a); (c) culturing the transformant of step b) to accumulate lecithin cholesterol acyltransferase in the culture; and (d) recovering the lecithin cholesterol acyltransferase from the culture.

[0011] US20010014319 relates to new recombinant viruses comprising an inserted gene encoding all or part of lecithin-cholesterol acyltransferase (LCAT) or a variant thereof.

[0012] EP2181190 provides modified LCAT proteins with increased enzymatic activity and / or stability and methods for treatment of coronary heart disease, atherosclerosis, inflammatory disorders and disorders associated with thrombosis using these modified LCAT proteins.

[0013] US7157259 relates to a novel protein having a lecithin-cholesterol acyltransferase-like activity or its salt, a precursor protein of the protein or its salt, a partial peptide of the protein or its salt; a DNA coding for the protein; a recombinant vector; a transformant and a method for producing the protein.

[0014] ERT therapy has some disadvantages such as recombinant LCAT has a short half-life in circulation, requiring frequent administration. ERT is expensive and difficult to scale for widespread clinical use and limiting its availability to patients. Long-term use of exogenous enzymes may lead to the development of neutralizing antibodies and reducing the efficacy of the therapy.

[0015] Gene therapy is also having its own limitations. Ensuring efficient and targeted delivery of the gene to appropriate tissues remains a significant hurdle. There are concerns about the longterm safety of gene therapy, including off-target effects, immune responses and potential oncogenic risks. The recombinant LCAT used in ERT has a short half-life, necessitating frequent administration. Furthermore, the high cost and scalability issues associated with ERT limit its accessibility for many patients. There is also a risk that the development of neutralizing antibodies may reduce the efficacy of the therapy over time. Additionally, some researchers have also developed synthetic compound(s)s such as small molecule activator that can enhance the LCAT enzyme's function. Specifically, a synthetic compound(s) that enhance LCAT activity by binding to allosteric sites on the enzyme. Particularly, Piperidinylpyrazolopyridine and piperidinylimidazopyridine are the compound(s)s were synthesized and confirmed to activate LCAT (Manthei et al. eLife 2018;7:e41604').

[0016] WO20 15 / 179293 discloses small molecule activators of lecithin cholesterol acyltransferase. This patent application describes the use of azetidine compound(s)s has a potential of LCAT activation.

[0017] WO / 2023 / 122185 describes trifluoromethyl molecule activators of LCAT that enhance lipid metabolism.

[0018] JP2006160757 discloses TCF-II, is an active ingredient and a known protein derived from human fibroblasts which increases LCAT activity.

[0019] However, there some of the drawbacks of these synthetic compound(s)s or small molecule activators. Many small-molecule activators or synthetic compound(s)s have demonstrated limited efficacy in significantly improving LCAT activity, leading to modest therapeutic effects. Small molecules may interact with other components of the lipid metabolism pathway which leading to undesirable side effects.

[0020] The abovementioned prior arts ERT, gene therapy and small molecule activators approaches lack the efficacy needed for long-term solutions or come with significant barriers such as cost, scalability, and safety.

[0021] Current therapies targeting cholesterol metabolism and LCAT function are often limited in efficacy due to low bioavailability or undesirable side effects. There is a need for more efficient bioactive therapeutic agents that can directly modulate LCAT activity, scalable and cost effective with fewer side effects.

[0022] LCAT activity is vital for maintaining cholesterol homeostasis, and understanding its activators could have therapeutic potential in managing lipid disorders.

[0023] The present invention addresses this need by providing a bioactive nucleotide compound(s), specifically designed to target and enhance LCAT activity. The invention further provides technically advanced synthesis for this preparation of a bioactive nucleotide compound that improves yield, purity, and scalability, making it viable for clinical and industrial applications.

[0024] Furthermore, the compound demonstrates enhanced bioavailability, minimal side effects, and overall safety for administration. OBJECTIVE OF THE INVENTION:

[0025] The primary objective of the present invention is to provide a bioactive nucleotide compound that can effectively modulate or ameliorate LCAT activity

[0026] Another objective of the invention is to provide method for treatment for conditions associated with Lecithin-Cholesterol Acyltransferase (LCAT) dysregulation in human.

[0027] Yet another object of the invention is to provide bioactive nucleotide compound(s) which ensures targeted action and improve bioavailability.

[0028] Yet another object of the invention is to provide a bioactive nucleotide compound(s) which minimizes systemic side effects, and provides prolonged effect.

[0029] Another object of the invention is to provide cost-effective, industrially viable in-situ process for the preparation of the bioactive nucleotide compound(s) ensuring high purity and yield.

[0030] Another object of the invention is to provide present bioactive nucleotide compound(s)-based composition and treatment option for conditions related to abnormal LCAT activity, including cardiovascular diseases, kidney diseases, liver disorders, and ocular conditions.

[0031] SUMMARY OF THE INVENTION

[0032] To achieve above objectives, the inventors conducted extensive experiments to demonstrate the significant effects of the bioactive nucleotide compound in enhancing therapeutic efficacy for the treatment of metabolic and neurological disorders through the regulation of LCAT activity. In one aspect, the present invention provides a bioactive nucleotide compound(s) which is prepared by commercially viable and non-hazardous process.

[0033] In certain aspect, the present invention provides a bioactive nucleotide compound(s) that has been developed to modulate LCAT activity, thereby addressing disorders related to cholesterol metabolism. The compound(s) shows promise in improving lipoprotein function, cholesterol esterification, and reverse cholesterol transport.

[0034] In another aspect, the invention presents an in-situ process for preparing bioactive nucleotide compound, which involves a streamlined series of steps, including esterification, phosphorylation, and deprotection. This method ensures high purity and yield of the final compounds, making them well-suited for pharmaceutical applications.

[0035] In yet another aspect, the invention provides therapeutically effective, non-toxic and safe bioactive nucleotide compound(s) with no major side effects.

[0036] In further aspect, the present invention provides pharmaceutical composition comprising bioactive nucleotide compound(s) along with pharmaceutically acceptable excipients / carriers is intended for the treatment of various health conditions associated with LCAT dysregulation, such as kidney and cardiovascular diseases, liver dysfunction, and ocular disorders.

[0037] Abbreviations:

[0038] LCAT: Lecithin-cholesterol acyltransferase

[0039] HDL: High-density lipoprotein

[0040] FLD: Familial LCAT deficiency

[0041] ERT: Enzyme Replacement Therapy

[0042] BRIEF DESCRIPTION OF THE FIGURES:

[0043] Fig. 1 shows the HPLC chromatogram of the standard and test sample of (2R,3S,4R,5R)- 5-(3-carbamoylpyridin- 1-ium- l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate.

[0044] Fig. 2 shows LCAT activity (Units / mL) for G1 (Negative control), G2 (Formula I compound 100 mg / kg), and G3 (Formula I compound 500 mg / kg).

[0045] DESCRIPTION OF THE INVENTION:

[0046] It is further to be understood that all terminology used herein is for the purpose of describing particular embodiment only and is not intended to be limiting in any manner or scope. Unless defined otherwise, all technical and scientific expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain.

[0047] In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below which are known in the state of art.

[0048] The singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Also, the term ‘composition’ does not limit the scope of the invention for multiple compositions that can be illustrated for best mode of the invention.

[0049] The term “pharmaceutically / nutraceutically acceptable salt,” as used herein, represents those salts which are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit / risk ratio.

[0050] Particularly the term “pharmaceutically-acceptable salts” refers to the relatively nontoxic, inorganic and organic acid addition salts of compound(s)s, amino acid salt, sugar-based salt, alkali or alkaline earth metal salts, as well as solvates, co-crystals, polymorphs and the like of the salts. All modifications and substitutions that come within the meaning of the description and the range of their legal equivalents are to be embraced within their scope. A description using the transition “comprising” allows the inclusion of other elements to be within the scope of the invention.

[0051] The inventors of the present invention have developed therapeutically significant bioactive nucleotide compound(s) in stable form.

[0052] In a preferred embodiment, the present invention provides the bioactive nucleotide compound(s) for disorder related to dysregulation of Lecithin-cholesterol acyltransferase (LCAT) activity.

[0053] In another embodiment, the present invention provides cost-effective, industrially viable process for the preparation of bioactive nucleotide compound(s) with high purity and high yield.

[0054] In another preferred embodiment the invention provides in-situ process for preparation of the bioactive nucleotide compound(s) with high purity and high yield.

[0055] Particularly, the process comprising steps of in-situ esterification followed by phosphorylation and deprotection.

[0056] The process involved in the present invention comprising green solvents, where the intermediates have acceptable and appropriate toxicity and ecotoxicity.

[0057] In a preferred embodiment, the present invention relates to in situ synthesis of a bioactive nucleotide compound(s) comprising steps a) esterification of ribofuranosyl compound (Formula- II): In the presence of boronic acid, compound (Formula- II) undergoes esterification to form an intermediate cyclic boron ester compound (Formula III); b) phosphorylation of compound (Formula III): compound (Formula-Ill) is then subjected to phosphorylation using a suitable phosphorylating agent, resulting in the formation of compound (Formula-IV); iii) deprotection of compound (Formula-IV): Finally, compound (Formula-IV) is deprotected using aqueous organic acid, yielding the final product, compound (Formula-I), with high yield and purity.

[0058] In some embodiment, the present invention provides in situ synthesis of a bioactive nucleotide compound(s) wherein the first esterification step comprises synthesize of a cyclic boron ester intermediate of formula-III from ribofuranosyl compound of formula II. The ribofuranosyl compound II is a sugar-based compound with hydroxyl groups. The hydroxyl groups on the ribose moiety of formula II react with boronic acid to form a cyclic boron ester, protecting the diol functional group from further reactions. formulae wherein ‘R’ is selected from H, (C1-C4) alkyl, (C2-C4) alkene, aryl, phenyl, benzyl, cycloalkyl, heterocyclic compound.

[0059] The temperature also influences the reaction outcomes. The esterification is accrued out at ambient temperatures to minimize decomposition reactions that occur during cyclization. The temperature is maintained between 20-25 °C.

[0060] The choice of solvent can impact the reactivity and stability of both formula II and boronic acid. Polar aprotic solvents are used to facilitate reactions involving boronic acids due to their ability to stabilize charged intermediates. Preferably the solvents are selected from acetone, chloroform, dichloromethane, ethyl acetate, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran in presence of catalytic amount of base is added to facilitate the esterification.

[0061] In some embodiment, the present invention provides in situ synthesis of a bioactive nucleotide compound(s) wherein the second phosphorylation step comprises introduction of a phosphate group into cyclic boron ester intermediate to produce phosphorylated compound of formula IV.

[0062] Particularly, the introduction of phosphate group is carried out in presence of suitable phosphorylating agent. Cyclic boron esters reacted with phosphorylating agent to form phosphorylated compounds. Typically, phosphorylation reaction is conducted at low temperatures at 0-5°C (ice bath) to control the exothermic reaction and avoid decomposition. In some embodiment, the present invention provides in situ synthesis of a bioactive nucleotide compound(s) wherein the final deprotection step comprises removal of the boron moiety and obtain the final product with high yield and purity.

[0063] The cyclic boron ester is deprotected using a mild and effective acidic medium to remove protecting groups without causing significant hydrolysis or degradation of sensitive structures, such as the nucleotide backbone. The deprotection is carried out at a mild temperature (30-35°C). This results in the formation of formula-I [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)- 3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate, the final product. The mild acidic agent hydrolyses the boron-oxygen bonds, leading to the removal of the boron moiety. This reaction restores the hydroxyl groups on the sugar ring while retaining the phosphate group introduced in phosphorylation reaction.

[0064] This synthetic pathway achieves selective functionalization of a sugar-based precursor through esterification, phosphorylation, and deprotection.

[0065] In another embodiment, the base used in the process is selected from sodium hydroxide, potassium hydroxide, ammonia hydroxide, sodium bicarbonate, sodium carbonate, lithium hydroxide, magnesium hydroxide, methylamine, ethylamine, aniline, dimethylamine, pyridine ammonia, triethylamine, dimethylformamide, pyrrolidine, N-N-dimethylacetamide and like thereof.

[0066] In some embodiment, the polar aprotic solvent used in the process is selected acetone, dimethyl sulfoxide, acetonitrile, dimethylformamide, tetrahydrofuran, (diethyl ether), chloroform, ether, dichloromethane, hexamethylphosphoramide, methyl ethyl ketone and like thereof.

[0067] In some embodiment, the phosphorylating agent used in the process is selected from phosphoryl chloride, diethyl phosphorochloridate, adenosine triphosphate, dimethyl phosphorochloridate, cytidine triphosphate, polyphosphates, diethyl phosphate, phosphoric acid, triethyl phosphate and like thereof. In some embodiment, the phosphorylating agent used is phosphoryl chloride in presence of triethyl phosphate. In preferred embodiment, the phosphorylation reaction is carried out using phosphoryl chloride (POCk) and tri ethyl phosphate (TEP), with the molar ratio at about 1:1 to 1:2.

[0068] In some embodiment, the acidic medium used in the process is selected from dilute or aqueous organic or inorganic acid but not limiting to acetic acid, tartaric acid, hydrochloric acid, formic acid, phosphoric acid, benzoic acid, sulfuric acid, boric acid, malic acid, triethylammonium acetate buffer, dichloroacetic acid, trifluoroacetic acid and like thereof. Preferably the deprotection is carried out in presence of aqueous acetic acid.

[0069] In some embodiment, the temperature maintained in the process is ranged from 0-40°C, preferably ambient temperature for esterification.

[0070] In some embodiments, the in-situ process produces the final product, [(2R,3S,4R,5R)-5- (3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate, with a yield of 80-90% (w / w). The crude product is then purified using column chromatography with a solvent mixture of polar and nonpolar solvents in the ratio of 1:1 to 1:2, preferably ethyl acetate / hexane in a 1:2 ratio, to eliminate impurities. The end product achieves a purity level exceeding 95%, as confirmed by known analytical techniques such as NMR, HPLC, TLC.UV, IR, FTIR, XRD, GC and like thereof.

[0071] In another embodiment, the present invention provides bioactive nucleotide compound which is specifically designed to regulate the activity of Lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for cholesterol esterification and lipoprotein metabolism.

[0072] In certain embodiments, the bioactive nucleotide compound, also known as the LCAT-activating compound, stabilizes the enzyme's active conformation, thereby enhancing substrate interaction and potentially improving therapeutic outcomes for metabolic disorders, including familial LCAT deficiency.

[0073] In another embodiment, the present invention provides bioactive nucleotide compound that effectively influence LCAT activity and ensures targeted action, improve bioavailability, minimizes systemic side effects, and provides prolonged effect.

[0074] In another embodiment, the present invention provides the bioactive nucleotide compound(s) has a unique molecular structure that enables it to interact with LCAT, enhancing its enzymatic activity. This results in the efficient conversion of free cholesterol into cholesteryl esters, which are crucial for reverse cholesterol transport.

[0075] In another embodiment, the present invention provides bioactive nucleotide compound for regulating Lecithin-cholesterol acyltransferase (LCAT) activity in human and in situ process for preparation thereof, wherein the bioactive nucleotide compound is [(2S,3R,4S,5S)-5-(3- carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate.

[0076] In another embodiment, the present bioactive compound(s) are useful in treating disorders associated with the dysregulation of Lecithin-Cholesterol Acyltransferase (LCAT) activity. Deficiency or dysregulation in LCAT leads to a range of conditions linked to impaired lipid metabolism, including corneal opacities, normochromic normocytic anemia, proteinuria, progressive renal failure, low HDL cholesterol, chronic kidney disease, cardiovascular disease, accumulation of unesterified cholesterol, dyslipidemia, diabetic retinopathy, and other related disorders.

[0077] The term "subject in need thereof" refers to a subject, preferably a mammal and more specifically a human, suffering from or suspected of having an LCAT-related disorder.

[0078] In the context of the present invention, the term “treatment” refers to alleviate, mitigate, prophylaxis, attenuate, manage, regulate, modulate, control, minimize, lessen, decrease, down regulate, up regulate, moderate, inhibit, restore, suppress, limit, block, decrease, prevent, inhibit, stabilize, ameliorate, cure, heal metabolic or nervous system related disorders observed in the patient. Notably, the present bioactive nucleotide compound(s) are non-hazardous, non-toxic, and safe for human consumption without any severe adverse effects, therefore the present medicinal composition can also be used as preventive therapy, adjuvant therapy, add-on therapy, combination, adjunctive therapy in a subject in need thereof.

[0079] In general, all physical forms are of use in the methods contemplated by the present invention and are intended to be within the scope of the invention.

[0080] Compound(s) or pharmaceutically acceptable salts includes, hydrates, polymorphs, solvates, enantiomers or racemates. Some of the crystalline forms of the compound(s) exist as polymorphs and as such are intended to be included in the present disclosure. In addition, some of the compound(s)s may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are intended to be encompassed by some embodiments.

[0081] In some of the embodiments, the present invention provides medicinal composition comprising the present bioactive nucleotide compound(s) which is present in therapeutically effective amount along with pharmaceutically acceptable excipients.

[0082] As used herein, the term “pharmaceutically acceptable carriers, diluents or excipients” is purported to mean, without limitation, any adjuvant, carrier, excipient, sweetening agent, diluents, preservative, dye / colorant, flavour enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, emulsifier, or encapsulating agent, encapsulating polymeric delivery systems or polyethylene glycol matrix, which is acceptable for use in the subject, preferably humans. Excipients also include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, fragrances, glidants (flow enhancers), lubricants, preservatives, sorbents, suspending or dispersing agents, sweeteners, surfactant, anticaking agent, food additives, or waters of hydration, salts.

[0083] In another embodiment, the present invention relates to medicinal composition of the bioactive nucleotide compound(s) prepared in a manner well known in the pharmaceutical art and administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.

[0084] The preferable route of administration includes but is not limited to sublingual, rectal, topical, parenteral, nasal, or oral.

[0085] In some embodiment, the medicinal compositions of the bioactive nucleotide compound(s) are administered to a subject in need thereof, in the form which is suitable for oral use, such as a tablet, capsule (in the form of delayed release, extended release, sustained release, enteric coated release); hard gelatin capsules, soft gelatin capsules in an oily vehicle, veg capsule, hard or soft cellulose capsule, granulate for sublingual use, effervescent or carbon tablets, aqueous or oily solution, suspension or emulsion, encapsulate, matrix, coat, beadlets, nanoparticles, caplet, granule, particulate, agglomerate, spansule, chewable tablet, lozenge, troche, solution, suspension, rapidly dissolving film, elixir, gel, tablets, pellets, granules, capsules, lozenges, aqueous or oily solutions, suspensions, emulsions, sprays or reconstituted dry powdered form with a liquid medium or syrup; for topical use including transmucosal and transdermal use, such as a cream, ointment, gel, aqueous or oil solution or suspension, salve, parch or plaster; for nasal use, such as a snuff nasal spray or nasal drops; for vaginal or rectal use, such as a suppository; for administration by inhalation, such as a finely divided powder or a liquid aerosol; for sub-lingual or buccal use, such as a tablet, capsule, film, spray.

[0086] In a further embodiment, the present composition is formulated in the form of age- appropriate pediatric oral dosage forms such as syrup, inhalation, spray minitablets, chewable formulations, orodispersible films and orodispersible tablets.

[0087] The magnitude of a prophylactic or therapeutic dose typically varies with the nature and severity of the condition to be treated and the route of administration. The dose, and perhaps the dose frequency, will also vary according to the age, body weight and response of the individual patient.

[0088] In some embodiment, the LCAT activator compound or pharmaceutically acceptable salts thereof, are formulated for medicaments, which preferably take the form of therapeutically effective individual doses adjusted to the form of administration.

[0089] In some embodiment, the daily dose of a pharmaceutical composition comprising present LCAT activator compound disclosed herein varies over a wide range from about 0.1 mg to about 5000 mg; preferably, the dose is in the range of about 1 mg to about 1000 mg per day for an average human.

[0090] In general, the total daily dose (in single or divided doses) ranges from about 0.1 mg per day to about 5000 mg per day, preferably about Img per day to about 1000 mg per day.

[0091] In some embodiment, the total daily dose can be administered in the range of about 1 mg to about 3000 mg per day, and preferably about 1 mg to about 1000 mg per day.

[0092] The term "therapeutically effective amount " denotes an amount that reduces the risk, potential, possibility or occurrence of a disease or disorder, or provides advanced alleviation, mitigation, and / or reduction or restoration or modulation, regulation of at least one indicator / biomarker (e.g., blood or serum CRP level), and / or minimize at least one clinical symptom related to cardiometabolic disorder like dyslipidaemia.

[0093] A "therapeutically effective amount" means the amount of the compound that, when administered to a subject to treat a disease or condition referred to herein, is sufficient to perform such treatment for the disease or condition.

[0094] The "therapeutically effective amount" will vary depending on the form of the compound (for example, the form of the salt), the disease or condition in question and its severity, as well as the age, weight, etc., of the subject to be treated.

[0095] The use of any and all examples, or exemplary language (e.g., such as) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0096] While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention. The present invention is not limited to the specific embodiments described, which serve as illustrations of its various aspects. The following examples are provided for illustrative purposes and should not be construed as limiting the invention’s scope. The scope is defined by the appended claims, and any modifications or equivalents fall within its scope.

[0097] Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiments thereof. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of invention.

[0098] EXAMPLES:

[0099] The in-situ preparation of a nucleotide compound involves esterifying a ribofuranosyl compound with boronic acid to form a cyclic boron ester intermediate, which is then phosphorylated and deprotected with aqueous acetic acid to yield a bioactive nucleotide compound with high yield and purity.

[0100] Example 1:

[0101] In situ synthesis of [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4- dihydroxyoxolan-2-yl] methyl hydrogen phosphate

[0102] In a round-bottom flask, 1 mmol of nicotinamide riboside chloride and 1 mmol of phenylboronic acid were combined with 20 mL of dichloromethane (DCM) as the solvent. Pyridine was added as a base in a stoichiometric amount of 1.0 to 1.2 equivalents to deprotonate the boronic acid, facilitating the formation of an ester. The mixture was stirred at a temperature of 20-25°C for 3-4 hours. Once the reaction was complete, the solvent was removed using rotary evaporation, yielding a cyclic boron ester.

[0103] Next, phosphorylating agents were introduced 0.5 equivalents of POCk per 1 mmol of cyclic boron ester and 1.5 equivalents of tri ethyl phosphate (P(OEt)3). This mixture was maintained in dry acetonitrile (20 mL per 1 mmol of substrate) and kept at 0-5°C in an ice bath to control the exothermic reaction and prevent decomposition. The reaction continued for 2-3 hours under a nitrogen atmosphere to minimize moisture and hydrolysis risks.

[0104] The phosphorylated intermediate was then treated with an aqueous solution of acetic acid (80:20 water / acetic acid) at a temperature of 30-35°C, stirring for 6-8 hours. Afterward, the mixture was neutralized with sodium bicarbonate and extracted with ethyl acetate, resulting in an 80-90% yield of the product [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan- 2-yl] methyl hydrogen phosphate. The crude product underwent purification using a water: acetonitrile mixture (30:70%) followed by methanol purification, achieving a final product purity typically exceeding 98%, confirmed by HPLC analysis.

[0105] Example 2: HPLC Analysis of [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4- dihydroxyoxolan-2-yl] methyl hydrogen phosphate

[0106] The High-Performance Liquid Chromatography (HPLC) method for the analysis of [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate purity generally involved using reverse-phase HPLC with appropriate conditions for detecting end product quantifying it, and ensuring its purity.

[0107] Column Type: C18 (250 mm x 4.6 mm, 5 pm particle size)

[0108] Mobile Phase: Water: Acetonitrile (70:30) (adjusted pH 6.0 with 0.1% phosphoric acid)

[0109] Detection Method: UV detection 260 nm

[0110] Column Temperature: 30°C

[0111] Flow Rate: 0.8 mL / min.

[0112] Injection Volume: 20 pL

[0113] Sample Preparation:

[0114] The [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate sample was dissolved in a suitable solvent (mixture of water and methanol) and filtered (e.g., 0.45 pm filter) before injection to avoid blockages or impurities from affecting the results.

[0115] The chromatogram showed a single peak corresponding to Formula-I , (2R,3S,4R,5R)- 5-(3-carbamoylpyridin- 1-ium- l-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate. with a retention time consistent with that of the standard. Standard purity 99.8%; ( Formula-I)

[0116] Test Sample Purity - 98.8%

[0117] Example 3:

[0118] Study Objective: To assess the effect of (2R,3S,4R,5R)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4- dihydroxyoxolan-2-yl] methyl hydrogen phosphate on LCAT activity in a preclinical animal model

[0119] Experimental Design:

[0120] Animal Condition

[0121] Lighting: 12 / 12-hour light-dark cycle

[0122] Temperature: 22 ± 3 °C

[0123] Relative Humidity: 30 to 70%

[0124] Animals had continuous access to fresh, potable, uncontaminated drinking water.

[0125] Feed: Normal chow diet

[0126] Study is approved by the Institutional Animal ethics committee and committee for the purpose of control and supervision of experiments on animals (CPCSEA).

[0127] Test system details:

[0128] Test Species: Mice

[0129] Strain: Swiss Albino Mice

[0130] Sex: Male / Female

[0131] Age: 8- 10 Weeks

[0132] Body Weight: 25-30gms

[0133] Source: In house breed

[0134] Animal Grouping:

[0135] • Group 1 (Control): Animals treated with saline (vehicle).

[0136] • Group 2 ((2R,3S,4R,5R)-5-(3-carbamoylpyridin- 1-ium- l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate: Animals treated with sample at dose (e.g., 100 mg / kg).

[0137] • Group 3 ((2R,3S,4R,5R)-5-(3-carbamoylpyridin- 1-ium- l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate: Animals treated with sample at dose (e.g., 500 mg / kg).

[0138] Number of Animals:

[0139] • 6 mice per group (total of 18 mice).

[0140] Study Duration:

[0141] • Duration: 2-4 weeks. LCAT activity was measured using an LCAT Activity Assay Kit. Blood was collected from animals via cardiac puncture or tail vein and centrifuged at 3000-4000 rpm for 10 minutes to isolate plasma. A negative control was prepared using reaction buffer without any sample.

[0142] 100 pL of substrate solution was added to each well, followed by 20-50 pL of diluted plasma. For the negative control, only the reaction buffer was added. 10 pL of calcium chloride (CaCh) solution (typically 1-5 mM) was then added to each well to activate LCAT. The reaction was incubated at 37°C for 30-60 minutes. After incubation, the reaction was stopped by adding 200 pL of chloroform and 100 pL of methanol to each sample. The mixture was briefly vortexed and allowed to separate into two phases: the lipid phase in the lower chloroform layer and the aqueous phase in the upper layer. The lower chloroform layer, containing the cholesterol esters, was carefully collected using a pipette. Cholesterol ester quantification was performed using an established analytical technique, such as fluorometry at 490 nm. Fluorescence counts corresponding to cholesterol ester formation were used to quantify LCAT activity. LCAT activity, expressed in units per mL, was calculated, and the activity in treated groups was compared to control groups using ANOVA. Significant differences in LCAT activity were observed between the experimental and control groups (p < 0.05).

[0143] Results

[0144] Table- 1

[0145] The study measures LCAT activity in units per milliliter (U / mL) of plasma from each mouse.

[0146] Discussion:

[0147] The LCAT activity assay provided a reliable method to measure LCAT enzyme activity in preclinical models, reflecting changes in cholesterol esterification and lipid transport. Bioactive nucleotide treatments (G2 / G3) significantly modulated LCAT activity, with G1 serving as the control for baseline activity. The nucleotide (G2 / G3) influenced lipid metabolism enzymes, including LCAT, with the compound (2R,3S,4R,5R)-5-(3-carbamoylpyridin- 1-ium- 1- yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate enhancing LCAT activity. Average LCAT activity (U / mL) for each group was calculated as the mean of individual mouse values.

[0148] Conclusion:

[0149] Increased LCAT activity in treated groups compared to control suggests that (2R,3S,4R,5R)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate positively affects cholesterol metabolism.

Claims

We Claim:

1. A bioactive nucleotide compound for regulating Lecithin-cholesterol acyltransferase (LCAT) activity in human and in situ process for preparation thereof.

2. The in-situ process as claimed in claim 1, wherein the process comprising steps of: a) Esterification- wherein a cyclic boron ester intermediate of formula-III is synthesized from a ribofuranosyl compound of formula-II in presence of boronic acid;wherein R is selected from H, (C1-C4) alkyl, (C2-C4) alkene, aryl, phenyl, benzyl, cycloalkyl, heterocyclic compound b) Phosphorylation- wherein a phosphate group is introduced into the cyclic boron ester intermediate in presence of phosphorylating agent to produce a phosphorylated compound of formula- IV ;c) Deprotection -wherein the boron moiety is removed using a mild acidic medium to obtain the final bioactive nucleotide compound of formula I with high yield and purity.

3. The in-situ process as claimed in claim 1 , wherein the esterification is carried out at ambient temperatures between 20-25 °C in the presence of a polar aprotic solvent selected from acetone, chloroform, dichloromethane, ethyl acetate, dimethylformamide, dimethyl sulfoxide, or tetrahydrofuran, chloroform, ether, dichloromethane, hexamethyl phosphoramide, or methyl ethyl ketone.

4. The in-situ process as claimed in claim 1 , wherein the base used in the esterification step is selected from sodium hydroxide, potassium hydroxide, ammonia hydroxide, sodium bicarbonate, sodium carbonate, lithium hydroxide, methylamine, ethylamine, aniline, dimethylamine, pyridine, triethylamine, or dimethylformamide.

5. The in-situ process as claimed in claim 1, wherein the phosphorylation is carried out at temperatures between 0-5 °C in the presence of a phosphorylating agent selected from phosphoryl chloride, diethyl phosphorochloridate, adenosine triphosphate, cytidine triphosphate, polyphosphates, or phosphoric acid either alone or in combination.

6. The in-situ process as claimed in claim 1, wherein the deprotection is carried out at temperatures between 30-35°C using an acidic medium selected from dilute acetic acid, tartaric acid, hydrochloric acid, formic acid, phosphoric acid, benzoic acid, sulfuric acid, triethylammonium acetate buffer either alone or in combination.

7. The in-situ process as claimed in claim 1, wherein the bioactive nucleotide compound is [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate.

8. The in-situ process as claimed in claim 7, wherein the bioactive nucleotide compound is formulated in oral dosage form with effective dose ranges from 1 mg to 1000 mg.

9. A method for treatment for conditions associated with Lecithin-Cholesterol Acyltransferase (LCAT) dysregulation in human, wherein the method comprising administration of a therapeutically effective amount of a bioactive nucleotide compound.

10. The method for treatment for conditions associated with Lecithin-Cholesterol Acyltransferase (LCAT) dysregulation, wherein the bioactive nucleotide compound is [(2S,3R,4S,5S)-5-(3-carbamoylpyridin-l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl] methyl hydrogen phosphate.

11. The method of treating conditions associated with Lecithin-Cholesterol Acyltransferase(LCAT) dysregulation, comprising administering an effective amount of a bioactive compound as claimed in claim 10, wherein the conditions are selected from opacities, anemia, proteinuria and progressive renal failure, low HDL cholesterol, proteinuria, chronic kidney disease, progressive loss of kidney function, renal impairment, cardiovascular disease, accumulation of unesterified cholesterol, dyslipidemia, diabetic retinopathy and like thereof.

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