Description:The present invention relates to the field of drug delivery systems, particularly lipid-based delivery systems for nucleic acid-based therapeutics such as messenger RNA (mRNA), small interfering RNA (siRNA), mRNA encoding firefly luciferase (FLuc), Renilla luciferase mRNA, single guide RNA (sgRNA), and such others. More specifically, it pertains to ionizable lipids, process for their preparation, and their application in lipid nanoparticles (LNPs) for the intracellular delivery of RNA.

BACKGROUND OF THE INVENTION

In the realm of drug delivery, lipid nanoparticles (LNPs) have become a revolutionary technology as they provide a versatile and efficient platform for the administration of a variety of therapeutic agents, especially nucleic acids. LNPs have gained global recognition since the success of mRNA vaccines for COVID-19, especially those developed by Pfizer-BioNTech and Moderna. These vaccines used a unique approach that enabled substantial protection against infection and quick immune activation by using LNPs to deliver mRNA encoding the SARS-CoV-2 spike protein.

In general, LNPs consist of four different lipids i.e., ionizable lipids, phospholipids, PEG lipids, and cholesterol. Among them, ionizable lipids are the major component in LNP formulation and play an important role in mRNA encapsulation and delivery. At low pH, the ionizable lipid molecules get protonated through tertiary amine center and shrink the long chain mRNA through electrostatic interactions. Upon formation of LNP, it encapsulates mRNA in the aqueous pockets and protects them from enzymatic degradation. LNP loaded with mRNA enters the intracellular matrix through endocytosis (phagocytosis or pinocytosis). The acidic environment of endo-lysosomal cavity protonates the ionizable lipids followed by destabilization of LNP architecture with electrostatic interaction with negatively charged lipids presents in periplasmic endo-lysosomal monolayers to release mRNA to the cytosol which then undergoes translation to express antibody. Amid the COVID-19 epidemic, two distinct ionizable lipids, ALC-0315 (((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)) and SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate), have received substantial attention. With just slight variations in chain length and branching, the two lipids have comparable structures.

ALC-0315 has proven to be one of the most efficient ionizable lipids and is widely used in mRNA therapeutics and delivery of mRNA vaccines. Several research methodologies have been adapted to develop ionizable lipids that could show similar or better efficiency as compared to the patented ALC-0315 in terms of mRNA encapsulation and delivery.

However, the synthesis of ALC-0315 has some drawbacks, such as long reaction times and complex steps. At the same time, there is a growing need for new ionizable lipids that offer improved performance or can serve as substitutes for ALC-0315.

OBJECTIVES OF THE PRESENT INVENTION
An objective of the present invention is to provide an ionizable lipid of Formula (I).

Another objective of the present invention is to provide a process of preparing the ionizable lipid of Formula (I).

Yet another objective of the present invention is to provide a lipid nanoparticle (LNP) comprising the ionizable lipid of Formula (I).

Further, objective of the present invention is to provide a process of preparing an ionizable lipid of Formula (I).

SUMMARY OF THE INVENTION
Accordingly, in an aspect, the present invention provides an ionizable lipid of Formula (I):

(I),
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof;
wherein,
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl;
R1 and R2 are independently alkyl or alkenyl; and
m and n are independently selected from 2 to 10.

In another aspect, the present invention provides a lipid nanoparticle comprising the ionizable lipid of Formula (I) or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.

In another aspect, the present invention provides a pharmaceutical composition comprising a lipid nanoparticle, a therapeutic agent, and optionally a pharmaceutically acceptable carrier or excipient, wherein the lipid nanoparticle comprises the ionizable lipid of Formula (I), or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.

In another aspect, the present invention provides a method of delivering a therapeutic agent to a cell, comprising administering to a subject a pharmaceutical composition comprising a lipid nanoparticle, a therapeutic agent, and optionally a pharmaceutically acceptable carrier or excipient; wherein the lipid nanoparticle comprises an ionizable lipid of Formula (I) or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.

In yet another aspect, the present invention provides a process of preparing an ionizable lipid of Formula (I):

(I),
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof,
wherein the process comprises:
reacting an alkyne precursor of Formula Id with an azide derivative (R-N3) in presence of a catalyst, a reducing agent and an amine in a solvent system at a temperature ranging from about 20ºC to 30ºC to obtain a crude mixture of Formula I,
wherein the alkyne precursor of Formula (Id) is

(Id),
R1 and R2 are independently alkyl or alkenyl.
m and n are independently selected from 2 to 10; and
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl.

The present invention further provides that the ionizable lipids of Formula (I) showed better or similar encapsulation efficiency as well as superior nano luciferase gene expression in vitro as compared to other FDA approved lipids ALC-0315 and/or SM-102.

BRIEF DESCRIPTION OF DRAWINGS OF THE INVENTION

Figure 1 illustrates mRNA encapsulated LNP formulation using Knauer Nanoscaler.

Figure 2 illustrates the hydrodynamic diameter of mRNA encapsulated LNPs.

Figure 3 illustrates (a) mRNA encapsulation efficiency values and (b) Nanoluciferase expression of mRNA encapsulated LNPs.

DETAILED DESCRIPTION OF THE INVENTION
At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a particular form of this invention. However, such a particular form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present disclosure.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods.

Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. In an embodiment, “about” can mean within one or more standard deviations, or within ± 30%, 25%, 20%, 15%, 10% or 5% of the stated value.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

For the purposes of the present invention, the following terms are defined below:

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By "pharmaceutically acceptable" it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

As used herein, the term “comprises” or “comprising” is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may occur or may not occur, and that the description includes instances where the event or circumstance occurs as well as instances in which it does not. For example, "optionally substituted alkyl" refers to the alkyl may be substituted as well as the event or circumstance where the alkyl is not substituted.

"Pharmaceutically acceptable" means that, which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use. These "pharmaceutically acceptable" substances are suitable for use in interact with bodily tissues without causing excessive irritation, allergic responses, or other complications, commensurate with a reasonable benefit/risk ratio.

"Pharmaceutically acceptable salt" refers to the salts of the compounds, that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. The pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Such salts include: salts derived from inorganic bases such as Li, Na, K, Ca, Mg, Fe, Cu, Zn, Al, Mn; salts of organic bases such as N,N'-diacetylethylenediamine, 2-dimethylaminoethanol, isopropylamine, morpholine, piperazine, piperidine, procaine, diethylamine, triethylamine, trimethylamine, tripropylamine, tromethamine, choline hydroxide, dicyclohexylamine, metformin, benzylamine, phenylethylamine, dialkylamine, trialkylamine, thiamine, aminopyrimidine, aminopyridine, purine, pyrimidine, spermidine, and the like; chiral bases like alkylphenylamine, glycinol, phenyl glycinol and the like, salts of natural amino acids such as glycine, alanine, valine, leucine, isoleucine, lysine, arginine, serine, threonine, phenylalanine; unnatural amino acids such as D-isomers or substituted amino acids; salts of acidic amino acids such as aspartic acid, glutamic acid; guanidine, substituted guanidine wherein the substituents are selected from nitro, amino, alkyl, alkenyl, alkynyl, ammonium or substituted ammonium salts. Salts may include acid addition salts where appropriate which are sulphates, nitrates, phosphates, perchlorates, borates, hydrohalides, acetates, tartrates, maleates, citrates, succinates, methanesulfonates, benzoates, salicylates, hydroxynaphthoates, benzenesulfonates, ascorbates, and the likes thereof.

The term “subject” includes mammals (especially humans) and other animals, such as domestic animals (e.g., household pets including cats and dogs) and non-domestic animals (such as wildlife).

The term "therapeutically effective amount" or "effective amount" refers to the quantity of the pharmaceutical composition disclosed herein that (i) treats or prevents a specific disease, disorder, or condition or syndrome; (ii) reduces, improves, or eliminates one or more symptoms associated with the disease, disorder, or syndrome; or (iii) delays the onset of one or more symptoms of the disease, disorder, or syndrome described herein. In cases of cancer, the therapeutically effective amount of the drug may reduce cancer cell proliferation, shrink tumor size, inhibit cancer cell migration to peripheral organs, suppress tumor metastasis, slow tumor growth to some extent, and/or alleviate one or more symptoms associated with cancer. For infectious diseases, the therapeutically effective amount is sufficient to reduce or alleviate the infection caused by bacteria, viruses, or fungi, and to alleviate symptoms associated with the infection.

The term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon that may be substituted or unsubstituted. In certain embodiments, the alkyl is C2-C15 alkyl that is either branched or linear aliphatic hydrocarbon. In certain embodiments, the alkyl is C6-C15 alkyl that is either branched or linear aliphatic hydrocarbon. In certain embodiments, the alkyl is a branched chain C6-C15 alkyl. Examples of "alkyl" include but are not limited to hexyl, heptyl, octyl, C9 alkyl, C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl and the likes thereof, wherein the alkyl is linear or branched.

The term "alkenyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon that may be substituted or unsubstituted. In certain embodiments, the alkenyl is C2-C15 alkenyl that is either branched or linear. In certain embodiments, the alkenyl is C6-C15 alkenyl that is either branched or linear, wherein the double bond is present at one or more positions. Examples of "alkenyl" include but are not limited to C6 alkenyl, C7 alkenyl, C8 alkenyl, C9 alkenyl, C10 alkenyl, C11 alkenyl, C12 alkenyl, C13 alkenyl, C14 alkenyl, C15 alkenyl and the likes thereof, wherein the alkenyl is linear or branched.

The term "alkynyl" refers to a straight or branched chain unsaturated aliphatic hydrocarbon that may be substituted or unsubstituted. In certain embodiments, the alkynyl is C2-C15 alkynyl that is either branched or linear. In certain embodiments, the alkenyl is C6-C15 alkynyl that is either branched or linear, wherein the double bond is present at one or more positions. Examples of "alkynyl" include but are not limited to C6 alkynyl, C7 alkynyl, C8 alkynyl, C9 alkynyl, C10 alkynyl, C11 alkynyl, C12 alkynyl, C13 alkynyl, C14 alkynyl, C15 alkynyl and the likes thereof, wherein the alkynyl is linear or branched.

The term "heterocyclyl" refers to a stable 3 to 15 membered ring that is either saturated or has one or more degrees of unsaturation or unsaturated. These heterocyclic rings contain one or more heteroatoms selected from the group consisting of nitrogen, sulfur, and oxygen where N-oxides, sulfur oxides and dioxides are permissible heteroatom substitutions. Such a ring is optionally fused to one or more of another heterocyclic ring(s), aryl ring(s) or cycloalkyl ring(s). Further, “heterocyclyl” refers to 4 to 7 membered rings that is either saturated or has one or more degrees of unsaturation or unsaturated. Examples of such groups are selected from the group consisting of azetidinyl, acridinyl, pyrazolyl, imidazolyl, triazolyl, pyrrolyl, thiophenyl, thiazolyl, oxazolyl, isoxazolyl, furanyl, pyrazinyl, tetrahydroisoquinolinyl, piperidinyl, piperazinyl, morpholinyl, thiomorphonilyl, pyridazinyl, indolyl, isoindolyl, quinolinyl, chromanyl and the likes thereof. "Heterocyclylalkyl" refers to a heterocyclic ring radical defined above, directly bonded to an alkyl group. The heterocyclylalkyl radical is attached to the main structure at carbon atom in the alkyl group that results in the creation of a stable structure.

The term "hydroxy" refers to -OH group. The term "hydroxyalkyl" refers to a hydroxy group containing bonded an alkyl group.

The term "alkoxy" refers to a group –O-alkyl, wherein alkyl is as defined above. Representative examples include, but are not limited to, C6 alkoxy, C7 alkoxy, C8 alkoxy, C9 alkoxy, C10 alkoxy, C11 alkoxy, C12 alkoxy, C13 alkoxy, C14 alkoxy, C15 alkoxy and the likes thereof, wherein the alkoxy is linear or branched. The term "alkoxy(alkoxy)alkyl" refers to a chemical compound or group containing one or more alkyl groups linked to an alkoxy group which is further linked to another alkoxy group. The term "hydroxyalkoxyalkyl" refers to a chemical compound or group containing one or more alkyl groups linked to an alkoxy group which is further linked to a hydroxy group. The term " alkoxyalkyl" refers to a chemical compound or group containing one or more alkyl groups linked to an alkoxy group. The term "phenoxyalkyl" typically refers to a chemical compound or group containing one or more alkyl groups linked to an phenoxy group.

The term "aryl" refers to optionally substituted unsaturated or partially saturated aromatic ring system having five to ten carbon atoms which are monocyclic, bicyclic, or polycyclic and may optionally be replaced by one or more hetero atoms selected from N, O and S. Exemplary aryl groups include phenyl, naphthyl, indanyl, biphenyl and the likes thereof. "Arylalkyl" refers to an aryl ring radical defined above, directly bonded to an alkyl group. The arylalkyl radical is attached to the main structure at carbon atom in the alkyl group that results in the creation of a stable structure.

The term "haloalkyl" typically refers to a chemical compound or group containing one or more alkyl groups (hydrocarbon chains) with one or more halogen atoms (fluorine, chlorine, bromine, or iodine) attached to them. In other words, "Haloalkyl" refers to a chemical moiety derived from an alkane by replacing one or more hydrogen atoms with halogen atoms. These halogen atoms may include, but are not limited to, fluorine (F), chlorine (Cl), bromine (Br), or iodine (I). The resulting haloalkyl group may be linear or branched and may contain one or more halogen atoms.

The term "stereoisomer" or "stereoisomers" refers to any enantiomers, diastereomers or geometrical isomers of the compounds of Formula (I), wherever they are chiral or when they bear one or more double bond. When the compounds of the Formula (I) and related formulae are chiral, they can exist in racemic or in optically active form. It should be understood that the invention encompasses all stereochemical isomeric forms, including diastereomeric, enantiomeric and epimeric forms, as well as -isomers and /-isomers and mixtures thereof. Individual stereoisomers of compounds can be prepared synthetically from commercially available starting materials which contain chiral centers or by preparation of mixtures of enantiomeric products followed by separation such as conversion to a mixture of diastereomers followed by separation or recrystallization, chromatographic techniques, direct separation of enantiomers on chiral chromatographic columns, or any other appropriate method known in the art. Starting compounds of particular stereochemistry are either commercially available or can be made and resolved by techniques known in the art. Additionally, the compounds of the present invention may exist as geometric isomers. The present invention includes all cis, trans, syn, anti, entgegen (E) and zusammen (Z) isomers as well as the appropriate mixtures thereof.

The stereoisomers of the compounds forming part of this invention may be prepared by using reactants in their single enantiomeric form in the process wherever possible or by conducting the reaction in the presence of reagents or catalysts in their single enantiomer form or by resolving the mixture of stereoisomers by conventional methods. Some of the preferred methods include use of microbial resolution, resolving the diastereomeric salts formed with chiral acids such as mandelic acid, camphorsulfonic acid, tartaric acid, lactic acid, and the like wherever applicable or chiral bases such as brucine, cinchona alkaloids and their derivatives and the like. Different polymorphs of a compound of Formula (I) of present invention may be prepared by crystallization of the corresponding compound of Formula (I) under different conditions. For example, making use of commonly used solvents or their mixtures for recrystallization, crystallization at different temperature ranges, different cooling techniques like very fast to very slow cooling during crystallization procedure, by exposing to room temp, by heating or melting the compound followed by gradual cooling and the like. The presence of polymorphs may be determined by one or more methods like solid probe NMR spectroscopy, DSC, TGA, Powder X-Ray diffraction and IR.

The term “tautomer” refers to structural isomers (constitutional isomers) of a chemical compound that are readily interconvertible to each other by relocating a proton.

Phospholipids are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, joined by an alcohol residue. Phospholipids are major membrane lipids that consist of lipid bilayers. The most common phospholipids are phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine.
Cholesterol is the principal sterol of all higher animals, distributed in body tissues, especially the brain and spinal cord, and in animal fats and oils. Cholesterol is biosynthesized by all animal cells and is an essential structural and signaling component of animal cell membranes. Cholesterol is biosynthesized by all animal cells and is an essential structural and signaling component of animal cell membranes. There are several types of cholesterol, with two main categories: LDL (low-density lipoprotein), often called bad cholesterol, and HDL (high-density lipoprotein), known as good cholesterol.

PEG lipid, also known as PEGylated lipid, are lipids modified with polyethylene glycol (PEG). PEG lipids have multiple effects on the properties of lipid nanoparticles. The amount of PEG lipids affect particle size. PEG lipids further contribute to particle stability by decreasing particle aggregation, and the optimization of PEG prolongs the blood circulation time of nanoparticles by reducing clearance mediated by the kidneys and the mononuclear phagocyte system. Finally, PEG lipids are used to conjugate specific ligands to the particle for targeted delivery.

Other objects, feature, and aspects of the present invention are disclosed in or are obvious from, the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not to be construed as limiting the broader aspects of the present invention.

The present invention provides a library of lipid molecules (triazole lipids) that show better or similar encapsulation efficiency and/or superior nano luciferase gene expression in vitro compared to the FDA approved lipids ALC-0315 and SM-102.

In an embodiment, the present invention provides an ionizable lipid of Formula (I):

(I)
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof;
wherein,
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl;
R1 and R2 are independently alkyl or alkenyl; and
m and n are independently selected from 2 to 10.

In certain embodiments, R1 and R2 are each alkyl.

In further embodiments, R1 and R2 are each C15 alkyl.

In some instances, R1 and R2 are each .

In an embodiment, the present invention provides ionizable lipid of Formula I wherein m and n are independently selected from 3 to 7.

Thus, in certain embodiments, the compound of Formula (I) is a compound of Formula (IA):

(IA),
or a pharmaceutically acceptable salt thereof; wherein R is same as defined above.

In certain embodiments, the present invention provides an ionizable lipid of Formula (I) or (IA); wherein R is selected from the group consisting of , , , , , , , , , and .

In certain embodiments, the present invention provides an ionizable lipid selected from:

CL1 CL2

CL3
CL4

CL5
CL6

CL7
CL8

CL9
CL10

CL11 ---

or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.

In certain embodiments, the ionizable lipid of Formula (I) or (IA) is Formula CL3:

CL3
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.

The present invention also provides a lipid nanoparticle comprising an ionizable lipid of Formula (I) or (IA) or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof, as defined in any one of the preceding embodiments. In certain embodiments, the ionizable lipid is present in an amount ranging from about 40 to about 60 wt %.

In certain embodiments, the lipid nanoparticle further comprises one or more phospholipid, a polyethylene glycol (PEG) lipid, and cholesterol.

In certain embodiments, the phospholipid is selected from the list comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and such others. In certain embodiments, the phospholipid is present in an amount ranging from about 10 to about 15 wt %.

In certain embodiments, the PEG lipid is selected from the list comprising 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DSPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DPPE-PEG2000), Ceramide-PEG2000 (Cer-PEG2000), Cholesterol-PEG2000 (Chol-PEG2000), DSPE-PEG1000, DSPE-PEG5000, and such others. In certain embodiments, the PEG lipid is present in an amount ranging from about 1 to about 3 wt %.

Examples of cholesterol include, but are not limited to, Cholesterol, Hydrogenated Cholesterol (Chol), Cholesteryl Hemisuccinate, Oxysterols (e.g. 7-ketocholesterol, 25-hydroxycholesterol), Chol-OH, Chol-NH₂, and such others. In certain embodiments, the cholesterol is present in an amount ranging from about 30 to about 40 wt %

In some embodiments, the lipid nanoparticle comprises the ionizable lipid, phospholipid, PEG lipid, and cholesterol in a ratio of 46.3:9.4:1.6:42.7 in a solvent.

The lipid nanoparticle disclosed herein may be formulated using conventional methods known to those skilled in the art. Examples include, but are not limited to, thin-film hydration method, solvent emulsification-evaporation, high-pressure homogenization, microfluidics, and rapid mixing methods.

The present invention further provides a pharmaceutical composition comprising a lipid nanoparticle, a therapeutic agent, and optionally a pharmaceutically acceptable carrier or excipient, wherein the lipid nanoparticle comprises an ionizable lipid of Formula (I) or (IA), or pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof. In certain embodiments, the RNA is encapsulated in the lipid nanoparticle. In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments is for delivering a therapeutic agent to a cell. In certain embodiments, the lipid nanoparticle is same as described in any of the preceding embodiments.

In an embodiment, the therapeutic agent present in the pharmaceutical composition is selected from the list comprising RNA selected from messenger RNA (mRNA), small interfering RNA (siRNA), mRNA encoding firefly luciferase (FLuc), Renilla luciferase mRNA, single guide RNA (sgRNA), and such others. In another embodiment, the RNA is encapsulated in the lipid nanoparticle. In an embodiment, the RNA is mRNA encoding firefly luciferase (FLuc).

In some embodiments, the lipid nanoparticle further comprises one or more phospholipid, a PEG lipid, and cholesterol, which are same as described in any of the preceding embodiments.

Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, and oils such as olive oil or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free or substantially pyrogen-free. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The pharmaceutical composition may be formulated as suspensions, emulsions, aerosols, or sterile injectable for primarily intravenous, intramuscular, subcutaneous, intranasal and solutions. Depending on the intended use, the composition can be administered to a subject by any of a number of routes of administration including, for example parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin or as an eye drop). The compound may also be formulated for inhalation. In yet another embodiment, a compound may be simply dissolved or suspended in sterile water.

The formulations can conveniently be presented in unit dosage form and can be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredients, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

In another embodiment, the pharmaceutical composition is for delivering a therapeutic agent to a cell.

In another embodiment, the pharmaceutical composition comprises a lipid nanoparticle, an mRNA encoding firefly luciferase (FLuc mRNA), and one or more pharmaceutically acceptable carrier or excipients, wherein lipid nanoparticle comprises an ionizable lipid of Formula I.

In another embodiment, the pharmaceutically acceptable excipients comprise phospholipid, cholesterol, polyethylene glycol (PEG)-lipid, a solvent and a buffer solution.

In certain embodiments, the phospholipid is selected from the list comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and such others. In certain embodiments, the the cholesterol is selected from the list comprising hydrogenated cholesterol, cholesteryl hemisuccinate, oxysterols (e.g. 7-ketocholesterol, 25-hydroxycholesterol), Chol-OH, Chol-NH₂, and such others. In certain embodiments, the PEG-lipid is selected from the list comprising 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), 1,2-distearoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DSPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DPPE-PEG2000), ceramide-PEG2000 (Cer-PEG2000), cholesterol-PEG2000 (Chol-PEG2000), DSPE-PEG1000, DSPE-PEG5000, and such others.

In another embodiment, the solvent is selected from the group comprising of ethanol chloroform, t-butanol, acetone, iso-propanol, methanol, and such others.

In another embodiment, the present invention provides that the mRNA is present in the buffer solution in a ratio ranging from about 3:1 to about 10:1 with the buffer solution.

The present invention further provides a method of delivering a therapeutic agent to a cell, comprising administering to a subject a pharmaceutical composition comprising a lipid nanoparticle, a therapeutic agent, and optionally a pharmaceutically acceptable carrier or excipient; wherein the lipid nanoparticle comprises an ionizable lipid of Formula (I) or (IA), as described in any of the preceding embodiments. In certain embodiments, the lipid nanoparticle and the therapeutic agent are same as described in any of the preceding embodiments. In an embodiment, the therapeutic agent present in the pharmaceutical composition is RNA selected from messenger RNA (mRNA), small interfering RNA (siRNA), mRNA encoding firefly luciferase (FLuc), Renilla luciferase mRNA, single guide RNA (sgRNA), and such others. In some instances, the therapeutic agent is mRNA encoding firefly luciferase (FLuc) mRNA. In certain embodiments, the lipid nanoparticle further comprises one or more phospholipid, a PEG lipid, and cholesterol.

In certain embodiments, the subject is a human in need of prevention or treatment for a disease, a disorder, or a condition including but not limited to those amenable to nucleic acid-based or drug-based therapy.

Thus, in certain embodiments, the present invention provides a method for treating or preventing a disease, disorder or a condition in a subject, the method comprises administering to the subject an effective of amount of a pharmaceutical composition comprising a lipid nanoparticle, a therapeutic agent, and optionally a pharmaceutically acceptable carrier or excipient, wherein the lipid nanoparticle comprises an ionizable lipid of Formula (I) or (IA). In certain embodiments, the lipid nanoparticle, the therapeutic agent, and the ionizable lipid of Formula (I) or (IA) are same as described in any of the preceding embodiments. In an embodiment, the therapeutic agent present in the pharmaceutical composition is RNA selected from the list comprising messenger RNA (mRNA), small interfering RNA (siRNA), mRNA encoding firefly luciferase (FLuc), Renilla luciferase mRNA, single guide RNA (sgRNA), and such others. In some instances, the therapeutic agent is mRNA encoding firefly luciferase (FLuc) mRNA. In certain embodiments, the lipid nanoparticle further comprises one or more phospholipid, a PEG lipid, and cholesterol.

In an embodiment, the disease, disorder or condition is selected from the group consisting of COVID-19, Influenza, RSV (Respiratory Syncytial Virus), Zika virus, Cytomegalovirus, HIV, Malaria, and such others.

The pharmaceutical compositions of the present invention are utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of Formula I and a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By "therapeutically effective amount" is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

The present invention also provides a process of preparing an ionizable lipid of Formula (I):

(I),
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof,
wherein the process comprises:
reacting an alkyne precursor of Formula Id with an azide derivative (R-N3) in presence of a catalyst, a reducing agent and an amine in a solvent system at a temperature ranging from about 20ºC to 30ºC to obtain a crude mixture comprising the ionizable lipid of Formula (I);
wherein the alkyne precursor of Formula (Id) is

(Id),
R1 and R2 are independently alkyl or alkenyl;
m and n are independently selected from 2 to 10; and
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl.

In certain embodiments, the process further comprises extracting the ionizable lipid from the crude reaction mixture. The extraction step is performed by contacting the crude mixture with one or more suitable solvents to separate the ionizable lipid.

Thus, in some embodiments, the process comprises:
reacting an alkyne precursor of Formula Id with an azide derivative (R-N3) in presence of a catalyst, a reducing agent and an amine in a solvent system at a temperature ranging from about 20ºC to 30ºC to obtain a crude mixture, and
extracting an ionizable lipid from the crude mixture followed by drying to obtain the pure ionizable lipid of Formula (I);
wherein the alkyne precursor of Formula (Id) is

(Id),
R1 and R2 are independently alkyl or alkenyl;
m and n are independently selected from 2 to 10; and
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl.

In certain embodiments, the crude mixture was extracted from the crude and dried using a drying agent. The crude mixture was then washed and purified to obtain the ionizable lipid product using chromatographic techniques.

In certain embodiment, the catalyst is a copper (II) salt selected from the group consisting of copper sulphate pentahydrate, copper (II) acetate, and copper (II) chloride. In certain embodiments, the reducing agent is selected from the group consisting of L-Ascorbic acid, sodium ascorbate, TCEP (Tris(2-carboxyethyl)phosphine), and combinations thereof. In certain embodiments, the amine is selected from the group consisting of Tris(3-Hydroxypropyltriazolylmethyl) amine (THPTA), Tris(benzyltriazolylmethyl)amine (TBTA), 2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetic acid (BTTA), Tris((1H-tetrazol-5-yl)methyl)amine (TTMA), Pentamethyldiethylenetriamine (PMDETA), Ethylenediaminetetraacetic acid / Nitrilotriacetic acid (EDTA/NTA), or combinations thereof. In an embodiment, the catalyst is copper sulphate pentahydrate, the reducing agent is L-Ascorbic acid, and the amine is Tris(3-Hydroxypropyltriazolylmethyl) amine (THPTA).

In certain embodiments, the solvent system is a combination of solvents selected from the group consisting of tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), water, methanol, ethanol, t-butanol, and dimethyl formamide (DMF). In an embodiment, the solvent system comprises a combination of THF, water and DMSO. In a further embodiment, the solvent system comprises a combination of THF, water and DMSO in a ratio of 4:1:0.05.

In certain embodiments, the alkyne precursor of formula (Id) is reacted with the azide derivative in presence of a copper based catalyst, a reducing agent and an amine. In certain embodiments, the copper based catalyst, the reducing agent and the amine are mixed together and solubilized in a solvent system. In certain embodiments, the azide derivative is added to the resulting mixture under stirring. After the reaction is completed, the crude mixture is extracted and dried using a drying agent and is subjected to further purification to obtain a compound of Formula (I).

In an embodiment, the process comprises reacting a compound of Formula Ia with propargyl amine in presence of a base in a solvent under reflux condition at a temperature ranging from 70ºC to 90ºC for a time ranging from about 36 to 48 hours to obtain the alkyne precursor of Formula Id,
wherein
the compound of Formula (Ia) is , and/or ,
R1 and R2 are independently alkyl or alkenyl,
m and n are independently selected from 2 to 10; and
X is chloro or bromo.

In certain embodiment, the solvent is selected from, but not limited to, ethanol, methyl cyanide, acetonitrile, DMF, Methanol, THF, DMSO, and such others. In certain embodiment, the base is selected from, but not limited to, potassium carbonate, potassium iodide, triethyl amine, and such others.

In another embodiment, the reaction is carried out at a temperature of about 70ºC, 80ºC, or 90ºC for a time of about 40 to about 48 hours. In certain embodiments, the reaction is performed under a stirring for about 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

In certain embodiments, the compound of formula (Ia) is reacted with propargyl amine in presence of a base and the resulting reaction mixture is dissolved in a solvent and subjected to reflux conditions for about 36 to about 48 hours. In certain embodiments, the crude mixture is dried over the rotor evaporator and extracted with a suitable solvent. In certain embodiments, the crude mixture is dried using a drying agent and is subjected to further purification to obtain a compound of Formula (Id). The resultant compound of Formula (Id) may be used in situ or isolated. In certain embodiments, said compound is used directly in reaction without further purification.

In an embodiment, the R1 and R2 present in the starting material and the intermediate products are each alkyl. In another embodiment, the R1 and R2 are each C15 alkyl. In yet another embodiment, the R1 and R2 are each .

In certain embodiments of the process, m and n are independently selected from 3 to 7.

In an embodiment, the compound of Formula Ia is 6-bromohexyl-2-hexyldecanoate.

In certain embodiments of the process, the compound of Formula Id is .

In an embodiment, the present invention provides a process comprising reacting a compound of 6-bromohexyl-2-hexyldecanoate (Formula Ia) with propargyl amine in presence of a base in a solvent under reflux condition at a temperature ranging from 70ºC to 90ºC for a time ranging from about 36 to 48 hours to obtain (Formula Id) as the alkyne precursor.

In certain embodiments of the process, the compound of Formula (I) is a compound of Formula (IA):

(IA),
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof; wherein R is same as defined above.

In an embodiment, the process comprises reacting a bromo derivative R-Br with an azide in presence of a catalyst in a solvent at a temperature ranging from about 50 to 70ºC for a time ranging from about 10 to 20 hours to obtain an azide derivative (R-N3), wherein R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl.

In certain embodiments, the catalyst is selected from the group comprising of sodium iodide, tetrabutylammonium iodide, potassium iodide, lithium iodide, and such others. In an embodiment, the catalyst is a sodium iodide.

In certain embodiments, the azide is selected from the group comprising of Trimethylsilyl azide (TMS-N₃), Diphenylphosphoryl azide (DPPA), Imidazole-1-sulfonyl azide, p-Toluenesulfonyl azide (TsN₃), and such others. In an embodiment, the azide is sodium azide.

In certain embodiments, the solvent in step (b) is selected from the group comprising of dimethylformamide (DMF), Acetonitrile, DMF, DMSO, THF, Acetone, and such others. In an embodiment, the solvent is dimethylformamide (DMF).

In certain embodiments, the reaction is carried out for about 10, 12, 14, 16, 18, or 20 hours. In certain embodiments, the reaction is carried out at a temperature of about 50 ºC, 60 ºC, or 70ºC, depending on the type of the solvent. In an embodiment, the process comprises reacting a bromo derivative R-Br with an azide in presence of sodium iodide catalyst in DMF at 60ºC for about 18 hours. In certain embodiments, the bromo derivative R-Br is reacted with an azide in presence of a catalyst in a solvent. The resulting reaction mixture is subjected to reflux conditions for about 10 to about 20 hours. In certain embodiments, the crude mixture is extracted and dried under controlled evaporation due to the volatile nature of the azides. The resultant azide may be used in situ or isolated. In certain embodiments, the resultant azide is used directly in reaction without further purification.

In an embodiment, the present invention provides a process of preparing an ionizable lipid of Formula (IA). The process comprises the steps of:
(a) reacting 6-bromohexyl-2-hexyldecanoate with propargyl amine in presence of a base in a solvent under reflux condition at a temperature of 80ºC for about 48 hours to obtain an alkyne precursor of Formula Id, wherein the compound of Formula Id is ;
(b) reacting a bromo derivative R-Br with an azide in presence of sodium iodide catalyst in DMF at a temperature of about 60ºC for about 18 hours to obtain an azide derivative R-N3;
(c) reacting the alkyne precursor with R-N3 in presence of a catalyst, a reducing agent and an amine in a solvent system at about 25ºC for about 4 hours to obtain a crude mixture, wherein the solvent system comprises THF, water and DMSO in the ratio of 4:1:0.05.

Examples
Although the content of the present invention is further specifically explained using Examples, the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded. Values of various manufacturing conditions and evaluation results in the following Examples mean preferable values of an upper limit or a lower limit in the embodiments of the present invention, and a preferable range may be a range defined by a combination of the above-described upper limit or the above-described lower limit and the values of the following Examples or a combination of the values of Examples.

The chemicals used during our experiments were procured from Tokyo Chemical Industry Co. Ltd, Spectrochem, BLDpharm, Sisco Research Laboratories Pvt. Ltd. (SRL)-India and SD Fine-Chem Ltd and were employed in experiments without any further purification. All the reactions were carried out using dried and distilled solvents unless mentioned otherwise. Dry DCM was obtained by drying over Calcium hydride in a Dean-Stark apparatus and THF was dried over Benzoquinone. Thin-layer chromatography was performed using Merck silica gel 60 F254 pre-coated plates (0.25 mm). Column chromatography was performed using 100-200 mesh silica gel.
NMR spectra were recorded on Bruker Ultrashield spectrometer at 400 MHz (for 1H-NMR) and 100 MHz (for 13C-NMR). Chemical shifts are reported in ppm from tetramethyl silane with the solvent resonance as internal standard (CDCl3: δ 7.26 for 1H-NMR and δ 77.16 for 13C-NMR). High resolution mass spectrometry was performed on XEVO G2-XS QToF instrument. Dynamic light scattering experiments and zeta potential measurements were recorded on Malvern Zetasizer Nano ZS.

Example 1: Synthesis of the alkyne precursor (Formula (Id) for the Click reaction:
6-bromohexyl-2-hexyldecanoate was prepared as per methods known in art as reported by Boldyrev et al. The as prepared 6-bromohexyl-2-hexyldecanoate (2.5 equivalents) was reacted with 1 equivalent propargyl amine and potassium carbonate (2.5 equivalents) along with potassium iodide (0.3 equivalents) acting as base as per Scheme 1. The reaction was carried out for 48 hours in methyl cyanide medium under reflux condition. Briefly, 2 gm of the 6-bromohexyl-2-hexyldecanoate (4.78 millimoles) were made to react with 0.105 gm of propargyl amine (1.91 millimoles, 122 µL) and potassium carbonate (2.5 equivalents) along with potassium iodide (0.3 equivalents) and the resulting reaction mixture was dissolved in 2 mL methyl cyanide solution and put under reflux condition for 48 hours. The resulting crude was then dried over the rotor evaporator and extracted thrice with ethyl acetate and water and finally dried over sodium sulphate. It was then subsequently loaded on a silica gel column and the alkyne precursor (prop-2-yn-1-ylazanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (1d) was separated by using EtOAc/Hexane solvent mixture. The column (diameter 8 cm) was packed to a height of 45 cm. The column was initially started with 1% EtOAc/Hexane and then the polarity gradually increased and finally the product starts eluting at 6% EtOAc/Hexane. Upon chromatographic purification, the desired product was obtained with a good yield of 58%.

Scheme 1: Synthesis of alkyne precursor of Formula (Id)

Example 2: Synthesis of azides from corresponding bromo derivatives
All bromo derivatives (500 mg, 1 equivalent) are bought commercially and were reacted with 1.5 equivalents of sodium azide to transform into the corresponding azide derivatives as per Scheme 2. Catalytic amount of sodium iodide (0.3 equivalent) was also employed to make the SN2 reaction more facile as iodide will act as a better leaving group than the corresponding bromides. The reaction was carried out in DMF under at 60ºC. Owing to the volatile nature of the formed azides, care was taken during the extraction and evaporation processes. The azide molecules were formed with 100% conversion of bromo precursors and were used directly for the final click reaction step without further purification.

Scheme 2: Azide precursors synthesized by varying R groups

Two bromo derivatives (3-bromopropan-1-amine (Br-3) and 3-bromo-N,N-dimethylpropan-1-amine (Br-8)) were available in the form of hydrobromide salts. Therefore, these two bromo derivatives particularly, were initially treated with potassium carbonate at room temperature to accomplish neutralization reaction and then are made to react with sodium azide and sodium iodide in the same solvent medium at 60ºC.

Example 3: Synthesis of lipid library based on Click Chemistry
All the lipid molecules of the library were synthesized by employing Click Chemistry as the core synthetic strategy. The starting alkyne precursor and the respective azides were subjected to react in the presence of copper sulphate pentahydrate (CuSO4.5H2O), L-Ascorbic acid and Tris(3-Hydroxypropyltriazolylmethyl) amine (THPTA) as per Scheme 3. The role of hydrated cupric sulphate was to act as a source of copper ion (Cu2+) which in the presence of suitable reducing agent like L-Ascorbic acid was converted into the corresponding cuprous (Cu+) ion that, in turn, act as an active catalyst in the click reaction. The cuprous ion facilitates the formation of the triazole ring between the azide and alkyne moiety, thereby enhancing the formation of desired product. The presence of THPTA ensured the stability of the cuprous ion by preventing its oxidation and maintaining the catalyst’s activity. Also, the reaction was carried out at an optimized solvent system which comprises the implication of THF, water and DMSO in the ratio of 4:1:0.05. The structure of THPTA also assisted in improving the solubility of the copper complexes in the solvent system and hence smoothened up the reaction to a greater extent.

100 mg (0.14 mmol, 1 equiv.) of 1d, 51.18 mg (0.21 mmol, 1.5 equiv.) of CuSO4.5H2O, 36.11 mg (0.21 mmol, 1.5 equiv.) of L-Ascorbic acid and 8.91 mg (0.02 mmol, 0.15 equiv.) of THPTA were taken in a 100 mL single neck round bottomed flask and were solubilized in the solvent mixture (THF: H2O: DMSO = 4:1:0.05). Following this, 0.16 mmol (1.2 equiv.) of Aza-1, 2, 3, 4, 5, 6, 8, 10 and 11 were added to the stirring mixture in each independent reaction. Owing to their high volatility, Aza-7 and Aza-9 were taken in 3 equivalents (0.41 mmol) and 2 equivalents (0.27 mmol) respectively. The reaction mixture was kept under stirring for 4 hours at a temperature of 25 ºC.

Scheme 3: Copper catalyzed Alkyne-Azide Click reaction

After the reaction was completed, the crude mixture was extracted over ethyl acetate/ saturated sodium chloride (brine) solution workup and was dried over sodium sulphate. Then, the crude was loaded on a silica gel column of diameter 3 cm, packed to a height of 28 cm. 100 mL of 10% triethyl amine in hexane was prepared and poured to saturate the active sites of silica and washed off again using DCM to remove the excess triethyl amine from the column. The column was initiated with 100% DCM and the product started eluting in the range of 0.5-3% MeOH in DCM. Instead of saturating the column with triethyl amine, a stock solution of 0.1% ammonium hydroxide (NH4OH) in DCM (Solution X) was made in case of CL-3 and CL-8. All the lipid molecules were obtained in moderate to good yield in the range of 52-78%. The library of the lipid molecules thus obtained along with their yield are shown below.

Scheme 4: A library of Click chemistry based ionizable lipids

Example 4: Formulation of the LNPs and characterization
mRNA encoding firefly luciferase (FLuc) mRNA in citrate buffer (pH 3) and ethanolic solution of the other lipid components in ethanol in their targeted concentration were injected to Knauer Nanoscaler IJM Mixer (Figure 1) to formulate the lipid nanoparticles. The LNPs were formulated using a standard molar ratio of 46.3:9.4:42.7:1.6 of the Ionizable lipid, DSPC, Cholesterol and PEG-lipid in ethanol and combined with a 50 mM sodium citrate buffer (pH 3) containing mRNA at a ratio of 3:1. The standard weight ratio of 10:1 of IL to FLuc mRNA was chosen for the LNP formulation. Formulations were finally buffer exchanged against phosphate-buffered saline (pH 7.4). Figure 1 shows mRNA encapsulated LNP formulation using Knauer Nanoscaler. The hydrodynamic diameter of the formulated mRNA-LNPs were determined by dynamic light scattering (DLS) measurement using the Malvern Zetasizer NanoZS and the results are shown in Figure 2.

Example 5: In vitro encapsulation and expression of mRNA-LNPs encoding nano luciferase
The percentage mRNA encapsulation efficiency was measured by mRNA binding to Ribogreen using the QuantiT Ribogreen RNA assay. All the LNPs exhibited very high mRNA encapsulation efficiency as shown in Figure 3a. The mRNA-LNPs encoding Nanoluciferase was transfected in HEK 293T cells. 12 hours post-transfection, the luminescence was measured using the Promega Nano-Glo® Luciferase assay system as shown in Figure 3b. The mRNA-LNPs made with ionizable lipids CL-1, 3, 5, 6 and 7 showed comparable expression to those with ALC-0315 (Pfizer) and SM102 (Moderna).

The following data further shows the improved mRNA encapsulation efficiency and Nanoluciferase expression of mRNA encapsulated LNPs synthesized in the present invention. The encapsulation efficiency for the best performing mRNA-LNPs was within the range of 90-98%. The error range of the assay used to measure encapsulation efficiency, was ~10%. So <5% increase in encapsulation efficiency was not considered as a significant improvement. The nanoluciferase expression of the mRNA encapsulated LNPs was expressed in terms of the luminescence intensity as presented in Table 1.

Table 1: The luminescence intensity obtained from the nanoluciferase assay.
Ionizable lipid Luminescence Intensity (105)
ALC-0315 5.344 ± 0.009
SM-102 7.815 ± 0.001
CL-1 1.526 ± 0.004
CL-2 0.010 ± 0.001
CL-3 10.700 ± 0.001
CL-4 1.240 ± 0.007
CL-5 2.490 ± 0.001
CL-6 2.470 ± 0.001
CL-7 3.573 ± 0.001
CL-8 0.317 ± 0.003
CL-9 0.108 ± 0.007
CL-10 0.169 ± 0.002
CL-11 0.005 ± 0.000 , Claims:1. An ionizable lipid of Formula I:

Formula I
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof; wherein,
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl;
R1 and R2 are independently alkyl or alkenyl; and
m and n are independently selected from 2 to 10.
2. The ionizable lipid as claimed in claim 1, wherein R1 and R2 are each alkyl.
3. The ionizable lipid as claimed in claim 1, wherein R1 and R2 are each C15 alkyl.
4. The ionizable lipid as claimed in claim 1, wherein R1 and R2 are each .
5. The ionizable lipid as claimed in claim 1, wherein m and n are independently selected from 3 to 7.
6. The ionizable lipid as claimed in claim 1, wherein R is selected from the group consisting of , , , , , , , , , and .
7. The ionizable lipid as claimed in claim 1, selected from

CL1 CL2

CL3
CL4

CL5
CL6

CL7
CL8

CL9
CL10

CL11 ---
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.

8. An ionizable lipid of Formula CL3
,
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof.
9. A lipid nanoparticle comprising an ionizable lipid as claimed in claim 1.
10. The lipid nanoparticle as claimed in claim 9, further comprises a phospholipid, a PEG lipid, and cholesterol.
11. The lipid nanoparticle as claimed in claim 10, wherein the ionizable lipid, phospholipid, PEG lipid, and cholesterol are present in a ratio of 46.3:9.4:1.6:42.7 in a solvent.
12. A pharmaceutical composition comprising a lipid nanoparticle as claimed in any one of the claims 9-11, a therapeutic agent, and optionally a pharmaceutically acceptable carrier or excipient.
13. The pharmaceutical composition as claimed in claim 12, wherein the therapeutic agent is RNA selected from messenger RNA (mRNA), small interfering RNA (siRNA), mRNA encoding firefly luciferase (FLuc), Renilla luciferase mRNA, and single guide RNA (sgRNA).
14. The pharmaceutical composition as claimed in claim 13, wherein the RNA is encapsulated in the lipid nanoparticle.
15. The pharmaceutical composition as claimed in any one of the claims is for delivering a therapeutic agent to a cell.
16. A process of preparing an ionizable lipid of Formula I:

Formula I,
or a pharmaceutically acceptable salt, stereoisomer, or a tautomer thereof,
wherein the process comprises:
reacting an alkyne precursor of Formula Id with an azide derivative (R-N3) in presence of a catalyst, a reducing agent and an amine in a solvent system at a temperature ranging from about 20ºC to 30ºC;
wherein the alkyne precursor of Formula (Id) is

(Id),
R1 and R2 are independently alkyl or alkenyl;
m and n are independently selected from 2 to 10; and
R is selected from hydroxyalkyl, aminoalkyl, alkoxy(alkoxy)alkyl, hydroxyalkoxyalkyl, alkoxyalkyl, phenoxyalkyl, heterocyclylalkyl, arylalkyl, and haloalkyl.
17. The process as claimed in claim 16, wherein the process comprises reacting a compound of Formula Ia with propargyl amine in presence of a base in a solvent under reflux condition at a temperature ranging from 70ºC to 90ºC for a time ranging from about 36 to 48 hours to obtain the alkyne precursor of Formula Id,
wherein
the compound of Formula (Ia) is , and/or ,
R1, R2, m, and n are same as defined in claim 16, and X is chloro or bromo.
18. The process as claimed in claim 16 or 17, wherein R1 and R2 are each alkyl.
19. The process as claimed in claim 17, wherein R1 and R2 are each C15 alkyl.
20. The process as claimed in claim 17, wherein R1 and R2 are each .
21. The process as claimed in claim 16, wherein the process comprises reacting a bromo derivative R-Br with an azide in presence of a catalyst in a solvent at a temperature ranging from about 50 to 70ºC for a time ranging from about 10 to 20 hours to obtain an azide derivative (R-N3), wherein R is same as defined in claim 16.
22. The process as claimed in claim 16, wherein the catalyst is a copper (II) salt selected from the group consisting of copper sulphate pentahydrate, copper (II) acetate, and copper (II) chloride.
23. The process as claimed in claim 16, wherein the reducing agent is selected from the group consisting of L-Ascorbic acid, sodium ascorbate, TCEP (Tris(2-carboxyethyl)phosphine), or a combination thereof.
24. The process as claimed in claim 16, wherein the amine is selected from the group consisting of Tris(3-Hydroxypropyltriazolylmethyl) amine (THPTA), Tris(benzyltriazolylmethyl)amine (TBTA), 2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]acetic acid (BTTA), Tris((1H-tetrazol-5-yl)methyl)amine (TTMA), Pentamethyldiethylenetriamine (PMDETA), Ethylenediaminetetraacetic acid / Nitrilotriacetic acid (EDTA/NTA), or combinations thereof.
25. The process as claimed in claim 16, wherein the solvent system is a combination of solvents selected from the group consisting of tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), water, methanol, ethanol, t-butanol, and dimethyl formamide (DMF).
26. The process as claimed in claim 25, wherein the solvent system comprises a combination of THF, water and DMSO.