Tetrahydropyrimidine (THP) ionizable lipids for efficient mRNA delivery

Abstract

The present disclosure is concerned with tetrahydropyrimidine (THP) ionizable lipids and lipid nanoparticle (LNP) formulations. The disclosed compounds and LNP formulations can be useful in the delivery of therapeutic agents such as, for example, mRNA for treatment of a viral infection, cancer, or a genetic disease or disorder. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Description

TETRAHYDROPYRIMIDINE (THP) IONIZABLE LIPIDS FOR EFFICIENT MRNA DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Application No. 63 / 684.835. filed onAugust 19, 2024, the contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0002] The field of mRNA technology is rapidly advancing, opening up new therapeutic possibilities from vaccines to gene editing (Kariko, K.; et ah, (2021) Cell Syst. 12 (8), 757- 758; Mukai, H.; et al., (2.022) Drug Metab. Pharmacokinet. 44, 100450; Qiu, M.; et al., (2021) Acc. Chem. Res. 54 (21), 4001-401 1). This technology leverages the body's own cellular machinery to produce proteins that combat diseases, repair damaged tissues, or replace missing proteins (Holt, C. E.; Bullock, S. L. (2009) Science 326 (5957), 1212-1216; Olszewska, M.; et al., (2012) Cell Biochem. Funct. 30 (3), 177-182; Arribas-Layton, et al., (2013) Biochim. Biophys. Acta BEtA - Gene Regul. Meeh. 1829 (6), 580-589). However, efficiently delivering mRNA into cells remains a. significant hurdle, primarily due to its inherent instability and susceptibility to degradation by nucleases (Ross, J. mRNA Stability in Mammahan Cells. (1995) MicrobioL Rev. 59 (3), 423-450). Developing safe and effective delivery systems is therefore crucial for unlocking the full potential of mRNA-based therapies. Lipid nanoparticles (LNPs) have risen to prominence as the leading vehicle for mRNA delivery (Lutz, J.; et al., (2017) Npj Vaccines 2 (1), 1-9; Geng, C.; et al., (2023) J.Controlled Release 364, 632-643; Schoenmaker, L.; et al., (2021) Int. J. Pharm. 601, 120586; Kiaie, S. H.; et. al., (2022.) J Nanobiotechnology 20 (1), 276). These nanoparticles play a crucial role in protecting the delicate mRNA strands, facilitating their entry' into target cells, and ensuring the release of mRNA into thecellular cytoplasm, where it can be translated into proteins (Cui, L,; et al ., (2.022) Nanoscale 14 (4), 1480-1491; Jiirgens, D, C.; et al., (2023) OpenNano 12, 100161; Eygeris, Y,; et al ., (2022) Acc. Chem. Res. 55 (1), 2-12), The successof mRNA LNP vaccines against COVID-19 has highlighted the transformative potential of this approach {Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine 1 New England Journal of Medicine. https / / www.nejm.org / doi / full / 101056 / nejmoa2034577 (accessed 2024-07-20). Advances in COVID-19 mRNA vaccine development I Signal Transduction and Targeted Therapy. https: / / wwwmature.com / articles / s41392-022-00950-y (accessed 2024- 07-20)).

[0003] LNPs typically consist of four main components: ionizable lipid, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipid. Among these, ionizable lipids are particularly important as they complex mRNA (Ndeupen, S.; et al., (2021) iScience 24 ( 12): A. Kulkami, J.; et al., (2019) Nanoscale 11 (45), 21733-21739; Zhang, L.; et al., (2023) Npj Vaccines 8 (1), 1-14; Hald Albertsen, C.; et al ., (2022) Adv. Drug Deliv. Rev. 188, 1 14416). Additionally, they are neutral at physiological pH to reduce toxicity, but in the acidic environment of the endosome, they become positively charged, facilitating the release of mRNA into the cytoplasm and thereby controlling the endosomal escape of the LNP. The lipid tail plays a crucial role in this process, affecting the lipophilicity, fusogenicity, and fluidity of the membrane (Zhang, Y.; et al., (2021) Chem. Rev. 121 (20), 12181 -12277).21 This dual functionality is a key factor in effective mRNA delivery (Ball, R. L.; et al., (2017) Int. J. Nanomedicine 2017, 12, 305-315; Koitabashi, K.; et al., (2021) Biochim. Biophys. Acta BBA - Biomembr. 1863 (8), 183627).

[0004] Recently, there have been significant efforts to develop new ionizable lipids that can enhance the delivery of mRNA with greater selectivity (Lam, K.; et ah, (2023) Adv. Mater 35 (15), 2209624; Schlich, M.; et al., (2021) Bioeng. Transl. Med. 6 (2), el 0213; Billingsley, M. M.; et al., (2020) Nano Lett. 20 (3), 1578-1589; Kulkami, J. A.; et al., (2018) ACS Nano 12 (5), 4787-4795). Traditionally, these lipids have been synthesized through multiple chemical reactions, resulting in diverse structures with varying levels of efficacy, often making the process time-consuming. (De, A.; Ko, Y. T. (2023) Expert Opin. Drug Deliv . 20 (2), 175- 187; Lamoot, A.; et. al., (2023) Biomater Sei. 11 (12), 4327-4334; Carrasco, M. J,; et al,, (2021) Commnn. Biol. 4 (1), 1-15). The emergence of multi-component reactions (MCRs) has remarkably accelerated the discovery of new ionizable lipids by facilitating the rapid and efficient synthesis of a wide range of compounds (Meanwell, M. W.; et al., (2017) Can. J. Chem. 95 (2), 120-129; Han, X.; et al ., (2024) Nat. Chem. 1-11; He, Z.; et al., (2024) ChemMedChem e202400199). One-pot MCRs are especially valuable because they create molecular diversity from basic starting materials in a single, rapid reaction step, thus conveniently expanding the chemical space (Han, X.; et al., (2024) Nat. Chem. 1-1 1 ; Farbiak,L.; et al., (2021) Adv. Mater. 33 (30), 2006619; Dong, W.; et al., (2024) J Am. Chem. Soc. 146 (22), 15085-15095; De, A.; Ko, Y. T. (2022) Drug Deliv. 29 (1), 2644-2657). This approach facilitates the systematic exploration of structure-activity relationships, aiding in the identification of lipids with optimal properties tor mRNA delivery. However, these reactions typically require several days to complete, highlighting the need for further exploration and optimization of MCR conditions.

[0005] Thus, there continues to remain a need for biodegradable ionizable lipids that can balance the hydrophobic and hydrophilic properties necessary for effective LNP formation and encapsulation of mRNA or other therapeutic agents.SUMMARY

[0006] In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to tetrahydropyrimidine (THP) ionizable lipids and lipid nanoparticle (LNP) formulations. The disclosed compounds and LNP formulations can be useful in the delivery' of therapeutic agents such as, for example, mRNA for treatment of a viral infection, cancer, or a genetic disease or disorder.

[0007] Thus, disclosed are compounds having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR10aR10b, -(C1-C20 alkylenefCy1, and -(C2.-C20 alkenylene)Cy1; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, --CN, - OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1 -C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR11aR11b, --(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R11aand R11bisindependently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0008] Also disclosed are compounds having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C7-C20 alkyl, a C7-C20 alkenyl, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy1; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1 , 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, Cl- C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C7-C20 alkyl, a C7-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialky larnino, or a pharmaceutically acceptable salt thereof.

[0009] Also disclosed are lipid nanoparticle (LNP) formulations comprising a plurality of nanoparticles, wherein each nanoparticle comprises a phospholipid and a disclosed compound.

[0010] Also disclosed are pharmaceutical compositions comprising a disclosed LNP formulation and a pharmaceutically acceptable carrier.

[0011] Also disclosed are methods for delivering an mRNA into a cell, the method comprising contacting the cell with a disclosed pharmaceutical composition.

[0012] Also disclosed are methods for delivering an mRNA into a subject in need thereof, the method comprising administering to the subject an effective amount of a disclosed pharmaceutical composition .

[0013] Also disclosed are kits comprising a disclosed pharmaceutical composition, and one or more selected from: (a) instructions for delivering an mRNA; and (b) an agent selected from an antiviral agent and a chemotherapeutic agent.

[0014] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.

[0016] FIG, 1 A-H show a representative schematic of a multicomponent reaction and the corresponding products.

[0017] FIG. 2A-H show a representative schematic of lipid nanoparticle (LNP) preparation and the corresponding physicochemical characterization and in vivo screening of THP LNPs.

[0018] FIG. 3 A-C show representative data illustrating the validation and evaluation of intramuscular versus intravenous administration of THP 1 LNPs formulations.

[0019] FIG. 4 A -D show representative data illustrating the pharmacokinetics and toxicity of THP1 LNPs.

[0020] FIG. 5A-C show representative data indicating that THP1 enabled highly efficient mRNA delivery and tissue-specific tdTomato expression in the liver.

[0021] FIG. 6 shows representative data illustrating the in vitro delivery efficacy of luciferase mRNA THP LNPs in HEK293 cells.

[0022] FIG. 7 show's representative data illustrating the in vitro delivery efficacy of luciferase mRNA THP LNPs in RAW 264.7 cells.

[0023] FIG. 8A-E show twenty-six exemplary THP ionizable lipids grouped into 5 batches depending on their chemical structure.

[0024] FIG. 9 shows the chemical structures of the different phospholipids used for formulating the LNPs.

[0025] FIG. 10 shows representative data illustrating the body weight of mice treated with THP1 POPE and DLin-MC3-DMA LNPs at a dose of 0.5 mg / kg (i.v.) for 21 days.

[0026] FIG. 11A and FIG. I IB show representative pKa measurements ofTHPl DOPE and MC3 nanoparticles using TNS fluorescence assay.

[0027] FIG. 12 shows representative data from the robustness study for THP1 to check for variation in transfection with different batches of synthesized THP1.

[0028] FIG. 13 shows representative data illustrating IVIS images and a graphical representation of the total flux 24 h after intravenous injection of FLuc mRNA-loaded THP1 POPE LNPs in male mice.

[0029] FIG. 14A and FIG. I4B show representative data from the stability study ofTHPl LNPs.

[0030] FIG. 15 shows representative data showing the size (in nm) of mTHP mRNA LNPs. Size was measured using dynamic light scatering (DLS).

[0031] FIG. 16 shows representative data showing the poly dispersity index (PDI) of mTHP mRNA LNPs.

[0032] FIG. 17 shows representative data illustrating the in vitro delivery efficacy of luciferase mRNA mTHP LNPs in HEK293 cells (150 ng mRNA per well, 96 well plate). Luminescence intensity was quantified 24 h after adding LNPs.

[0033] FIG. 18 show's representative in vivo imaging system (IVIS) images at 18 h postinjection and graphical representation of total flux of Flue mRNA mTHP LNP pool based on six different diesters (DI, D2, D3, D4, D5, and D6). Dlin-MC3 DMA was used as a control. C57BL / 6 mice were injected intravenously w'ith 0.5 mg kg - 1 of pooled mTHP LNPs. (n:::2 biologically independent mice, ± SD). Organs are arranged left to right as: brain, heart, lung, spleen, pancreas, liver, spleen, and kidneys.

[0034] FIG. 19 shows representative IVIS images at 24 h post-injection and graphical representation of total flux of FLuc mRNA mTHP LNP pool based on R1 amines (CIO, C12, and Cl 6). Liver targeting DI and D4 LNPs were pooled and batched based on R1 amines (CIO, C12, and C16) and injected intravenously. Dlin-MC3-DMA was used as a control. C57BL / 6 mice were injected intravenously with 0.5 mg kg-1 of pooled mTHP LNPs. (n = 2 biologically independent mice, ± SD). Organs are arranged left to right as: brain, heart, lung, spleen, pancreas, liver, spleen, and kidneys.

[0035] FIG. 20 shows representative IVIS images at 18 h post-injection and graphical representation of total flux of Flue mRNA mTHP LNP pool based on R2 amines (Al, A2, A3, A4, A5, and A6). Liver targeting DI and D4 with C16 LNPs were pooled and batched based on R2 amines (Al, A2, A3, A4, A5, and A6) and injected intravenously. Dlin-MC3- DMA was used as a control. C57BL / 6 mice were injected intravenously with 0.5 mg kg-1 of pooled mTHP LNPs. (n:::2 biologically independent mice, i SD). Organs are arranged left to right as: brain, heart, lung, spleen, pancreas, liver, spleen, and kidneys.

[0036] FIG. 21 A shows representative IVIS images at 18 h post-injection and graphical representation of total flux of mTHP14 (D1C16A2) and mTHP68 (D4C16A2) Flue mRNA LNPs were injected intravenously. Dlin-MC3-DMA was used as a control. C57BL / 6 mice were injected intravenously with 0.5 mg kg-1 mTHP 14 and 68 LNPs. (n = 2 biologically independent mice, ± SD). Organs are arranged left to right as: brain, heart, lung, spleen, pancreas, liver, spleen, and kidneys.

[0037] FIG. 21B shows the structures of mTHP14 (D1C16A2) and mTHP68 (D4C16A2).

[0038] FIG. 22 shows a representative 'HNMR spectra of dioctyl l-(2-(lH-imidazol-4- yl)ethyl)-3-hexadecyl-l,2,3,6~tetrahydropyrimidme-4,5-dicarboxylate (mTHP 14).

[0039] Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and atained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.DETAILED DESCRIPTION

[0040] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0041] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0042] While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0043] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.A. DEFINITIONS

[0044] As used in the specification and the appended claims, the singular forms “a,” "‘an’1and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.

[0045] As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0046] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0047] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and oilier factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0048] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0049] A weight percent (wi. %) of a component, unless specifically stated to the contrary', is based on the total weight of the formulation or composition in which the component is included.

[0050] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0051] As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g. , a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0052] As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below . The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g. , a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary', individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0053] In defining various terms, “A1,” “A2,” “Ad,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

[0054] Hie term “aliphatic” or “aliphatic group,” as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spirofused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-20 carbon atoms. Aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl .

[0055] Tire terms “alkyl” and “alkylene” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s -pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl , hexadecyl, eicosyl, tetracosyl, and the like. Tire alkyl group can be cyclic or acyclic. Tire alkyl group can be branched or unbranched. The alkyl group can also be substituted or unsubstituted. For example, the alkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein. A “lower alkyl” group is an alkyl group containing from one to six (e.g., from one to four) carbon atoms. The term alkyl group can also be a C l alkyl, C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl, and the like up to and including a C 1 -C24 alkyl.

[0056] Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group.For example, the term “halogenated alkyl” or “haloalkyl’ specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. Alternatively, the term “rnonohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g. fluorine, chlorine, bromine, or iodine. The term “polyhaloalkyl” specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon . The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “aminoalkyl” specifically refers to rm alkyl group that is substituted with one or more amino groups. The term “hydroxyalkyl” specifically refers to rm alkyl group that is substituted with one or more hydroxy groups. When “alkyl” is used in one instance and a specific term such as “hydroxy alkyl” is used inanother, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “hydroxyalkyl” and the like .

[0057] This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenyl alcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that tire general term does not also include the specific term.

[0058] The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is a type of cycloalkyl group as defined above, and is included within the meaning of the term “cycloalkyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0059] The term “polyalkylene group” as used herein is a group having two or more CH’ groups linked to one another. The polyalkylene group can be represented by the formula — (CH2)a— , where “a” is an integer of from 2 to 500.

[0060] The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as --OA1where A1is alkyl or cycloalkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as — OA1— OA2or — OA1---(OA2)a --- ()A3, where “a” is an integer of from 1 to 200 and A1, A2, and A3are alkyl and / or cycloalkyl groups.

[0061] The terms “alkenyl” and “alkenylene” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A1A2)C=C(A3A4) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. Thealkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyi, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0062] The term ■‘cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one carbon-carbon double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, norbornenyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyi, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, ammo, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0063] The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be unsubstituted or substituted with one or more groups including, but not limited to, alkyl, cycloalkyi, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, as described herein.

[0064] The term “cycloalkynyl” as used herein is a non-aromatic carbon-based ring composed of at least seven carbon atoms and containing at least one carbon-carbon triple bound. Examples of cycloalkynyl groups include, but are not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and the like. The term “heterocycloalkynyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkynyl,” where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkynyl group and heterocycloalkynyl group can be substituted or unsubstituted. The cycloalkynyl group and heterocycloalkynyl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyi, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

[0065] The term “aromatic group” as used herein refers to a ring structure having cyclic clouds of delocalized re electrons above and below the plane of the molecule, where the n clouds contain (4n+2 ) re electrons. A further discussion of aromaticity' is found in Morrison and Boyd, Organic Chemistry, (5th Ed., 1987), Chapter 13, entitled “Aromaticity,” pages 477-497, incorporated herein by reference. The term “aromatic group” is inclusive of both aryl and heteroaryl groups.

[0066] The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, anthracene, and the like. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyk cycloalkynyl, aryl, heteroaryl, aldehyde, — NH2, carboxylic acid, ester, ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol as described herein. The term “biatyl” is a specific type of aryl group and is included in the definition of “aryl.” In addition, the aryl group can be a single ring structure or comprise multiple ring structures that are either fused ring structures or attached via one or more bridging groups such as a carboncarbon bond. For example, biaryl can be two and groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

[0067] The term “aldehyde” as used herein is represented by the formula - -- C(O)H. Throughout this specification “C(O)” is a short hand notation for a carbonyl group, i.e., C=O.

[0068] The terms “amine” or “amino” as used herein are represented by the formula — NA kA2, where A1and A2can be, independently, hydrogen or alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. A specific example of amino is —NHz.

[0069] The term “alkylamino” as used herein is represented by the formula — NH(-alkyl) where alkyl is a described herein. Representative examples include, but are not limited to, methylamino group, ethylamino group, propylamino group, isopropylamino group, butylamino group, isobutylamino group, (sec-butyl)amino group, (tert-butyl)amino group, pentylamino group, isopentylamino group, (tert-pentyl)amino group, hexylamino group, and the like.

[0070] The term “dialkylamino” as used herein is represented by the formula --N(-alkyl)2 w here alkyl is a described herein. Representative examples include, but are not limited to,dimethylamino group, diethylamino group, dipropylamino group, diisopropylamino group, dibutylamino group, diisobutylamino group, di(sec-butyl)amino group, di(tert-butyd)amino group, dipentylamino group, diisopentylamino group, di(tert-pentyl)ammo group, dihexylamino group, N-ethyl-N-methylamino group, N-methyl-N-propylamino group, N- ethyl-N-propylamino group and the like.

[0071] The term “carboxylic acid” as used herein is represented by the formula — C(O)OH.

[0072] The term “ester” as used herein is represented by the formula — OC(O)A!or — C(O)OA\ where A!can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “polyester” as used herein is represented by the formula — (A1O(O)C-A2-C(O)O)3— or — (A!O(O)C-A2-OC(O))a— , where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer from 1 to 500. “Polyester” is as the term used to describe a group that is produced by the reaction between a compound having at least two carboxy lic acid groups with a compound having at least two hydroxyl groups.

[0073] The term “ether” as used herein is represented by the formula A1OA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein. The term “polyether” as used herein is represented by the formula — (A1O-A2O)a— , 'where A!and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and “a” is an integer of from 1 to 500. Examples of polyether groups include polyethylene oxide, polypropylene oxide, and polybutylene oxide.

[0074] The terms “halo,” “halogen,” or “halide” as used herein can be used interchangeably and refer to F, Cl, Br, or I.

[0075] The terms “pseudohalide,” “pseudohalogen,” or “pseudohalo” as used herein can be used interchangeably and refer to functional groups that behave substantially similar to halides. Such functional groups include, by way of example, cyano, thiocyanate, azido, trifluoromethyl, trifluoromethoxy, perfluoroalkyl, and perfluoroalkoxy groups.

[0076] The term “heteroalkyl,” as used herein refers to an alkyl group containing at least one heteroatom . Suitable heteroatoms include, but are not limited to, O, N, Si, P and S, wherein the nitrogen, phosphorous and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. Heteroalkyls can be substituted as defined above for alkyl groups.

[0077] Hie term “heteroaryl,” as used herein refers to an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. The heteroaryl group can be substituted or unsubstituted. The heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol as described herein. Heteroaryl groups can be monocyclic, or alternatively fused ring systems. Heteroaryl groups include, but are not limited to, furyl, imidazolyl, pyrimidinyl, tetrazolyl, thienyl, pyridinyl, pyrrolyl, JV-methylpyrrolyl, quinolinyl, isoquinolinyl, pyrazolyl, triazolyl, thiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, isothiazolyl, pyridazinyl, pyrazinyl, benzofuranyl, benzodioxolyl, benzothiophenyl, indolyl, indazolyl, benzimidazolyl, imidazopyridinyl, pyrazolopyridinyl, and pyrazolopyrimidinyl . Further not limiting examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, pyrazolyl, imidazolyl, benzo [rij oxazolyl, benzo[<fjthiazolyl, quinolinyl, quinazolinyl, indazolyl, imidazo[l,2-b]pyridazinyl, imidazo[l,2-a]pyrazinyl, benzo[c][l ,2,5]thiadiazolyl, benzo[c][l,2,5]oxadiazolyl, and pyrido[2,3-b]pyrazinyl.

[0078] The terms “heterocycle” or “heterocyclyl,” as used herein can be used interchangeably and refer to single and multi-cyclic aromatic or non-aromatic ring systems in which at least one of the ring members is other than carbon. Thus, the term is inclusive of, but not limited to, “heterocycloalkyl,” “heteroaryl,” “bicyclic heterocycle” and “polycyclic heterocycle ” Heterocycle includes pyridine, pyrimidine, furan, thiophene, pyrrole, isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, oxazole, including, 1,2,3- oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole, thiadiazole, including, 1,2,3 -thiadiazole, 1,2,5-thiadiazole, and 1, 3, 4-thiadiazole, triazole, including, 1,2,3-triazoIe, 1,3,4-triazole, tetrazole, including 1,2,3,4-tetrazole and 1,2,4,5-tetrazole, pyridazine, pyrazine, triazine, including 1,2,4-triazine and 1,3,5-triazine, tetrazine, including 1, 2,4,5 -tetrazine, pyrrolidine, piperidine, piperazine, morpholine, azetidine, tetrahydropyran, tetrahydrofuran, dioxane, and the like. The term heterocyclyl group can also be a C2 heterocyclyl, C2-C3 heterocyclyl, C2- C4 heterocyclyl, C2-C5 heterocyclyl, C2-C6 heterocyclyl, C2-C7 heterocyclyl, C2-C8 heterocyclyl, C2-C9 heterocyclyl, C2-C10 heterocyclyl, C2-C11 heterocyclyl, and the like up to and including a C2-C18 heterocyclyl. For example, a C2 heterocyclyl comprises a group which has two carbon atoms and at least one heteroatom, including, but not limited to, aziridinyl, diazetidinyl, dihydrodi azetyl, oxiranyl, thiiranyl, and the like. Alternatively, forexample, a C5 heterocyclyl comprises a group which has five carbon atoms and at least one heteroatom, including, but not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, diazepanyl, pyridinyl, and the like. It is understood that a heterocyclyl group may be bound either through a heteroatom in the ring, where chemically possible, or one of carbons comprising the heterocyclyl ring.

[0079] The term “bicyclic heterocycle” or “bicyclic heterocyclyl,” as used herein refers to a ring system in which at least one of the ring members is other than carbon. Bicyclic heterocyclyl encompasses ring systems wherein an aromatic ring is fused with another aromatic ring, or wherein an aromatic ring is fused with a non-aromatic ring. Bicyclic heterocyclyl encompasses ring systems wherein a benzene ring is fused to a 5- or a 6- membered ring containing 1, 2 or 3 ring heteroatoms or wherein a pyridine ring is fused to a 5- or a 6-membered ring containing 1, 2 or 3 ring heteroatoms. Bicyclic heterocyclic groups include, but are not limited to, indolyl, indazolyl, pyrazolo[l,5-a]pyridinyl, benzofuranyl, quinolinyl, quinoxalinyl, 1,3-benzodioxolyl, 2,3-dihydro-l,4-benzodioxinyl, 3,4-dihydro-2H- chromenyl, lH~pyrazolo[4,3~c]pyridm-3-yl; lH-pyrrolo[3,2-b]pyridin~3-yl; and 1H- pyrazolo [3 ,2-b]pyridin-3 -y 1.

[0080] The term “heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated or hilly saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems. The heterocycloalkyl ring-systems include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolinyl, pyrazolidmyl, imidazolinyl, imidazolidmyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0081] The term “hydroxyl” or “hydroxyl” as used herein is represented by the formula — OH.

[0082] The term “ketone” as used herein is represented by the formula A’CCOfA2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0083] The term “azide” or “azido” as used herein is represented by the formula — N3.

[0084] The term “nitro” as used herein is represented by the formula --NO2.

[0085] The term “nitrile” or “cyano” as used herein is represented by the formula — -CN.

[0086] The term “silyl” as used herein is represented by the formula — SiA1A2AJ, where A1, A2, and A-’ can be, independently, hydrogen or an alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0087] The term “sulfo-oxo” as used herein is represented by the formulas — S(O)A\ — S(O)2A1, --OS(O)2A1, or --OS(O)2OA1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyi, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. Throughout this specification “S(O)” is a short hand notation for S=O. The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula — S(O)2A1, where A1can be hydrogen or an alkyl, cycloalkyl, alkenyl, cycloalkenyi, alkynyl, cycloalkynyl, aryl, or heteroaiyl group as described herein. The term “sulfone” as used herein is represented by the formula A5S(O)2A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyi, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. The term “sulfoxide” as used herein is represented by the formula AlS(O)A2, where A1and A2can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyi, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.

[0088] Tire term “thiol” as used herein is represented by the formula — SH.

[0089] “R1,” “R2,” “R2,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally' be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be atached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

[0090] As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogen of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those thatresult in the formation of stable or chemically feasible compounds. In is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0091] The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain aspects, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0092] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen;which may besubstituted with which may be substituted withwhich may be substituted with-1-pyridyl which may be substitutedbranched alkylene)O-N(R°)2; or — (C1-4 straight or branched alkylene)wherein each R° may be substituted as defined below and is independently hydrogen, Ci 6 aliphatic,, -CH2-(5-6 membered heteroaryl ring), or a 5-6- membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12- membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0093] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen,wherein each R® is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic,or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =8.[0094 j Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following:wherein each independent occurrence of R is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include:wherein each independent occurrence of R’ is selected from hydrogen. Ci s aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0095] Suitable substituents on the aliphatic group of R’ include halogen, -or -NO2, wherein each R® is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently Cj -4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0096] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -wherein each R1is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R1, taken together withtheir intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono--- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0097] Suitable substituents on the aliphatic group of RTare independently halogen, -or -NO2, wherein each R* is unsubstituted or where preceded by ’‘halo” is substituted only with one or more halogens, and is independently Cu aliphatic, or a5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0098] The term ‘leaving group” refers to an atom (or a group of atoms) with electron withdrawing ability that can be displaced as a stable species, taking with it the bonding electrons. Examples of suitable leaving groups include halides and sulfonate esters, including, but not limited to, triflate, mesylate, tosylate, and brosylate.

[0099] The terms “hydrolyzable group” and “hydrolyzable moiety” refer to a functional group capable of undergoing hydrolysis, e.g., under basic or acidic conditions. Examples of hydrolysable residues include, without limitation, acid halides, activated carboxylic acids, and various protecting groups known in the art (see, for example, “Protective Groups in Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience, 1999).

[0100] The term “organic residue” defines a carbon-containing residue, i.e., a residue comprising at least one carbon atom, and includes but is not limited to the carbon-containing groups, residues, or radicals defined hereinabove. Organic residues can contain various heteroatoms, or be bonded to another molecule through a heteroatom, including oxygen, nitrogen, sulfur, phosphorus, or the like. Examples of organic residues include but are not limited alkyl or substituted alkyls, alkoxy or substituted alkoxy, mono or di-substituted amino, amide groups, etc. Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15, carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In a further aspect, an organic residue can comprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbon atoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

[0101] A very close synonym of the term “residue” is the term “radical,” which as used in the specification and concluding claims, refers to a fragment, group, or substructure of a molecule described herein, regardless of how the molecule is prepared. For example, a 2,4- thiazolidinedione radical in a particular compound has the structure:regardless of whether thiazolidmedione is used to prepare tire compound. In some embodiments the radical (for example an alkyl) can be further modified (i.e., substituted alkyl) by having bonded thereto one or more “substituent radicals.” The number of atoms in a given radical is not critical to the present invention unless it is indicated to the contrary elsewhere herein.[00102 j “Organic radicals,” as the term is defined and used herein, contain one or more carbon atoms. An organic radical can have, for example, 1-26 carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms, 1-6 carbon atoms, or 1-4 carbon atoms, hi a further aspect, an organic radical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbon atoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organic radicals often have hydrogen bound to at least some of the carbon atoms of the organic radical. One example, of an organic radical that comprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2- naphthyl radical. In some embodiments, an organic radical can contain 1-10 inorganic heteroatoms bound thereto or therein, including halogens, oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organic radicals include but are not limited to an alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, mono-substituted amino, disubstituted amino, acyloxy, cyano, carboxy, carboalkoxy, alkylcarboxamide, substituted alkyl carboxamide, dialkylcarboxamide, substituted dia.lkylca.rboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl, thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl, substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclic radicals, wherein the terms are defined elsewhere herein. A few- non-limiting examples of organic radicals that include heteroatoms include alkoxy radicals, trifluorom ethoxy radicals, acetoxy radicals, dimethylamino radicals and the like.

[0103] Compounds described herein can contain one or more double bonds and, thus, potentially give rise to cis / trans (E / Z) isomers, as well as other conformational isomers. Unless stated to the contrary, the invention includes all such possible isomers, as well as mixtures of such isomers.

[0104] Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemicmixture. Compounds described herein can contain one or more asymmetric centers and, thus, potentially give rise to diastereomers and optical isomers. Unless stated to the contrary, the present invention includes ali such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof. Mixtures of stereoisomers, as well as isolated specific stereoisomers, are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.

[0105] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L. or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed withor d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non- superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist m different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the disclosed formulas, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Ingold-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[0106] When the disclosed compounds contain one chiral center, the compounds exist in twzo enantiomeric forms. Unless specifically stated to the contrary7, a disclosed compound includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixture. The enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts which may be separated, for example, by cry stallization (see, CRC Handbook of Optical Resolutions viaDiastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step can liberate the desired enantiomeric form. Alternatively, specific enantiomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.[00107 j Designation of a specific absolute configuration at a chiral carbon in a disclosed compound is understood to mean that the designated enantiomeric form of the compounds can be provided in enantiomeric excess (e.e). Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%, for example, greater than 60%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99%. In one aspect, the designated enantiomer is substantially free from the other enantiomer. For example, the "‘R” forms of the compounds can be substantially free from the “S” forms of the compounds and are, thus, in enantiomeric excess of the “S” forms. Conversely, “S” forms of the compounds can be substantially free of “R” forms of the compounds and are, thus, in enantiomeric excess of the “R” forms.

[0108] When a disclosed compound has two or more chiral carbons, it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to four optical isomers and two pairs of enantiomers ((S,S) / (R,R) and (R,S) / (S,R)). The pairs of enantiomers (e.g., (S,S) / (R,R)) are mirror image stereoisomers of one another. The stereoisomers that are not mirror-images (e.g., (S,S) and (R,S)) are diastereomers. The diastereoisomeric pairs can be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. Unless otherwise specifically excluded, a disclosed compound includes each diastereoi somer of such compounds and mixtures thereof.

[0109] The compounds according to this disclosure may form prodrugs at hydroxyl or amino functionalities using alkoxy, amino acids, etc., groups as the prodrug formingmoieties. For instance, the hydroxymethyl position may form mono-, di-, or triphosphates and again these phosphates can form prodrugs. Preparations of such prodrug derivatives are discussed in various literature sources (examples are: Alexander et al., J. Med, Chem. 1988, 31, 318; Aligas-Martin et ah, PCT WO 2000 / 041531, p. 30). The nitrogen function converted in preparing these derivatives is one (or more) of the nitrogen atoms of a compound of the disclosure.

[0110] “Derivatives” of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-active! y labeled forms, isomers, solvates and combinations thereof. The “combinations” mentioned in this context refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, and solvates. Examples of radio- actively labeled forms include compounds labeled with tritium, phosphorous-32, iodine- 129, carbon- 1 1, fluorine- 18, and the like.

[0111] Compounds described herein comprise atoms in both their natural isotopic abundance and in non-natural abundance. The disclosed compounds can be isotopically- labeled or isotopically-substituted compounds identical to those described, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, such as2H,3H,13C,14C,15N,18O,17O,35S,18F and36Cl, respectively. Compounds further comprise prodrugs thereof, and pharmaceutically acceptable salts of said compounds or of said prodrugs which contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labeled compounds of the present invention, for example those into which radioactive isotopes such as3’ H and34C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e..,3H, and carbon-14, i.e. ,14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically labeled compounds of the present invention and prodrugs thereof can generally be prepared by carrying out the procedures below, by substituting a readily available isotopically labeled reagent for a non- isotopically labeled reagent.

[0112] The compounds described in the invention can be present as a solvate. In some cases, the solvent used to prepare the solvate is an aqueous solution, and the solvate is then often referred to as a hydrate , The compounds can be present as a hydrate, which can be obtained, for example, by crystallization from a solvent or from aqueous solution. In this connection, one, two, three or any arbitrary number of solvent or water molecules can combine with the compounds according to the invention to form solvates and hydrates. Unless stated to the contrary, the invention includes all such possible solvates.

[0113] Tire term “co-crystal” means a physical association of two or more molecules which owe their stability through non-covalent interaction. One or more components of this molecular complex provide a stable framework in the crystalline lattice, hi certain instances, the guest molecules are incorporated in the crystalline lattice as anhydrates or solvates, see e.g. “Crystal Engineering of the Composition of Pharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a New Path to Improved Medicines?” Almarasson, O., et. al.. The Royal Society of Chemistry, 1889-1896, 2004. Examples of co-crystals include p- toluenesulfonic acid and benzenesulfonic acid,

[0114] It is also appreciated that certain compounds described herein can be present as an equilibrium of tautomers. For example, ketones with an a-hydrogen can exist in an equilibrium of the keto form and the enol fonn.keto form mol form amide form imidic acid form

[0115] Uikewise, amides with an N-hydrogen can exist in an equilibrium of the amide form and the imidic acid form. As another example, pyrazoles can exist in two tautomeric forms, N1-unsubstituted, 3-A3and N1-unsubstituted, 5-A3as shown below.Unless stated to the contrary, the invention includes all such possible tautomers.

[0116] It is known that chemical substances form solids, which are present in different states of order which are termed polymorphic forms or modifications. The differentmodifications of a polymorphic substance can differ greatly in their physical properties. The compounds according to the invention can be present in different polymorphic forms, with it being possible for particular modifications to be metastable. Unless stated to the contrary, the invention includes all such possible polymorphic forms.|00117] In some aspects, a structure of a compound can be represented by a formula:which is understood to be equivalent to a formula:wherein n is typically an integer. That is, R” is understood to represent five independent substituents, By “independent substituents,” it is meant that eachR substituent can be independently defined. For example, if in one instance is halogen,then is not necessarily halogen in that instance,

[0118] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Moms Plains, N.J,), Stem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the ait foliowing procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry' of Carbon Compound s, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).[00119 j Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly,where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: maters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.

[0120] Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and pennutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and even- combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.

[0121] It is understood that the compounds and compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.B. COMPOUNDS

[0122] In one aspect, the invention relates to tetrahydropyrimidine (THP) ionizable lipids and lipid nanoparticle (LNP) formulations. The disclosed compounds and L.NP formulations can be useful in the delivery of therapeutic agents such as, for example, mRNA for treatment of a viral infection, cancer, or a genetic disease or disorder.

[0123] It is contemplated that each disclosed derivative can be optionally further substituted. It is also contemplated that any one or more derivative can be optionally omitted from the invention. It is understood that a disclosed compound can be provided by the disclosed methods. It is also understood that the disclosed compounds can be employed in the disclosed methods of using.1. STRUCTURE

[0124] In one aspect, disclosed are compounds having a structure represented by a formula:

[0125] wherein each of R1 and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R" is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(Cl-C20 alkyl)NR10aR10b, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy1; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxy alkyl; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, - NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, CI-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)N R11aR11b, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R11aand R11bis independently selected from C 1-C4 alkyl and C1-C4 hydroxyalkyl; and wherein Cy2is selected from a 5 - to 7-membered heterocyclyl, and a 5 -membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH,-SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1 -C4) dialkylamino, or a pharmaceutically acceptable salt thereof. In various aspects, R2is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(Cl-C'20 alkylene )Cy\ and -(C2-C20 alkenylenejCy1and R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2,

[0126] In one aspect, disclosed are compounds having a a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R5is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenyIene)wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroary 1, and is substituted with 0, 1 , 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, Cl- C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(Cl-C'20 alkylene )Cy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof,

[0127] In various aspects, the compound has a structure represented by a formula:wherein each of R3and R4is a C8-C20 alkyl, or a pharmaceutically acceptable salt thereof.

[0128] hr various aspects, the compound has a structure represented by a formula:wherein R4is selected from -(C1-C20 alkylene)Cy2and -(C2-C20 alkenylene)Cy2, or a pharmaceutically acceptable salt thereof.

[0129] In various aspects, the the compound is selected from:or a pharmaceutically acceptable salt thereof.1001301 In various aspects, the compound is:or a pharmaceutically acceptable salt thereof.

[0131] In various aspects, the compound is:or a pharmaceutically acceptable salt thereof.

[0132] In various aspects, each of r and q is independently an integer selected from 2 to 8. In a further aspect, each of r and q is independently an integer selected from 3 to 8. In a still further aspect, each of r and q is independently an integer selected from 4 to 8. In yet a further aspect, each of r and q is independently an integer selected from 5 to 8. In an even further aspect, each of r and q is independently an integer selected from 6 to 8. In an even still further aspect, each of r and q is independently an integer selected from 7 to 8.

[0133] in various aspects, r is an integer selected from 2. to 8. In a further aspect, r is an integer selected from 3 to 8. In a still further aspect, r is an integer selected from 4 to 8. In yet a further aspect, r is an integer selected from 5 to 8. In an even further aspect, r is independently an integer selected from 6 to 8. In an even still further aspect, r is independently an integer selected from 7 to 8.

[0134] In various aspects, q is an integer selected from 2 to 8. In a further aspect, q is an integer selected from 3 to 8. In a still further aspect, q is an integer selected from 4 to 8.In yet a further aspect, q is an integer selected from 5 to 8. In an even further aspect, q is independently an integer selected from 6 to 8. In an even still further aspect, q is independently an integer selected from 7 to 8.

[0135] In various aspects, each of r and q is 8.

[0136] In various aspects, each of s and t is independently an integer selected from 2 to 8. In a further aspect, each of s and t is independently an integer selected from 3 to 8. In a still further aspect, each of s and t is independently an integer selected from 4 to 8. In yet a further aspect, each of s and t is independently an integer selected from 5 to 8. In an even further aspect, each of s and t is independently an integer selected from 6 to 8. In an even still further aspect, each of s and t is independently an integer selected from 7 to 8.

[0137] In various aspects, s is an integer selected from 2 to 8. In a further aspect, s is an integer selected from 3 to 8. In a still further aspect, s is an integer selected from 4 to 8. In yet a further aspect, s is an integer selected from 5 to 8. In an even further aspect, s is an integer selected from 6 to 8. In an even still further aspect, s is an integer selected from 7 to 8.

[0138] In various aspects, t is an integer selected from 2 to 8. In a further aspect, t is an integer selected from 3 to 8. In a still further aspect, t is an integer selected from 4 to 8. In yet a further aspect, t is an integer selected from 5 to 8. hr an even further aspect, t is an integer selected from 6 to 8. In an even still further aspect, t is an integer selected from 7 to 8.

[0139] In various aspects, each of s and t is 8. a. R1AND R2GROUPS

[0140] In one aspect, each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl. In a further aspect, each of R1and R2is independently selected from C2-C8 alkyl and C2-C8 alkenyl. In a still further aspect, each of R’ and R2is independently selected from C2-C4 alkyl and C2-C4 alkenyl. In yet a further aspect, each of R1and R2is independently selected from ethyl, propyl, isopropyl and 2-propene. In an even further aspect, each of R!and R2is ethyl.

[0141] hi one aspect, each of R1and R2is independently a C2-C21 alkyl, hi a further aspect, each of R1and R2is independently a C2-C8 alkyl. In a still further aspect, each of R1and R2is independently a C2-C4 alkyl. In yet a further aspect, each of R1and R2isindependently selected from ethyl, propyl, and isopropyl. In an even further aspect, each of R5and R2is ethyl.

[0142] In various aspects, each of R1and R2is ethyl. b. R3GROUPS

[0143] In one aspect, R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR10aR10b, -(C1-C20 alkylenelCy1, and -(C2-C20 alkenylenelCy1. In a further aspect, R3is selected from a C8-C18 alkyl, a C8-C18 alkenyl, a -(C8-C18 alkyl)NR1OaR!Ob, - (Cl- C18 alkylenelCy1, and -(C2-C18 alkenylene)Cy1. In a still further aspect, R3is selected from a C8-C16 alkyl, a C8-C16 alkenyl, a -(C8-C16 alkyl)NR10aR10b, -(C1-C16 alkylenelCy1, and -(C2-C16 alkenylenelCy1. In yet a further aspect, R5is selected from a C8-C14 alkyl, a C8- C14 alkenyl, a . — (C1-C14 alkyl)NRlOaRlub, -(C1-C14 alkylenelCy5, and -(C2-C14 alkenylenelCy1. In an even still further aspect, R3is selected from a C8-C12 alkyl, a C8-C12 alkenyl, a -(Cl-C12 alkyl)NR10aR10b, --(C1-C12 alkylenelCy1, and -(C2-C12 alkenylenelCy1. In an even still further aspect, R3is selected from a C8-C10 alkyl, a C8-C10 alkenyl, a-(C8- C10 alkyl)NR10aR10b, -(C1-C10 alkylenelCy1, and -(C2-C10 alkenylenelCy1. In yet an even further aspect, R' is selected from a C8-C10 alkyl, a C8-C10 alkenyl, a -(C8-C10 alkyl)NR103R10s’, -(C1-C6 alkylene)Cy5, and -(C2-C6 alkenylene)Cy1. In yet an even further aspect, R3is selected from a C1-C4 alkyl, a C1-C4 alkenyl, a -(C1-C4 alkyl)NR10aR5(>b, - (C1-C4 alkylene)Cy!, and -(C2-C4 alkenylenelCy1.

[0144] In one aspect, R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy!, and -(C2-C20 alkenylenelCy1. In a further aspect, R3is selected from a C8- C18 alkyl, a C8-C18 alkenyl, -(C1-C18 alkylenelCy5, and -(C2-C18 alkenylenelCy5, In a still further aspect, R" is selected from a C8-C16 alkyl, a C8-C16 alkenyl, -(Cl -C16 alkylenelCy1, and -(C2-C16 alkenylenelCy1. In yet a further aspect, R3is selected from a C8-C14 alkyl, a C8-C14 alkenyl, -(C1-C14 alkylenelCy5, and -(C2-C14 alkenylenelCy5. In an even still further aspect, R3is selected from a C8-C12 alkyl, a C8-C12 alkenyl, -(Cl -Cl 2. alkylenelCy1, and -(C2-C12 alkenylenelCy1. In an even still further aspect, R ' is selected from a C8-CI0 alkyl, a C8-C10 alkenyl, -(C1-C10 alkylene)Cy5, and -(C2-C10 alkenylenelCy1. In yet an even further aspect, R3is selected from a C8-C10 alkyl, a C8-C10 alkenyl, -(C1-C8 alkylene)Cy1, and -(C2-C8 alkenylenelCy1. In yet an even further aspect, R3is selected from a C8-C10 alkyl, a C8-C10 alkenyl, -(C1-C6 alkylenelCy1, and -(C2-C6alkenylene)Cy1. In yet an even further aspect, R3is selected from a C8-C10 alkyl, a C8-C10 alkenyl, -(C1-C4 alkylene)Cy’, and -(C2-C4 alkenylenejCy1.[0014S] In various aspects, R? is selected from a C8-C20 alkyl and a C8-C20 alkenyl. In a further aspect, Rf is selected from a C8-C 18 alkyl and a C8-C18 alkenyl. In a still further aspect. R' is selected from a C8-C16 alkyl and a C8-C16 alkenyl. In yet a further aspect, RJis selected from a C8-C14 alkyl and a C8-C14 alkenyl. In an even still further aspect, R2 is selected from a C8-C12 alkyl and a C8-C12 alkenyl. In an even still further aspect, R3is selected from a C8-CI0 alkyl and a C8-C10 alkenyl.

[0146] In various aspects, R3is a C8-C20 alkyl. Examples of C8-C20 alkanes include, but are not limited to, straight chain alkanes and branch chain alkanes. In a further aspect, R3is a C8-C 18 alkyl . In a still further aspect, R3is a C8-C 16 alkyl . In yet a further aspect, R3is a C8-C14 alkyl. In an even still further aspect, R3is a C8-C12 alkyl. In an even still further aspect, R' is C8-C10 alkyl. In yet an even further aspect, R3is a C8 alkyl.

[0147] In various aspects, R3is selected from:

[0148] In a further aspect, R3 is selected from:

[0149] In a further aspect, R3is:

[0150] In various aspects, R3is a C8-C20 alkenyl. Examples of C8-C20 alkenyls include, but are not limited to, straight chain alkenes, branched chain alkenes, terminal alkenes, internal alkenes, cis-alkenes, trans-alkenes, dienes, and trienes. In a further aspect, R3is a C8-C18 alkenyl. In a still further aspect, R3is a C8-C16 alkenyl. In yet a further aspect, R' is a C8-C14 alkenyl. In an even still further aspect, R3is a C8-C12 alkenyl. In an even still further aspect, R3is a C8-C10 alkenyl.

[0151] In various aspects, R3is selected from 3-octene, 4-octene, 5-octene, 6-octene,7-octene, 3-nonene, 4-nonene, 5-nonene, 6-nonene, 7 -nonene, 8-nonene, 3-decene, 4-decene, 5-decene, 6-decene, 7-decene, 8-decene, 9-decene, 3-undecene, 4-undecene, 5-undecene, 6- undecene, 7 -undecene, 8-undecene, 9-undecene, 10-undecene, 3-dodecene, 4-dodecene, 5- dodecene, 6-dodecene, 7-dodecene, 8-dodecene, 9-dodecene, 10-dodecene, 11-dodecene, 3- tridecene, 4-tridecene, 5-tridecene, 6-tridecene, 7-tridecene, 8-tridecene, 9-tridecene, 10- tridecene, 11-tridecene, 12-tridecene, 3 -tetradecene, 4-tetradecene, 5 -tetradecene, 6- tetradecene, 7 -tetradecene, 8-tetradecene, 9-tetradecene, I O-tetradecene, 11 -tetradecene, 12- tetradecene, 13 -tetradecene, 3 -pentadecene, 4-pentadecene, 5 -pentadecene, 6-pentadecene, 7- pentadecene, 8-pentadecene, 9-pentadecene, l()-pentadecene, 11 -pentadecene, 12- pentadecene, 13-pentadecene, 14-pentadecene, 3-hexadecence, 4-hexadecence, 5- hexadecence, 6-hexadecence, 7-hexadecence, 8-hexadecence, 9-hexadecence, 10- hexadecence, 11-hexadecence, 12-hexadecence, 13-hexadecence, 14-hexadecence, 15- hexadecence, 3 -heptadecene, 4-heptadecene, 5-heptadecene, 6-heptadecene, 7-heptadecene,8-heptadecene, 9-heptadecene, 10-heptadecene, 11 -heptadecene, 12-heptadecene, 13- heptadecene, 14-heptadecene, 15-heptadecene, 16-heptadecene, 3~octadecene, 4~octadecene, 5-octadecene, 6-octadecene, 7-octadecene, 8-octadecene, 9-octadecene, 10-octadecene, 1 1-octadecene, 12-octadecene, 13-octadecene, 14-octadecene, 15 -octadecene, 16-octadecene, 17-octadecene, 3-nonadecene, 4-nonadecene, 5-nonadecene, 6-nonadecene, 7-nonadecene, 8- nonadecene, 9-nonadecene, 10-nonadecene, 11 -nonadecene, 12-nonadecene, 13-nonadecene, 14-nonadecene, 15-nonadecene, 16-nonadecene, 17-nonadecene, 18-nonadecene, 3-icosene, 4-icosene, 5-icosene, 6-icosene, 7-icosene, 8-icosene, 9-icosene, 10-icosene, 11-icosene, 12- icosene, 13-icosene, 14-icosene, 15-icosene, 16-icosene, 17-icosene, 18-icosene, and 19- icosene. In a further aspect, R3 is selected from 4-octene, 5-decene, 6-dodecene, 7- tetradecene, 8-hexadecence, 9-octadecene, and 10-icosene.

[0152] In various aspects, the C8-C20 alkenyl has a structure represented by a formula:wherein each of r and q is independently an integer selected from 2. to 8.

[0153] In various aspects, R3is:

[0154] In various aspects, R3is selected from -(C1-C20 alkylene)Cy1and -(C2-C20 alkenylene)Cy1. In a further aspect, R3is selected from -(C l -C l 8 alkyleneJCy1and -(C2- C18 alkenyl eneJCy1. In a still further aspect, R3is selected from -(C1-C16 alkylene)Cy1and -(C2-C16 alkenylene)Cy1. In yet a further aspect, R3is selected from -(C1-C14 alkylene)Cy1and -(C2-C14 alkenylene)Cy1. In an even still further aspect, R3is selected from -(C1-C12 alkylene)Cy1and -(C2-C12 alkenylene)Cy1. In yet an even still further aspect, R' is selected from -(Cl -CIO alkyleneJCy1and -(C2-C10 alkenylene)Cy1. In a further aspect, R3is selected from -(C1-C8 alkylene)Cy1and -(C2-C8 alkenylene)Cy1. In a still further aspect, R3is selected from -(C1-C6 alkylene)Cy1and -(C2-C6 alkenylene)Cy1. In yet a further aspect, Rdis selected from -(C1-C5 alkylene)Cy1and -(C2-C5 alkenylene)Cy1. In an even further aspect, R3is selected from -(C1-C4 alkylene)Cy1and - (C2-C4 alkenylene)Cy1. In an even still further aspect, R3is selected from -(C1-C3 alkylene)Cy1and -(C2-C3 alkenylene)Cy1.

[0155] In various aspects, R3is -(C1-C20 alkylene)Cy1. In a further aspect, R3is - (C1-C18 alkylene)Cy1. In a stiil further aspect, R3is --(C1-C16 alkylene)Cy1. In yet a further aspect, R3is -(C1-C14 alkylene)Cy1. In an even still further aspect, R3is -(C1 -C12 alkylene)Cy1. In yet an even still further aspect, R3is -(Cl -Cl 0 alkylene)Cy1. In a further aspect, R3is -(C1-C8 alkylene)Cy1. In a still further aspect, R3is -(C1-C6 alkyleneJCy1. In yet a further aspect, R3is -(C1-C5 alkylene)Cy1. In an even further aspect, R3is -(C1-C4 alkylene)Cy1. In an even still further aspect, R3is -(C1-C3 alkyleneJCy5,

[0156] In various aspects, R3is -(C2-C20 alkenylene)Cy1. Tn a further aspect, R3is - (C2-C18 alkenylene)Cy1. In a still further aspect, R3is -(C2-C16 alkenylene)Cy1. In yet a further aspect, R3is -(C2-C14 alkenylene)Cy1. In an even still further aspect, R’ is -(C2-C12 alkenylene)Cy1. In yet an even stiil further aspect, R3is -(C2-C10 alkenyl eneJCy1. In a further aspect, R3is -(C2-C8 alkenyleneJCy1. In a still further aspect, R3is -(C2-C6 alkenyleneJCy1. In yet a further aspect, R3is -(C2-C5 alkenylene)Cy1. In an even further aspect, R3is -(C2-C4 alkenylene)Cy5. In an even still further aspect, R3is -(C2-C3 alkenyleneJCy5. c. R4GROUPS|00157[ In one aspect, R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a — (C 1- C2.0 alkyl)NR11aR11b’, -(C1-C20 alkyleneJCy2, and -(C2-C20 alkenyleneJCy2. In a further aspect, R4is selected from a C8-C18 alkyl, a C8-C18 alkenyl, a -(C8-C 18 alkyl)NR11aR11b, - (C1-C18 alkylene)Cy1, and -(C2-C18 alkenylene)Cy1. In a still further aspect, R4is selected from a C8-CI6 alkyl, a C8-C16 alkenyl, a -(C1-C16 alkylJNR11aR11b, -(C1-C16 alkyleneJCy5, and -(C2-C16 alkenylene)Cy1. In yet a further aspect, R4is selected from a C8-C14 alkyl, a C8-C14 alkenyl, a -(C1-C14 alkylJNR11aR11b, -(C1-C14 alkylene)Cy1, and - (C2-C14 alkenylene)Cy1. In an even still further aspect, R4is selected from a C1-C12 alkyl, a C1-C12 alkenyl, a --(Cl-C12 alkyl)NR11aR11b, -(C1-C12 alkylene)Cy1, and -(C2-C12 alkenyleneJCy5. In an even still further aspect, R4is selected from a C8-C10 alkyl, a C8-C10 alkenyl, a-(C8-C10 alkylJNRllaR11D, -(C8-C10 alkylene)Cy1, and -(C8-C10 alkenylene)Cy1. In yet an even further aspect, R4is selected from a C1-C8 alkyl, a C2-C8 alkenyl, a -(C1-C8 alkyl)NRllaRUb, -(C1-C8 alkylene)Cy1, and -(C2-C8 alkenyleneJCy5. In yet an even further aspect, R4is selected from a C1-C4 alkyl, a C2-C4 alkenyl, a -(C1 -C4 alkyl)NR11aR11b, - (C1-C4 alkylene)Cy1, and -(C2-C4 alkenyleneJCy1.

[0158] In one aspect, R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(Cl- C20 alkyleneJCy2, and -(C2-C20 alkenyleneJCy2. In a further aspect, R4is selected from a C8-C18 alkyl, a C8-C18 alkenyl, -(Cl -Cl 8 alkylene)Cy1, and -(C2-C18 alkenylene)Cy1. In a still further aspect, R4is selected from a C8-C16 alkyl, a C8-C16 alkenyl, -(CI-C16 alkylene)Cy1, and -(C2-C16 alkenylene)Cy1. In yet a further aspect, R4is selected from a C8-C14 alkyl, a C8-C14 alkenyl, -(C1-C14 alkylene)Cy1, and -(C2-C14 alkenylene)Cy3. In an even still further aspect, R4is selected from a C8-C12 alkyl, a C8-C12 alkenyl, -(Cl -Cl2 alkylene)Cy1, and -(C2-C12 alkenylene)Cy1. In an even still further aspect, R4is selected from a C8-C10 alkyl, a C8-C10 alkenyl, -(C1-C10 alkylene)Cy1, and -(C2-C10 alkenylene)Cy1. In yet an even further aspect, R4is selected from a C8-C10 alkyl, a C8-C10 alkenyl, -(C1-C8 alkylene)Cy1, and -(C2-C8 alkenylene)Cy1. In yet an even further aspect, R4is selected from a C8-C10 alkyl, a C8-C10 alkenyl, -(C1-C6 alkyleneJCy1, and -(C2-C6 alkenyleneJCy1. In yet an even further aspect, R4is selected from a C1-C4 alkyl, a C2-C4 alkenyl, -(C1-C4 alkylene)Cy1, and -(C2-C4 alkenylene)Cy1.

[0159] In various aspects, R4is selected from a C8-C20 alkyl and a C8-C20 alkenyl. In a further aspect, R4is a. C8-C18 alkyl and a. C8-C18 alkenyl. In a still further aspect, R4is selected from a C8-CI6 alkyl and a C8-C16 alkenyl. In yet a further aspect, R4is selected from a C8-C14 alkyl and a C8-C14 alkenyl. In an even still further aspect, R4is selected from a C8-C12 alkyl and a C8-C12 alkenyl. In an even still further aspect, R4is selected from a C8-C10 alkyl and a C8-C 10 alkenyl.

[0160] In various aspects, R4is a C8-C20 alkyl. In a further aspect, R4is a C8-C18 alkyl . In a still further aspect, R4is a C8-C 16 alkyl . In yet a further aspect, R4is a C8-C 14 alkyl . In an even still further aspect, R4is a C8-C12 alkyl . In an even still further aspect, R4is a C8-CI0 alkyl. In yet an even further aspect, R4is a C8 alkyl.

[0161] In various aspects, R4is selected from:

[0162] In a further aspect, R4is selected from:

[0163] In a further aspect, R4is:

[0164] In various aspects, R4is a C8-C20 alkenyl. In a further aspect, R4is a C8-C18 alkenyl. In a still further aspect, R4is a C8-C16 alkenyl. In yet a further aspect, R4is a C8- C14 alkenyl. In an even still further aspect, R4is a C8-C12 alkenyl. In an even still further aspect, R4is a C8-C10 alkenyl.

[0165] In various aspects, R4is a C8-C20 alkenyl. Examples of C8-C20 alkenyls include, but are not limited to, straight chain alkenes, branched chain alkenes, terminal alkenes, internal alkenes, cis-alkenes, trans-alkenes, dienes, and trienes. In a further aspect, R4is a C8-C 18 alkenyl. In a still further aspect, R4is a C8-C16 alkenyl. In yet a further aspect, R4is a C8-C14 alkenyl. In an even still further aspect, R4is a C8-C12 alkenyl. In an even still further aspect, R4is a C8-C10 alkenyl.

[0166] In various aspects, R4is selected from 3-octene, 4-octene, 5-octene, 6-octene,7-octene, 3-nonene, 4-nonene, 5-nonene, 6-nonene, 7-nonene, 8-nonene, 3-decene, 4-decene, 5-decene, 6-decene, 7-decene, 8-decene, 9-decene, 3-undecene, 4-undecene, 5-undecene, 6- undecene, 7-undecene, 8-undecene, 9-undecene, 10-undecene, 3-dodecene, 4-dodecene, 5- dodecene, 6-dodecene, 7 -dodecene, 8-dodecene, 9-dodecene, 10-dodecene, 11-dodecene, 3- tridecene, 4-tridecene, 5-tridecene, 6-tridecene, 7-tridecene, 8-tridecene, 9-tridecene, 10- tridecene, 11-tridecene, 12-tridecene, 3 -tetradecene, 4-tetradecene, 5-tetradecene, 6- tetradecene, 7-tetradecene, 8-tetradecene, 9-tetradecene, 10-tetradecene, 11 -tetradecene, 12- tetradecene, 13 -tetradecene, 3 -pentadecene, 4-pentadecene, 5-pentadecene, 6-pentadecene, 7- pentadecene, 8-pentadecene, 9-pentadecene, 10-pentadecene, 11 -pentadecene, 12- pentadecene, 13-pentadecene, 14-pentadecene, 3-hexadecence, 4-hexadecence, 5- hexadecence, 6-hexadecence, 7-hexadeeence, 8-hexadecence, 9-hexadecence, 10- hexadecence, 11-hexadecence, 12-hexadecence, 13-hexadecence, 14-hexadecence, 15- hexadecence, 3 -heptadecene, 4-heptadecene, 5 -heptadecene, 6-heptadecene, 7-heptadecene,8-heptadecene, 9-beptadecene, 10-heptadecene, 11 -heptadecene, 12-heptadecene, 13- heptadecene, 14-heptadecene, 15-heptadeeene, 16-heptadecene, 3-octadecene, 4-octadecene, 5-octadecene, 6-octadecene, 7-octadecene, 8-octadecene, 9-octadecene, 10-octadecene, 1 1- octadecene, 12-octadecene, 13-octadecene, 14-octadecene, 15 -octadecene, 16-octadecene, 17-octadecene, 3-nonadecene, 4-nonadecene, 5-nonadecene, 6-nonadecene, 7-nonadecene, 8- nonadecene, 9-nonadecene, 10-nonadecene, 11 -nonadecene, 12-nonadecene, 13 -nonadecene, 14-nonadecene, 15-nonadecene, 16-nonadecene, 17-nonadecene, 18-nonadecene, 3-icosene, 4-icosene, 5-icosene, 6-icosene, 7-icosene, 8-icosene, 9-icosene, 10-icosene, 11-icosene, 12- icosene, 13-icosene, 14-icosene, 15-icosene, 16-icosene, 17-icosene, 18-icosene, and 19- icosene. In a further aspect, R4is selected from 4-octene, 5-decene, 6-dodecene, 7- tetradecene, 8-hexadecence, 9-octadecene, and 10-icosene.

[0167] In various aspects, the C8-C20 alkenyl has a structure represented by a formula:wherein each of s and t is independently an integer selected from 2 to 8.

[0168] In various aspects, R4is:

[0169] In various aspects, R4is selected from -(C1-C20 alkyleneJCy2and -(C2-C20 alkenyleneJCy2In a further aspect, R4is selected from - (Cl -Cl 8 alkyleneJCy2and -(C2- C 18 alkenyleneJCy2In a still further aspect, R4is selected from -(C1-C16 alkylene)Cy2and -(C2-C16 alkenyleneJCy2. In yet a further aspect, R4is selected from —(C1 -C14 alkyleneJCy2and -(C2-C14 alkenyleneJCy2. In an even still further aspect, R4is selected from -(C1-C12 alkyleneJCy2and -(C2-C12 alkenyleneJCy2. In yet an even still further aspect, R4is selected from -(C 1 -CIO alkyleneJCy2and -(C2-C 10 alkenyleneJCy2. In a further aspect, R4is selected from -(Cl -C8 alkyleneJCy2and -(C2-C8 alkenyleneJCy2. In a still further aspect, R4is selected from -(Cl -C6 alkyleneJCy2and -(C2-C6 alkenyleneJCy2. In yet a further aspect, R4is selected from -(C1-C5 alkyleneJCy2and -(C2-C5 alkenyleneJCy2. In an even further aspect, R4is selected from -(C1-C4 alkyleneJCy2and - (C2-C4 alkenyleneJCy2. In an even still further aspect, R4is selected from -(Cl -C3 alkyleneJCy2and -(C2-C3 alkenyleneJCy2.

[0170] In various aspects, R4is ~(C 1-C20 alkyleneJCy2. In a further aspect, R4is - (C 1 -C 18 alkyleneJCy2. In a still further aspect, R4is -(C 1 -C 16 alkyleneJCy2. In yet a further aspect, R4is -(C1-C14 alkyleneJCy2. In an even still further aspect, R4is -(C1 -C12 alkyleneJCy2. In yet an even still further aspect, R4is -(C1-C10 alkyleneJCy2. In a further aspect, R4is -(C1-C8 alkyleneJCy2. In a still further aspect, R4is -(C1-C6 alkyleneJCy2. In yet a further aspect, R4is -(C1-C5 alkyleneJCy2, In an even further aspect, R4is -(Cl -C4 alkyleneJCy2. In an even still further aspect, R4is -(C1-C3 alkyleneJCy2. d. R10AANDR10B

[0171] In one aspect, each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl. In a further aspect, each of R10aand R10bis independently selected from methyl, ethyl, n-propyl, isopropyl, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, and -CH(CH3)CH2OH. In a still further aspect, each of R10aand R10bis independently selected from methyl, ethyl, -CH2OH, and -CH2CH2OH. In yet a further aspect, each of R10aand R10bis independently selected from methyl and -CH2OH.

[0172] In various aspects, each of R10aand R10b is independently C1-C4 hydroxyalkyl. In a further aspect, each of R10aand R10bis independently selected from -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, and -CH(CH3)CH2OH. In a still further aspect, each of R10aand R10bis independently selected from -CH2OH, and -CH2CH2OH. In yet a further aspect, each of R10aand R10bis -CH2OH.

[0173] In various aspects, each of R10aand R10bis independently C 1 -C4 alkyl. In a further aspect, each of R10aand R10bis independently selected from methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, each of R10aand R10bis independently selected from methyl and ethyl . In yet a further aspect, each of R10aand R10bis methyl. e. R11AAND R11B

[0174] In one aspect, each of R11aand R11bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl. In a further aspect, each of R11aand R11bis independently selected from methyl, ethyl, n-propyl, isopropyl, -CH2OH, -CH2CH2OH, -CH2CH2CH2OH, and ~CH(CH3)CH2OH. In a still further aspect, each of R11aand R11bis independently selected from methyl, ethyl, -- CH2OH, and --CH2CH2OH. In yet a further aspect, each of R11aand R11bis independently selected from methyl and -CH2OH.

[0175] In various aspects, each of R11aand R11bis independently C1-C4 hydroxyalkyl. In a further aspect, each of R11aand R11bis independently selected from -CH2OH, - CH2CH2OH, CH2CH2CH2OH, and -CH(CH3)CH2()H. In a still further aspect, each of R11aand R11bis independently selected from -CH2OH, and -CH2CH2OH. In yet a further aspect, each of R11aand R11bis is -CH2OH.

[0176] In various aspects, each of R11aand R11bis independently C 1-C4 alkyl . In a further aspect, each of R11aand R11bis independently selected from methyl, ethyl, n-propyl, and isopropyl. In a still further aspect, each of R11aand R11bis independently selected from methyl and ethyl. In yet a further aspect, each of R11aand R11bis methyl. f. CY1GROUPS

[0177] In one aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1 , 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalky 1, C1-C4 cyanoalkyl, C1-C4 hydroxy alkyl, C 1 -C4 haloalkoxy, C1- C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, or2 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1- C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a still further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0 or 1 group selected from halogen, -NO2, -CN, --OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1 -C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C 1 -C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialky lamino. In yet a further aspect, Cy!is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is monosubstituted with a group selected from halogen, -NO2, -CN, -OH, - SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, hr an even further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N- linked heteroaryl, and is un substituted.

[0178] In various aspects, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO 2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1 -C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. Examples of 5- to 7-membered N-linked heterocyclycls include, but are not limited to, pyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl. In a further aspect, Cy1is a 5- to 7- membered N-linked heterocyclyl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C 1 -C4 haloalkoxy, C1- C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a still further aspect, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted with 0 or 1 group selected from halogen, -NO2, -CN, -OH, - SH, -NH2, C1 -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Cy' is a 5- to 7-membered N-linked heterocyclyl monosubstituted with a group selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, Cl- C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an even further aspect, Cy!is an unsubstituted 5- to 7- membered N-linked heterocyclyl.

[0179] In various aspects, Cy1is a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, Cl- C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1 -C4 cyanoalkyl, C1-C4 hydroxyalky], C 1 -C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1 -C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialky lamino. Examples of N-linked heteroaryls include, but are not limited to, pyrrolyl, imidazolyl, and triazolyl. In a further aspect, Cy5is a 5-membered N-linked heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1 -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl. C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C 1 -C4)(C1 -C4) dialkylamino. In a still further aspect, Cy5is a 5-membered N-linked heteroary l, and is substituted with 0 or 1 group selected from halogen, -NO2, -CN,OH, SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol. C1-C4 aminoalkyl, C1-C4 alkylamino, and (Cl -C4)(C1 -C4) dialkylamino. In yet a further aspect, Cy1is a 5-membered N-linked heteroaryl monosubstituted with a group selected from halogen, -NO2, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, Cl- C4 alkoxy, C1-C4 thioalkyl, C1 -C4 alkylthiol, CI-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an even further aspect, Cy1is an unsubstituted 5- membered N-linked heteroaryl.

[0180] In various aspects, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, I, 2, or 3 groups independently selected from CI-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a further aspect, Cy1is selected from a 5- to 7- membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a still further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0 or 1 group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl.C 1 -C4 hydroxyalkyl, and C1-C4 aminoalkyl. In yet a further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is monosubstituted with a group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, Cl- C4 hydroxyalkyl, and C1-C4 aminoalkyl. In an even further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is unsubstituted.

[0181] In various aspects, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted with 0, 1, 2, or 3 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a further aspect, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted with 0, 1, or 2 groups independently selected from CI-C4 alkyl, C2.-C4 alkenyl, C2-C4 alkynyl, CI-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a still further aspect, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted with 0 or 1 group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, Cl- C4 hydroxyalkyl, and C 1-C4 aminoalkyl. In yet a further aspect, Cy5is a 5- to 7-membered N-lmked heterocyclyl monosubstituted with a group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1 -C4 aminoalkyl. In an even further aspect, Cy1is an unsubstituted 5- to 7-membered N-linked heterocyclyl.

[0182] In various aspects, Cy1is a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a further aspect, Cy1is a 5 -membered N- linked heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a still further aspect, Cy1is a 5-membered N-linked het.eroaryl, and is substituted with 0 or I group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In yet a further aspect, Cy1is a 5 -membered N-linked heteroaryl monosubstituted with a group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl. Cl- C4 hydroxyalkyl, and C1-C4 aminoalkyl. In an even further aspect, Cy1is an unsubstituted 5-membered N-linked heteroaryl.|00183] In various aspects, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0 or 1 methyl group. In a further aspect, Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5- membered N-linked heteroaiyl, and is substituted a methyl group. In a still further aspect, Cy1is selected from a 5 - to 7-membered N-linked heterocyclyl and a 5-rnernbered N-linked heteroaryl, and is unsubstituted.

[0184] In various aspects, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted with 0 or 1 methyl group. In a further aspect, Cy1is a 5- to 7-membered N-linked heterocyclyl, and is substituted a methyl group. In a still further aspect, Cy1is an unsubstituted 5- to 7-membered N-linked heterocyclyl.

[0185] In various aspects, Cy1is a 5-membered N-linked heteroaryl, and is substituted with 0 or 1 methyl group. In a further aspect, Cy1is a 5-membered N-linked heteroaryl, and is substituted a methyl group. In a still further aspect, Cy1is an unsubstituted 5-membered N- linked heteroaryl.

[0186] In various aspects, Cy1is selected from pyrrolidinyl, morpholinyl, piperazinyl, and imidazolyl, and is unsubstituted. g. CY2GROUPS

[0187] In one aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5- membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2.-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C 1 -C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, CI-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkyl amino. In a further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, CI-C4 hydroxyalkyl, CI-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, CI-C4 aminoalkyl, C1-C4 alkylamino, and (CI-C4)(CI-C4) dialkylamino. In a still further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is substituted with 0 or 1 group selected from halogen, - NOz, -CN, -OH, Si L -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CI-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5- membered heteroaryl, and is monosubstituted with a group selected from halogen, -NOz, - CN, -OH, -SH. -NH2. C1-C4 alkyl. C2-C4 alkenyl, C2-C4 alkynyl. C1-C4 haloalkyl, CI-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C 1 -C4)(C 1 -C4) dialkylamino. In aneven further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is unsubstituted.

[0188] In various aspects, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NHz, C1 -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. Examples of 5- to 7- membered N-linked heterocyclycls include, but are not limited to, pyrrolidinyl, piperidinyl, morpholinyl, and piperazinyl. In a further aspect, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -NO2, -CN, - OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1 -C4 alkoxy, C1 -C4 thioalkyl, C1 -C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a still further aspect, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0 or 1 group selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, CI -C4 hydroxyalkyl, CI -C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In yet a further aspect, Cy2is a 5- to 7- membered heterocyclyl monosubstituted with a group selected from halogen, -NO?, -CN, - OH, -SH, -KHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, CI-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an even further aspect, Cy2is an unsubstituted 5- to 7-membered heterocyclyl.

[0189] In various aspects, Cy2is a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, --NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1 -C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1 -C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. Examples ofN-linked heteroaryls include, but are not limited to, pyrrolyl, imidazolyl, and triazolyl. In a further aspect, Cy2is a 5-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In a still further aspect, Cy2is a 5-memberedheteroaryl, and is substituted with 0 or 1 group selected from halogen, -NO2, -CN, -OH, - SH, M b. C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, CI-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. Tn yet a further aspect, Cy2is a 5 -membered heteroaryl monosubstituted with a group selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1 -C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1 -C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino. In an even further aspect, Cy2is an unsubstituted 5 -membered heteroaryl.

[0190] In various aspects, Cy2is selected from a 5- to 7 -membered heterocyclyl and a 5- membered heteroaryl, and is substituted with 0, I, 2, or 3 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1 -C4 aminoalkyl . In a further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is substituted with 0, 1, or 2 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a still further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5 -membered heteroaryl, and is substituted with 0 or 1 group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In yet a further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is monosubstituted with a group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, Cl- C4 hydroxy alkyl, and Cl-C'4 arninoalkyl. In an even further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is unsubstituted.

[0191] In various aspects, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0, 1, 2, or 3 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and Cl-C'4 arninoalkyl. In a further aspect, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0, 1, or 2. groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 arninoalkyl. In a still further aspect, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0 or 1 group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 arninoalkyl. In yet a further aspect, Cy2is a 5- to 7-membered heterocyclyl monosubstituted with a group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In an even further aspect, Cy2is an unsubstituted 5- to 7-membered heterocyclyl.

[0192] In various aspects, Cy2is a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a further aspect, Cy2is a 5-membered heteroaryl, and is substituted with 0, 1 , or 2 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In a still further aspect, Cy2is a 5-membered heteroaryl, and is substituted with 0 or 1 group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl.In yet a further aspect, Cy2is a 5-membered heteroaryl monosubstituted with a group selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl. In an even further aspect, Cy2is an unsubstituted 5-membered heteroaryl.

[0193] In various aspects, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5- membered heteroaryl, and is substituted with 0 or 1 methyl group. In a further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is substituted a methyl group. In a still further aspect, Cy2is selected from a 5- to 7-membered heterocyclyl and a 5-membered heteroaryl, and is un substituted.

[0194] In various aspects, Cy2is a 5- to 7-membered heterocyclyl, and is substituted with 0 or 1 methyl group. In a further aspect, Cy2is a 5- to 7-membered heterocyclyl, and is substituted a methyl group. In a still further aspect, Cy2is an unsubstituted 5- to 7-membered heterocyclyl.

[0195] In various aspects, Cy2is a 5-membered heteroaryl, and is substituted with 0 or I methyl group. In a further aspect, Cy2is a 5-membered heteroaryl, and is substituted a methyl group. In a still further aspect, Cy2is an unsubstituted 5-membered heteroaryl,

[0196] In various aspects, Cy2is selected from pyrrolidinyl, morpholinyl, piperazinyl, and imidazolyl, and is substituted with 0 or 1 methyl group. In a further aspect, Cy2is selected from pyrrolidinyl, morpholinyl, piperazinyl, and imidazolyl, and is substituted a methyl group. In a still further aspect, Cy2is selected from pyrrolidinyl, morpholinyl, piperazinyl, and imidazolyl, and is unsubstituted.2. EXEMPLARY COMPOUNDS

[0197] In one aspect, a compound can be present as:or a pharmaceutically acceptable salt thereof.C. METHODS OF MAKING TETRAHYDROPYRMIDINE LIPID COMPOUNDS

[0198] The compounds of this invention can be prepared by employing reactions as shown in the following schemes, in addition to other standard manipulations that are known in the literature, exemplified in the experimental sections or clear to one skilled in the art. For clarity, examples having a single substituent are shown where multiple substituents are allowed under the definitions disclosed herein.

[0199] Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the following Reaction Schemes, as described and exemplified below. In certain specific examples, the disclosed compounds can be prepared by Route I, as described and exemplified below. The following examples are provided so that the invention might be more fully understood, are illustrative only, and should not be construed as limiting.1. ROUTE I

[0200] In one aspect, tetrahydropyrmidine lipid compounds can be prepared as shownbelow.SCHEME 1A.

[0201] Compounds are represented in generic form with substituents as noted in compound descriptions elsewhere herein. A more specific example is set forth below.SCHEME I B.

[0202] In one aspect, compounds of type 1.8, and similar compounds, can be prepared according to reaction Scheme IB above. Thus, compounds of type 1.8 can be prepared by reaction of an appropriate but-2-ynedioic acid diester, e.g., 1.5 as shown above, a first appropriate amine, e.g., 1.6 as shown above, a second appropriate amine, e.g., 1.7 as shownabove, and formaldehye. Appropiate but-2-ynedioic acid diesters and appropriate amines are commercially available or prepared by methods known to person of ordinary skill in the art. The reaction can be earned out in an appropriate solvent, e.g., dimethyl formamide, at an appropriate temperature, e.g. , 100 °C, for an appropriate period of time, e.g. , 3 h. As can be appreciated by one skilled in the art, the above reaction provides an example of a generalized approach wherein compounds similar in structure to the specific reactants above (compounds similar to compounds of type 1.1, 1.2, and 1.3), can be substituted in the reaction to provide tetrahydropyrmidine lipid compounds similar to Formula 1.4,D. LIPID NANOPARTICLE (LNP) FORMULATIONS

[0203] In one aspect, disclosed are lipid nanoparticle (LNP) formulations comprising a plurality' of nanoparticles, wherein each nanoparticle comprises a phospholipid and a disclosed compound.

[0204] Thus, in one aspect, disclosed are lipid nanoparticle (LNP) formulations comprising a plurality of nanoparticles, wherein each nanoparticle comprises a phospholipid and a compound having a structure represented by a formula:wherein each of Rland R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR1OaR10b, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)CyI; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxy alkyl; wherein Cy1is selected from a 5- to 7 -membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO?„ -CN, - OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalky 1, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylammo; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR11aR11b, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R11aand R11bis independently selected from C1-C4 alkyl and C1-C4 hydroxy alkyl; and wherein Cyzis selected from a 5 - to 7-membered heterocyclyl, and a 5 -membered heteroaryl, and issubstituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0205] In one aspect, disclosed are lipid nanoparticle (LNP) formulations comprising a plurality of nanoparticles, wherein each nanoparticle comprises a phospholipid and a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylenejCy1, and -(C2-C20 alkenylenelCy1; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1 , 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, Cl-C-4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, Cl- C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylenelCy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0206] In various aspects, the phospholipid is selected from 1 ,2-dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamme (POPE), 1 -stearoyl -2~oleoyl-sn-glycero-3 -phosphoethanolamine (SOPE), l-stearoyl-2- oleoyl-sn-glycero-3-phosphocholine (SOPC), l,2-dioieoyl-3 -trimethylammonium propane (DOTAP), and l,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4Me).

[0207] In various aspects, the phospholipid is POPE.

[0208] In various aspects, the phospholipid is present in an amount of from about 10 wt% to about 20 wt%. In a further aspect, the phospholipid is present in an amount of from about 11 wt% to about 19 wt%. In a still further aspect, the phospholipid is present in an amount of from about 12 wt% to about 18 wt%. In yet a further aspect, the phospholipid is present in an amount of from about 13 wt% to about 17 wt%. in an even further aspect, the phospholipid is present in an amount of from about 10 wt% to about 20 wt%.

[0209] In various aspects, the phospholipid is present in an amount of about 16 wt%.

[0210] In various aspects, LNP formulations further comprises cholesterol.

[0211] hr various aspects, cholesterol is present in an amount of from about 40 wt% to about 50 wt%. In a further aspect, cholesterol is present in an amount of from about 41 wt% to about 49 wt%. In a still further aspect, cholesterol is presen t in an amount of from about 42 wt% to about 48 wt%. In yet a further aspect, cholesterol is present in an amount of from about 43 wt% to about 47 wt%. In an even further aspect, cholesterol is present in an amount of from about 44 wt% to about 46 wt%.

[0212] In various aspects, cholesterol is present in an amount of about 47 wt%.

[0213] In various aspects, LNP formulations further comprises a PEGylated lipid.

[0214] hr various aspects, the PEGylated lipid is selected from 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol (DMG-PEG), l,2-distearoyl-sn-glycero-3~ phosphoetlianolamine-N-[amino(polyethylene glycol) (DSPE-PEG), 1,2-distearoyl-sn- Glycero-3 -Phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG- DSPE), l,2-dimyristoyl~sn~glycero~3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and l ,2~dioleoyl-sn-glycero-3-phosphoethanolamme-N-[amino(polyethylene glycol)] (DOPE), or pharmaceutically acceptable salts thereof.

[0215] In various aspects, the PEGylated lipid is DMG-PEG.

[0216] In various aspects, each nanoparticle comprises the phospholipid, a disclosed compound, cholesterol, and a PEGylated lipid.

[0217] In various aspects, the nanoparticle has a diameter of from about 80 nm to about 200 nm.

[0218]

[0219] In various aspects, the nanoparticle further comprises a messenger ribonucleic acid (mRNA) that encodes a protein or peptide, wherein the mRNA is encapsulated within the nanoparticle.

[0220] In various aspects, the protein or peptide is a viral antigen. In a still further aspect, the viral antigen is from a vims selected from human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), chicken pox, infectious mononucleosis, mumps, measles, rubella, shingles, ebola, viral gastroenteritis, viral hepatitis, viral meningitis, human metapneumovirus, human parainfluenza virus type 1, parainfluenza vims type 2, parainfluenza virus type 3, respiratory syncytial virus, viral pneumonia, Chikungunya virus (CHIKV), Venezuelan equine encephalitis (VEEV), dengue (DENV), influenza. West Nile virus (WNV), human coronavirus, and zika (ZIKV).The nanoparticle of claim 41, wherein the protein or peptide is a tumor antigen.

[0221] In various aspects, the protein or peptide is a tumor antigen. In a further aspect, the tumor antigen is selected from CD 19, BCMA, CD36, CD71 , CD41 a, C-D61 , CD4, and CD7.

[0222] In various aspects, the protein or peptide is a growth factor. In a further aspect, the growth factor is selected from an epidermal growth factor (EGF), a transforming growth factor (TGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a bone morphogenic protein (BMP), a hepatocyte growth factor (HGF), an insulinlike growth factor (IGF), a platelet derived growth factor (PDGF), and a keratinocyte growth factor (KGF) .

[0223] In various aspects, the protein or peptide is a gene-editing tool. In a further aspect, the gene-editing tool is selected from a zine-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), Cas9, Cas9 nickase, and Casl 2a.E, PHAMACEUTICAL COMPOSITIONS

[0224] In one aspect, disclosed are pharmaceutical compositions comprising a plurality of disclosed nanoparticles.

[0225] Thus, in one aspect, disclosed are pharmaceutical compositions comprising a nanoparticle comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NRIOaRlOb, -(Cl -C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy{; wherein each of R10aand R,0Bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, - OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylarnino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NRllaRllb, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R1,aand R, ,Bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; and wherein Cyzis selected from a 5 - to 7-membered heterocyclyl, and a 5 -membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, --NO2, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylarnino, or a pharmaceutically acceptable salt thereof

[0226] In one aspect, disclosed are pharmaceutical compositions comprising a nanoparticle comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R’ is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy!, and -(C2-C20 alkenylene)Cyl; wherein Cy’ is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, Cl- C4 alkylamino, and (Cl -C4)(C1 -C4) dialkylarnino; wherein R4is selected from a C8-C20alky], a C8-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0227] In various aspects, the plurality of nanoparticles has a poly dispersity index of less than about 0.2.

[0228] in various aspects, the plurality of nanoparticles has a zeta potential of from about 2 mV to about 6 mV.

[0229] In various aspects, the composition is formulated for intravenous administration, pulmonary administration, or intramuscular administrationF. DELIVERING AN MRNA INTO A CELL

[0230] In one aspect, disclosed are methods for delivering an mRNA into a cell, the method composing contacting the cell with a disclosed pharmaceutical composition.

[0231] Tirus, in one aspect, disclosed are methods for delivering an mRNA into a cell, the method comprising contacting the cell with a pharmaceutical composition comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C2.1 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR10aR10b, -(C1-C20 alkylene)Cyl, and -(C2-C20 alkenylene)Cy!; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N -linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, ~CN, - OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino;wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR11aR11b, “(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R1,aand R”” is independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; and wherein Cy2is selected from a 5 - to 7-membered heterocyclyl, and a 5 -membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, --NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyi, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1 -C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0232] In one aspect, disclosed are methods for delivering an mRNA into a cell, the method comprising contacting the cell with a pharmaceutical composition comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R’ is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cyl; wherein Cy’ is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyi, C1-C4 cyanoalkyl, C1-C4 hydroxy alkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, Cl- C4 alkylamino, and (C1 -C4)(C1 -C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyi, C1 -C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1 -C4 alkoxy, C1-C4 thioalkyl, C1 -C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0233] In various aspects, the cell exhibits aberrant expression or activity' of the protein or peptide that is encoded by the mRNA prior to the contacting step. In a further aspect, the aberrant expression or activity comprises expression of a non-fimctionai variant of the protein or peptide. In a still further aspect, the aberrant expression or activity is associated with a genetic disease or disorder. In yet a further aspect, the genetic disease or disorder is selected from cystic fibrosis, citrin deficiency, hemophilia, phenylketonuria, classic galactosemia, arginase 1 deficiency, Crigler-Najjar syndrome, alpha- 1 antitrypsin deficiency, ornithine transcarbamylase deficiency, progressive familial intrahepatic cholestasis type 3, familial hypercholesterolemia, thyhnalonic academia, Fabry disease, glycogen storage disease, acute intermittent porphyria, argininosuccinic aciduria, surfactant protein B deficiency, and propionic acidemia.

[0234] In various aspects, upon contacting the cell the pharmaceutical composition, the mRNA is expressed in the cell to produce a functional variant of the protein or peptide.

[0235] In various aspects, the functional variant of the protein or the peptide is produced m an amount that is greater than an amount of the functional vanant of the protein or peptide generated in the absence of the contacting step.

[0236] In various aspects, contacting is in vivo.

[0237] hi various aspects, the ceil is in a tissue or organ of a subject. In a further aspect, the tissue or organ is a functionally compromised tissue or organ. In a still further aspect, contacting comprises administering the pharmaceutical composition to the subject.

[0238] In various aspects, the method further comprises repeating the contacting step.

[0239] In various aspects, contacting comprises contacting a plurality of cells with the pharmaceutical composition. In a further aspect, upon contacting the plurality' of cells, the mRNA is expressed in at least 40% of the plurality of cells to produce a functional variant of the protein or peptide that is encoded by the mRNA.G. DELIVERING AN MRNA INTO A SUBJECT

[0240] hi one aspect, disclosed are methods for delivering an mRNA into a subject in need thereof, the method comprising contacting the cell with a disclosed pharmaceutical composition.

[0241] Thus, in one aspect, disclosed are methods for delivering an mRNA into a cell, the method comprising administering to the subject an effective amount a pharmaceutical composition comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3 is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR10aR10b, -(Cl -C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy1; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; wherein Cy1is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, - OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylammo, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR11aR11b, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R11aand R11bis independently selected from C1-C4 alkyl and C1-C4 hydroxy alkyl; and wherein Cyzis selected from a 5 - to 7-membered heterocyclyl, and a 5 -membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylammo, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof,

[0242] In one aspect, disclosed are methods for delivering an mRNA into a cell, the method comprising administering to the subject an effective amount a pharmaceutical composition comprising a compound having a structure represented by a formula:wherein each of Rland R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy1; wherein Cy1is selected from a 5- to 7-membered N-linkedheterocyclyl and a 5-membered N-linked heteroary 1, and is substituted with 0, 1 , 2, or 3 groups independently selected from halogen, --NO2, -CN, -OH, --SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haioaikyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1 -C4 alkoxy, C1 -C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1- C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haioaikyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1 -C4 aminoalkyl, C1-C4 alkylamino, and (C1 -C4)(C1 -C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

[0243] In various aspects, the subject is a mammal. In a further aspect, the mammal is a human.

[0244] In various aspects, prior to the administering step, the subject has been diagnosed as having a viral infection, cancer, or a genetic disease or disorder.

[0245] In varous aspects, the viral infection is due to a virus selected from human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), chicken pox, infectious mononucleosis, mumps, measles, rubella, shingles, ebola, viral gastroenteritis, viral hepatitis, viral meningitis, human metapneumovirus, human parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza vims type 3, respiratory syncytial virus, viral pneumonia, Chikungunya virus (CHIKV), Venezuelan equine encephalitis (VEEV), dengue (DENY), influenza. West Nile virus (WNV), human coronavirus, and zika (ZIKV).

[0246] In various aspects, cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma).

[0247] In various aspects, the genetic disease or disorder is selected from cystic fibrosis, citrin deficiency, hemophilia, phenylketonuria, classic galactosemia, arginase 1 deficiency, Crigler-Najjar syndrome, alpha- 1 antitrypsin deficiency, ornithinetranscarbamylase deficiency, progressive familial intrahepatic cholestasis type 3, familial hypercholesterolemia, thylmalonic academia, Fabry disease, glycogen storage disease, acute intermittent porphyria, argininosuccinic aciduria, surfactant protein B deficiency, and propionic acidemia.

[0248] In various aspects, the subject has been diagnosed with a need for treatment of a viral infection, cancer, or a genetic disease or disorder prior to the administering step.

[0249] In various aspects, the method further comprises the step of identifying a subject in need of treatment of a viral infection, cancer, or a genetic disease or disorder.

[0250] In various aspects, the method further comprises administering to the subject an effective amount of at least one agent selected from an antiviral agent and a chemotherapeutic agent. In a further aspect, the composition and the agent are administered simultaneously. In a still further aspect, the composition and the agent are administered simultaneously. In yet a further aspect, the composition and the agent are administered sequentially.

[0251] In various aspects, the antiviral agent, is selected from acemannan, acyclovir, acyclovir sodium, adamantanamine, adefovir, adenine arabinoside, alovudine, alvircept sudotox, amantadine hydrochloride, aranotin, arildone, atevirdine mesylate, avridine, cidofovir, cipamfylline, cytarabine hydrochloride, BMS 806, C31G, carrageenan, cellulose sulfate, cyclodextrins, dapivirine, delavirdine mesylate, desciclovir, dextrin 2-sulfate, didanosine, disoxaril, dolutegravir, edoxudine, enviradene, envirozime, etravirine, famciclovir, famotine hydrochloride, fiacitabine, fialuridine, fosarilate, foscarnet sodium, fosfonet sodium, FTC, ganciclovir, ganciclovir sodium, GSK 1265744, 9-2 -hydroxy-ethoxy methylguanine, ibalizumab, idoxuridine, interferon, 5-iodo-2'-deoxyuridine, IQP-0528, kethoxal, lamivudine, lobucavir, maraviroc, memotine pirodavir, penciclovir, raltegravir, ribavirin, rimantadine hydrochloride, rilpivirine (TMC-278), saquinavir mesylate, SCH-C, SCH-D, somantadine hydrochloride, sorivudine, statolon, stavudme, T20, tilorone hydrochloride, TMC120, TMC125, trifluridine, trifluorothymidine, tenofovir, tenofovir alefenamide, tenofovir disoproxyl fumarate, prodrugs of tenofovir, UC-781, UK-427, UK- 857, valacyclovir, valacyclovir hydrochloride, vidarabine, vidarabine phosphate, vidarabine sodium phosphate, viroxime, zalcitabene, zidovudine, and zinviroxime.

[0252] In various aspects, the chemotherapeutic agent is selected from an alkylating agent, an antimetabolite agent, an antineoplastic antibiotic agent, a mitotic inhibitor agent, and a mTor inhibitor agent.

[0253] In a further aspect, the antineoplastic antibiotic agent is selected from doxorubicin, mi toxan irone, bleomycin, daonorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt thereof.|00254] In a further aspect, the antimetabolite agent is selected from gemcitabine, 5- fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof

[0255] In a further aspect, the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharmaceutically acceptable salt thereof.

[0256] In a further aspect, the mitotic inhibitor agent is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or a pharmaceutically acceptable salt thereof.

[0257] In a further aspect, the mTor inhibitor agent is selected from everolimus, siroliumus, and ternsirolimus, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

[0258] In various aspects, administering is via intravenous administration, pulmonary' administration, or intramuscular administration.

[0259] In various aspects, the effective amount is a therapeutically effective amount.

[0260] In various aspects, the effective amount is a prophylactical ly effective amount.H. ADDITIONAL METHODS OF USING THE COMPOUNDS

[0261] The compounds and pharmaceutical compositions of the invention are useful in treatment of a viral infection, cancer, or a genetic disease or disorder.

[0262] To treat or control the disorder, the compounds and pharmaceutical compositions comprising the compounds are administered to a subject in need thereof, such as a vertebrate, e.g. , a mammal, a fish, a bird, a reptile, or an amphibian , The subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The subject is preferably a mammal, such as a human . Prior to administering the compounds or compositions, the subject can be diagnosed with a need for treatment of cancer.

[0263] The compounds or compositions can be administered to the subject according to any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermitent. A preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. A preparation can also be administered prophylactically; that is, administered for prevention of cancer.

[0264] The therapeutically effective amount or dosage of the compound can vary within wide limits. Such a dosage is adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg or more, a dailydosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although tire upper limit may be exceeded. lire daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, as a continuous infusion. Single dose compositions can contain such amounts or submultiples thereof of the compound or composition to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.1. USE OF COMPOUNDS

[0265] In one aspect, the invention relates to the use of a disclosed compound or a product of a disclosed method. In a further aspect, a use relates to the manufacture of a medicament for the treatment of a viral infection, cancer, or a genetic disease or disorder in a subject.

[0266] Also provided are the uses of the disclosed compounds and products. In one aspect, the invention relates to use of at least one disclosed compound; or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof. In a further aspect, the compound used is a product of a disclosed method of making.

[0267] In a further aspect, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, for use as a medicament.

[0268] In a further aspect, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, wherein a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of the compound or the product of a disclosed method of making.

[0269] In various aspects, the use relates to a treatment of viral infection in a subject. In one aspect, the use is characterized in that the subject is a human. In one aspect, the use is characterized in that the viral infection is due to a virus selected from human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), chicken pox, infectious mononucleosis, mumps, measles, rubella, shingles, ebola, viral gastroenteritis, viral hepatitis, viral meningitis, human metapneumovirus, human parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus, viral pneumonia, Chikungunya virus (CHIKV), Venezuelan equine encephalitis (VEEV), dengue (DENV), influenza, West Nile virus (WNV), human coronavirus, and zika (ZIKV).The nanoparticle of claim 41, wherein the protein or peptide is a tumor antigen.

[0270] In various aspects, the use relates to a treatment of cancer in a subject. In one aspect, the use is characterized in that the subject is a human. In one aspect, the use is characterized in that the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma).

[0271] In various aspects, the use relates to a treatment of a genetic disease or disorder in a subject. In one aspect, the use is characterized in that the subject is a human. In one aspect, the use is characterized in that the genetic disease or disorder is selected from cystic fibrosis, citrin deficiency, hemophilia, phenylketonuria, classic galactosemia, arginase1 deficiency, Crigler-Najjar syndrome, alpha- 1 antitrypsin deficiency, ornithine transcarbamylase deficiency, progressive familial intrahepatic cholestasis type 3, familial hypercholesterolemia, thylmalonic academia, Fabry disease, glycogen storage disease, acute intermittent porphyria, argininosuccinic aciduria, surfactant protein B deficiency, and propionic acidemia.

[0272] In a further aspect, the use relates to the manufacture of a medicament for the treatment of a viral infection, cancer, or a genetic disease or disorder in a subject,

[0273] It is understood that the disclosed uses can be employed in connection with the disclosed compounds, products of disclosed methods of making, methods, compositions, and kits. In a further aspect, the invention relates to the use of a disclosed compound or a disclosed product in the manufacture of a medicament for the treatment of a disorder of uncontrolled cellular proliferation in a mammal.2. MANUFACTURE OF A MEDICAMENT

[0274] In one aspect, the invention relates to a method for the manufacture of a medicament for treating viral infection, cancer, or a genetic disease or disorder in a subject having the disorder, the method comprising combining a therapeutically effective amount of a disclosed compound or product of a disclosed method with a pharmaceutically acceptable earner or diluent.

[0275] As regards these applications, the present method includes the administration to an animal, particularly a mammal, and more particularly a human, of a therapeutically effective amount of the compound effective in the treatment a viral infection, cancer, or a genetic disease or disorder. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the animal over a reasonable period. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition of the animal and the body weight of the animal.

[0276] The total amount of the compound of the present disclosure administered in a typical treatment is preferably between about 0.05 mg / kg and about 100 mg / kg of body weight tor mice, and more preferably between 0.05 mg / kg and about 50 mg / kg of body weight for mice, and between about 100 mg / kg and about 500 mg / kg of body weight, and more preferably between 200 mg / kg and about 400 mg / kg of body weight for humans per daily dose. This total amount is typically, but not necessarily, administered as a series ofsmaller doses over a period of about one time per day to about three times per day tor about 24 months, and preferably over a period of twice per day for about 12 months.

[0277] The size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of the compound and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations.

[0278] Thus, in one aspect, the invention relates to the manufacture of a medicament comprising combining a disclosed compound or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, with a pharmaceutically acceptable carrier or diluent.3. KITS

[0279] In one aspect, the invention relates to kits comprising the disclosed pharmaceutical composition, and one or more selected from: (a) instractions for delivering an mRNA; and (b) an agent selected from an antiviral agent and a chemotherapeutic agent.

[0280] Tirus, in one aspect, disclosed are kits comprising a pharmaceutical composition further comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein IV is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR10aR10b’, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy’!; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; wherein Cy!is selected from a 5- to 7-membered N-Iinked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, - OH, ~SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1 -C4 alkoxy, C1-C4 thioalkyl, C1 -C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (CT-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(C1-C20 alkyl)NR11 aR11b’,-(C1-C20 a1kylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R11aand R11bis independently selected from C1-C4 alkyl and C1-C4 hydroxyalkyl; and wherein Cy2is selected from a 5- to 7 -membered heterocyclyl, and a 5 -membered heteroaryl, and is substituted with 0, 1 , 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cy anoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof, and one or more selected from: instructions for delivering an mRNA; and an agent selected from an antiviral agent and a chemotherapeutic agent.

[0281] In one aspect, disclosed are kits comprising a pharmaceutical composition further comprising a compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3 is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy1; wherein Cy’ is selected from a 5- to 7-membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxy alkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1- C4 alkylamino, and (C1 -C4)(C1 -C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NOz, -CN, -OH, -SH, -NHz, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1 -C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1 -C4 alkoxy, C1-C4 thioalkyl, C1 -C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof, and one or more selected from: instructions fordelivering an mRNA; and an agent selected from an antiviral agent and a chemotherapeutic agent.

[0282] In various aspects, the composition and the agent are co-packaged.

[0283] The foregoing description illustrates and describes the disclosure.Additionally, the disclosure shows and describes only the preferred embodiments but, as mentioned above, it is to be understood that it is capable to use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the invention concepts as expressed herein, commensurate with the above teachings and / or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to explain best modes known by applicant and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the invention to the form disclosed herein. Also, it is intended to the appended claims be construed to include alternative embodiments.

[0284] All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between tire present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls.I. EXAMPLES

[0285] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are intended to be purely exemplary' of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0286] The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way. Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way.1. EXPERIMENTAL a. GENERAL INFORMATION

[0287] All starting materials used for the reaction were purchased from Sigma Aldrich and Ambeed. Unless otherwise mentioned, commercially available reagents were used without any further purification. The reactions were carried out using oven-dried glassware with magnetic stirring. Reactions were monitored by thin-layer chromatography (TLC) using precoated silica plates with the help of U V-light, and KMnCL stains. Column chromatography was performed on silica gel 60 -120 mesh) using EtOAc / Hexane or DCM / Methanol as eluents. Evaporation of solvents was done under reduced pressure at temperature less than 40 °C.1H and13C NMR spectra were recorded in CDCh solvent using 400 MHz spectrometers at ambient temperature. Chemical shifts δ and coupling constants J are given in ppm (parts per million) and Hz (hertz) respectively. Chemical shifts are reported relative to residual solvent as an internal standard (CDCh;1H: δ = 7.26 and13C: 3 = 77.16 ppm). Peak splitting patterns are designated as follows: s = singlet, d = doublet, dd = doublet of doublet, dt = doublet of triplet, t:=:triplet, q = quartet, m = multiplet. b. ABBREVIATIONS

[0288] LNP, Lipid Nanoparticle; mRNA, messenger RNA; MCR, multicomponent reaction; THP, tetrahydropyrimidine; DLin-MC3, DMA, 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z, 12Z)-9, 12-octadecadien-l-y 1-10, 13-nonadecadien-l-y 1 ester; COVID-19, Corona Virus disease of 2019; PEG, Polyethylene glycol; CRISPR, Clustered regularly interspaced palindromic repeats; DOPE, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine;DMG-PEG 2000, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000; FLuc, firefly luciferase; DSPC, l,2.-distearoyl-sn~glycero-3-phosphocholine; PDI, polydispersity index; PC, phosphatidylcholine; SOPC, 1 -stearoyl -2 -oleoyl-sn-glycero-3 -phosphocholine; PE, phosphatidylethanolainine; POPE, l-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine; SOPE, l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine;DOTAP, 1,2-dioleoyl-3- trimethylammonium; 4Me, l,2-di-O-phytanyl-sn-glycero-3- phosphoethanolamine; ALT, alanine transaminase; AST, Aspartate aminotransferase; FDR, False Discovery Rate; ApoE, apolipoprotein E; CAG, Chicken 0 Actin.c. SYNTHESIS i. GENERAL PROCEDURE FOR THE THREE COMPONENT MCR (ONE AMINE; R4NPb = R3NHz)

[0289] To a mixture of but-2-ynedioic acid diethyl ester (1 eq.) and R4NH2 (2. 1 eq.) in DMF (2 ml), formaldehyde (4 eq.) was added and resulting reaction mixture was stirred at 100 °C for 3 hours. After completion of reaction, it was diluted with water and extracted 3 times with 50 ml of EtOAc using separating funnel. The organic layer was dried over Na2SO4 and solvent was removed using rotary' evaporator to obtain crude product, which was purified by flash column chromatography to obtain pure product. ii. GENERAL PROCEDURE FOR THE FOUR COMPONENT MCR (TWO DIFFERENT AMINE; R4NH? AND R3NH1) ARE DIFFERENT)

[0290] To a mixture of but-2-ynedioic acid diethyl ester (1 eq.) and R4NH2 (1 eq.) in DMF (2 ml), formaldehyde (4 eq.) and R3NH2.) (1.1 eq.) was added and resulting reaction mixture was stirred at 100 °C for 3 hours. After completion of reaction, it was diluted with water and extracted 3 times with 50 ml of EtOAc using separating funnel. The organic layer was dried over Na2SO4, and solvent was removed using rotary evaporator to obtain crude product, which was purified by flash column chromatography to obtain pure product. iii. GENERAL PROCEDURE FOR THE SYNTHESIS OF DIESTERS

[0291] Six different diesters were synthesized by the esterification of acetylene dicarboxylic acid with six distinct alcohols. In a typical procedure, acetylene dicarboxylic acid (1.0 equiv.) and the respective alcohol (2.2 equiv.) were combined in dry toluene under an inert atmosphere. The reaction mixture was refluxed overnight to ensure complete esterification. Upon completion, the mixture was diluted with a saturated NaCl solution and extracted twice with Hexane (2 * 20 ml.,) using a separating funnel. The combined organic layers were dried over anhydrous sodium sulfate ( Na2SO4), filtered, and concentrated under reduced pressure using a rotary evaporator. The etude product was purified by flash column chromatography to afford the pure desired compound.iv. GENERAL PROCEDURE FOR THE FOUR-COMPONENT MULTICOMPONENT REACT! ON (MCR)

[0292] In a representative reaction, diester (1.0 equiv.) and the first amine, R'NHz (1.0 equiv.), were dissolved in ethanol (1 mL) and sonicated for 15 minutes. Subsequently, 37% aqueous formaldehyde (4.0 equiv.) and the second amine, R;NH? (1.0 equiv.), and AcOH (2equiv.) were added to the mixture. The resulting reaction mixture was sonicated for 8 hours. Upon completion, the mixture was concentrated under reduced pressure using a centrifugal rota-evaporator. The crude product was obtained. d. NMR DATA i. DIETHYL 1,3-DIOCTYL-1,2,3,6-TETRAHYDROPYR!M!BINE- 4,5-DICARBOXYLATE (THP1)

[0293] ' XMR (400 MHz, CDCl3) 54.35 (q, J ----- 7.2 Hz, 2H), 4.10 (q, J--- 7.1 Hz,2H), 3.96 (s, 2H), 3.51 (s, 2H), 3.06 - 2.97 (m, 2H), 2.59 - 2.50 (m, 2H), 1.57 - 1.48 (m, 4H), 1 .40 - 1.21 (m, 26H), 0.88 (td, J= 6.9, 1.7 Hz, 6H);!3C NMR (100 MHz, CDCh) 8 166.40, 165.05, 148.13, 91.44, 67.65, 61.93, 59.55, 52.95, 51.25, 48.48, 31.95, 31.88, 29.63, 29.39, 29.29, 28.03, 27.47, 26.85, 22.78, 22.73, 14.56, 14.21, 14.18, 14.08. FIRMS (m / z): [M+H] + calcd for C26H49N2O4453.3692, found 453.3692; 0 ppm. ii. DIETHYL 1, 3-DIDODECYL-1, 2,3,6- TETR / \HYDROPYRLVIIDINE-4,5-DICARBOXYLATE (THP2)

[0294] :H NMR (400 MHz, CDCl3) 54.35 (q, . / 7.2 Hz, 2H), 4.10 (q, J == 7.1 Hz, 2H), 3.97 (s, 2H), 3.51 (s, 2H), 3.03 - 3.00 (m, 2H), 2.56 - 2.52 (m, 2.H), 1.53 (s, 4H), 1.38 - 1.20 (m, 42H), 0.91 - 0.85 (m, 6H);13C NMR (100 MHz, CDCl3) 8 166.33, 164.98, 148.09, 91.42, 67.65, 61.80, 59.42, 52.90, 51.13, 48.46, 31.97, 31.96, 29.73, 29.70, 29.68, 29.62, 29.56, 2.9.41, 29.39, 29.36, 29.33, 28.02, 27.41, 26.78, 2.2.73, 22.73, 14.48, 14.15, 14.01.HRMS (m / z): [M+H] + calcd for C34H55N2O4565.4944, found 565.4944; 0 ppm. iii. DIETHYL 1,3-DIHEXADECYL-1, 2,3,6- TETRAHYDROPYRTMTDTNE-4,5-DICARBOXYLATE (THP3)

[0295] 'HNMR (400 MHz, CDCh) 54.35 (q, J= 7.2 Hz, 2H), 4.10 (q, J= 7.1 Hz,2.H), 3.96 (s, 2H), 3.51 (s, 2H), 3.03 - 3.00 (m, 2H), 2.56 - 2.52 (m, 2H), 1.53 - 1 .49 (m, 4H),1 .37 - 1 .20 (m, 58H), 0.92 - 0.84 (m, 6H);13C NMR (100 MHz, CDCh) 5 166.44, 165.09, 148.16, 91.49, 67.72, 61.91, 59.54, 52.99, 51.24, 48.54, 32.06, 29.84, 29.79, 29.77, 29.71, 29.65, 29.49, 29.45, 29.41, 28.11, 2.7.50, 26.87, 22.82, 14.57, 14,24, 14.10. HRMS (m / z): [M+H] + calcd for C42H81N2O4677.6196, found 677.6196; 0 ppm. iv. DIETHYL 1,3-DI((Z)-OCTADEC-9-EN-1-YL)-1, 2,3,6- TETRAHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP4)

[0296] SH NMR (400 MHz, CDCh) 55.38 - 5.30 (m, 4H), 4.36 (q, J= 7.2 Hz, 2.H), 4.10 (q, J= 7.1 Hz, 2H), 3.98 (s, 2H), 3.52 (s, 2H), 3.04 - 3.02 (m, 2H), 2.55 (s, 2H), 1.99 (dd, J- 12.3, 7.2 Hz, 8H), 1.54 (s, 4H), 1.38 - 1.24 (m, 51H), 0.90 - 0.86 (m, 6H);i3C NMR (100 MHz, CDCI3) 5 166.21 , 164.88, 147.96, 129.98, 129.92, 12.9.79, 129.68, 91.32, 67.48, 61.81 , 59.44, 52.80, 51.14, 48.30, 32.59, 31 .89, 2.9.75, 29.68, 29.64, 29.51, 29.42, 29.30, 29.26, 29.19, 27.85, 27.34, 27.20, 27.16, 26.72, 22.66, 14.42, 14.09, 13.96. HRMS (m / z): [M+H] + calcd for C46H85N2O4729.6509, found 729.6509; 0 ppm. v. DIETHYL 3-(3-(1H-IMIDAZOL-1-YL)PROPYL)-1-OCTYL- 1,2,3,6-TETRAHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP5)

[0297] 'l l NMR (400 MHz, CDCh) 5 7.48 (s, 1 H), 7.06 (d. J == 0.8 Hz, 1H). 6.90 (t, J = 1 .2 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.97 (t, J = 7.0 Hz, 2H), 3.83 (s, 2H), 3.43 (s, 2H), 3.09 - 2.98 (m, 2H), 2.53 - 2.44 (m, 2H), 2.04 (dt, J = 14.2, 7.0 Hz, 2H), 1.52 - 1.46 (m, , 21 1 ). 1.33 - 1.19 (m, 16H), 0.87 (t, J - 6.9 Hz, 3H);13C NMR (100 MHz, CDCh) 8 166.15, 165.22, 147.54, 137.01, 118.87. 94.84, 77.48, 77.16, 76.84, 68.19, 62.29, 59.90, 53.40, 48.68, 48.44, 44.59, 31 .97, 30.48, 29.63, 29.41, 27.94, 27.49, 22.79, 14.53, 14.24, 14.08. HRMS (m / z): [M+H] + calcd for C24H41N4O4449.3127, found 449.3127; 0 ppm. vi. DIETHYL 3-(3-(l H-IMIDAZOL4-YL)PROPYL)-1-DODECYL- 1,2,3,6-TETRAHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP6)

[0298] :1 1 NMR (400 MHz, CDCh) 8 7.47 (s, 1H). 7.06 (s, 1H), 6.90 (t, J ---- 1.2 Hz,1H), 4.28 (q, J= 7.2 Hz, 2.H), 4.10 (q, J = 7.1 Hz, 2H), 3.97 (t, J= 7.0 Hz, 2H), 3.83 (s, 2.H), 3.43 (s, 2H), 3.06 - 3.00 (m, 2H), 2.52 - 2.43 (m, 2H), 2.10 - 1.98 (m, 2H), 1 .57 - 1.44 (m.2H), 1.33 - 1.19 (m, 24H), 0.90 - 0.84 (m, 3H);13C NMR (100 MHz, CDCh) 5 166.16, 165.18, 147.55, 137.10, 129.83, 118.72, 94.68, 68.20, 62.24, 59.85, 53.36, 48.65, 48.43, 44.42, 32.04, 30.51 , 29.80, 29.77, 29.75, 29.68, 29.48, 27.99, 27.49, 22.81, 14.52, 14.25, 14.07.. HRMS (m / z): [M+H] + calcd for C28H49N4O4505.3753, found 505.3753; 0 ppm vii. DIETHYL 3-(3-( IH-IMTOAZOL-I-YPPROPYL)-1 - HEXA»ECYL-l,2,3,6-TETRAHYDROPYRIMroiNE-4,5- DiCARBOXYLATE (THP7)

[0299] 1H NMR (400 MHz, CDCh) 3 7.46 (s, 1H), 7.06 (s, 1H), 6.89 (t, J= 1 .2 Hz,1 H), 4.27 (q, ■ / 7.2 Hz, 2H), 4.10 (q, J 7.1 Hz, 2H), 3.97 (t, J ------ 7.0 Hz, 2H), 3.83 (s, 2H), 3.43 (s, 2H), 3.06 - 2.99 (m, 2H), 2.51 - 2.43 (m, 2H), 2.07 - 2.00 (m, 2H), 1.50 (s, 2H), 1.34 - 1.19 (m, 32H), 0.90 - 0.82 (m, 3H);13C NMR (100 MHz, CDCI3) 3 166.14, 165.18, 147.53, 137.06, 129.57, 118.76, 94.68, 53.35, 48.64, 48.42, 44.47, 32.04, 30.48, 29.82, 29.77, 29.75, 29.67, 29.48, 27.96, 27.49, 22.81, 14.51, 14.24, 14.05. HRMS (m / z): [MH 1] + calcd for C32H57N4O4 561 .4379, found 561 .4380; 0 ppm viii. DIETHYL (Z)-1-(3-(1H-IMIDAZOL-1-YL)PROPYL)-3-(OCT ADEC-9-EN- 1 - YL)- 1 ,2,3,6-TETRAHYDROPYRIMIDINE- 4,5-DiCARBOXYLATE (THP8)

[0300] 1H NMR (400 MHz, CDCh,) 8 7.53 (s, 1H), 7.08 (s, 1H), 6.93 (s, 1H), 5.35(dd, J= 9.1 , 3.4 Hz, 2H), 4.35 (q, .7= 7.1 Hz, 2H), 4.18 - 4.03 (m, 4H), 3.91 (s, 2H), 3.48 (s, 2H), 3.02 - 2.92 (m, 2H), 2.46 (t, J ------ 6.4 Hz, 2H), 1.98 (dt, J --- 13.0, 6.3 Hz, 6H), 1.49 (s, 2H), 1.43 - 1.10 (m, 28H), 0.87 (t, J = 6.7 Hz, 3H);!3C NMR (100 MHz. CDCl3) 8 166.31, 164,85, 148.12, 137.32, 130.11, 129.80, 128.96, 119.08, 90.78, 77.48, 77.16, 76.84, 68.27, 62.01 , 59.64, 51.19, 48.63, 47.45, 44.42, 31.98, 29.84, 29.80, 29.60, 29.53, 29.40, 29.28, 28.81, 27.30, 26.79, 22.76, 14.53, 14.20, 14.06. HRMS (m / z): [M+H] + calcd for C34H59N4O4 587.4536, found 587.4536; 0 ppm. ix. DIETHYL 3-(3-(1H-IMIDAZOL-1-YL)PROPYL)-1- TETR / \DECYL-1,2,3,6-TETR / \HYDROPYRIYIIDINE-4,5- DSCARBOXYLATE (THP9)

[0301] H NMR (400 MHz, CDCh,) 8 7.50 (s, 1 H), 7.08 (s, 1H), 6.91 (s, 1H), 4.2.9 (q, .7= 7.2, 7.2, 7.1 Hz, 2H), 4.11 (q, J= 7.1 , 7.1 , 7.1 Hz, 2H), 3.98 (t, J= 7.0, 7.0 Hz, 2H), 3.84(s, 2H), 3.44 (s, 2H), 3.07 - 3.01 (m, 2H), 2.52 - 2.46 (m, 2H), 2.05 (dd, J= 7.8, 6.4 Hz, 2H), 1.50 (d, J ----- 7.5 Hz, 2H). 1.32 (s, 4H), 1.24 (d, J -- 11.7 Hz, 24H), 0.90 - 0.86 (m, 3H);13C NMR ( 100 MHz, CDCh) 5 166.16, 165.18, 147.55, 137.10, 129.87, 118.71 , 94.66, 68.20, 62.24, 59.85, 53.36, 48.65, 48.43, 44.41, 32.05, 30.51, 29.81, 29.80, 29.78, 29.76, 29.68, 29.48, 27.99, 27.49, 22.81, 14.52, 14.25, 14.06. HRMS (m / z): [M+H] + calcd for C30H53N4O4 533.4066, found 533.4047; 0 ppm. x. DIETHYL (Z)-3-(3-(1H-IMIDAZOL-1-YL)PROPYL)-1- (OCTADEC-9-EN-1-YL)-1,2,3,6-TETRAHYDROPYRIMIDINE- 4,5-DICARBOXYL ATE (THP10)

[0302] 1H NMR (400 MHz, CDCh) 5 7.52 (s, 1H), 7.08 (s, H I). 6.91 (s, H I). 5.40 - 5.31 (m, 2H), 4.29 (q, J = 7.2 Hz, 2H), 4.11 (q, J= 7.1 Hz, 2H), 3.99 (t, J= 7.0 Hz, 2.H), 3.84 (s, 2H), 3.44 (s, 2H), 3.08 - 2.99 (m, 2H), 2.53 - 2.45 (m, 2H), 2.11 - 1.92 (m, 6H), 1.51 (s, 2H), 1.37 - 1.19 (m, 28H), 0.88 (t, J--- 6.8 Hz, 3H);l3C NMR (100 MHz, CDCh) 8 166.15, 165.17, 147.54, 137.08, 130.08, 129.91, 129.75, 1 18.72, 94.63, 68.19, 62.2.4, 59.85, 53.34, 48.62, 48.43, 44.43, 32.73, 32.02, 30.49, 29.89, 29.82, 29.78, 29.64, 29.44, 29.40, 29.10, 27.97, 27.49, 27.34, 22.80, 14.51, 14.24, 14.05. HRMS (m / z): [M+H] + calcd for C34H59N4O4 587.4536, found 587.4536; 0 ppm. xi. DIETHYL 1,3-DITETRADECYL-1, 2,3,6-TETRAHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP11)

[0303] 1H NMR (400 MHz, CDCh) 34.36 (q, J ----- 7.2 Hz, 2H), 4.10 (q, J--- 7.1 Hz, 2H), 3.98 (s, 2H), 3.52 (s, 2H), 3.05 - 2.98 (m, 2H). 2.55 (s, 2H). 1.53 (s, 4H), 1.37 R . / 7.2 Hz, 31 H. 1.27 - 1 .20 (m, 47H), 0.90 - 0.86 (m, 61 H:13C NMR (100 MHz, CDCh,) 8 166.40, 165.05, 148.13, 91.45, 67.69, 61.85, 59.48, 52.94, 51.18, 48.50, 32.01, 29.79, 29.78, 29.75, 29.73, 29.66, 29.61, 29.45, 29.45, 29.40, 29.37, 28.07, 27.46, 26.83, 22.78, 14.53, 14.20, 14.05. HRMS (m / z): [M+H] + calcd for C38H73N2O462.1.5570. found 62.1.5569; 0.2 ppm. xii. DIETHYL (Z)-1-(3-MORPHOLINOPROPYL)-3-(OCTADEC-9- EN-1-YL)-1,2,3,6-TETRAHYDROPYRIMIDINE-4,5- DICARBOXYLATE (THP12)

[0304] 1H NMR (400 MHz, CDCh) 55.40 - 5.29 (m, 2H), 4.35 (q, J= 7.2 Hz, 2.H), 4.09 (q, J= 7.1 Hz, 2H), 3.96 (s, 2H), 3.75 (s, 4H), 3.49 (s, 2H), 3.05 - 2.95 (m, 2H), 2.63 -2.30 (m, 8H), 1.99 (dd, J= 13.2, 6.3 Hz, 4H), 1.74 (d, J= 6.5 Hz, 2H), 1.58 - 1.48 (m, 2H), 1.43 - 1.1 1 (m, 281 1). 0.87 (t, J 6.8 Hz, 3H);13C NMR ( 100 MHz, CDCh) 8 166.38, 165.00, 148.13, 130.11, 12.9.80, 91.2.5, 67.84, 66.87, 61.94, 59.57, 56.88, 53.79, 51.26, 50.78, 48.36, 32.00, 29.86, 29.82, 29.62, 29.55, 29.41 , 29.31, 27.32, 27.28, 26.84, 24.92, 22.78, 14.55, 14.22, 14.08. HRMS (m / z): [M+H] + calcd for C35H64N3O5606.4845, found 606.4847; 0.2 ppm. xiii. DIETHYL 1-DO!>ECYL-3-(3-MORPHOLINOPROPYL)-1 ,2,3,6- TETTLIHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP13)J00305]1H NMR (400 MHz, CDCh) 84.34 (q, J ----- 7.2 Hz, 2H), 4.10 (q, J--- 7.1 Hz, 2.H), 3.94 (s, 2H), 3.74 - 3.64 (m, 4H), 3.47 (s, 2H), 3.14 - 3.05 (m, 2H), 2.53 - 2.47 (m, 2H), 2.42 (s, 4H), 2.32 (t, J= 6.8 Hz, 2H), 1.73 (p, J= 7.2 Hz, 2H), 1 .50 (q, J= 6.4 Hz, 2H), 1.37 (t, J --- 7.2 Hz, 3H), 1.25 (td, J--- 14.9, 7.1 Hz, 21H), 0.90 - 0.84 (m, 3H);13C NMR (100 MHz, CDCI3) 8 166.42, 165.19, 148.05, 68.08, 67.03, 62.03, 59.68, 55.90, 53.78, 53.16, 49.28, 48.55, 32.09, 29.84, 29.80, 29.75, 29.52, 28.14, 27.56, 22.86, 14.61, 14.29, 14.18. HRMS (m / z): [M+H] + calcd for C29H54N3O552.4.4063, found 524.4063; 0 ppm. xiv. DIETHYL 3-(3-MORPHOLINOPROPYL)-1-TETRADECYL- l,2,3,6-TETRAHYDROPYRiMIDINE-4,5-DICARBOXYLATE (THP14)

[0306] 1H NMR (400 MHz, CDCh,) 34.34 (q, J= 7.2 Hz, 2H), 4.10 (q, ,7= 7.1 Hz,2H), 3.95 (s, 2H), 3.73 - 3.65 (m, 4H), 3.47 (s, 2H), 3.13 -- 3.07 (m, 2H), 2.53 - 2.48 (m, 2H), 2.40 (s, 4H), 2.30 (d, . / 6.2 Hz, 2H), 1.77 - 1.68 (m, 2H), 1.55 - 1.47 (m, 2H), 1.37 (t, J-= 1.2 Hz, 3H), 1 .2.5 (s, 22H), 1 .22. (t, J = 7. 1 Hz, 3H), 0.91 - 0.84 (m, 3H);13C NMR (100 MHz, CDCh) 8 166.40, 165.15, 148.03, 68.07, 67.07, 61.99, 59.64, 55.89, 53.78, 53.12, 49.26, 48.52, 32.07, 29.83, 29.82, 29.80, 29.78, 29.73, 29.50, 28.13, 27.53, 26.33, 22.83, 14.58. 14.26, 14.16. HRMS (m / z): [M+H] + calcd for C31H58N3O5552.4376, found 552.4376; 0 ppm.xv. DIETHYL 1-HEXADECYL-3-(3-MORPHOLINOPROPYL)- 1,2,3,6-TETRAHYDROPYRLMIDINE-4,5-DICARBOXYLAri’E (THP15)

[0307] 1H NMR (400 MHz, CDC13) 84.34 (q, J = 7.2 Hz, 2H), 4.10 (q, . / 7.1 Hz, 2H), 3.95 (s, 2H), 3.74 - 3.66 (m, 4H), 3.47 (s, 2H), 3.13 - 3.07 (m, 2H), 2.53 - 2.48 (m, 2H), 2.41 (s, 4H), 2.32 (t, J= 6.5 Hz, 2H), 1.73 (p, J= 7.1 Hz, 211), 1 .55 - 1.47 (m, 2H), 1 .39 - 1.19 (m, 32H), 0.91 - 0.84 (m, 3H);13C NMR (100 MHz, CDCI3) 8 166.37, 165.14, 148.00, 92.41. 68.05, 67.00, 61.99, 59.63, 55.86, 53.74, 53.12. 49.23, 48.51, 32.05. 29.83, 29.79, 29.77, 29.71, 29.49, 28.10, 27.52, 2.6.26, 22.82, 14.57, 14.25, 14.14. HRMS (m / z): [M+H] + calcd for C33H62N3O5 580.4689, found 580.4689; 0 ppm. xvi. DIETHYL (Z)-3-(3-MORPHOLINOPROPYL)-1-(OCTADEC-9- EN-1-YL)-1,2,3,6-TETRAHYDROPYRIMIDINE-4,5- DICARBOXYLATE (THP16)

[0308] 1H NMR (400 MHz, CDCI3) 8 5.41 - 5.30 (m, 2H), 4.35 (q, J ------ 7.2 Hz, 2H), 4.10 (q, J--- 7.1 Hz, 2.H), 3.94 (s, 2H), 3.71 (s, 4H), 3.47 (s, 2H), 3.14 - 3.07 (m, 2H). 2.54 - 2.30 (m, 8H), 2.06 - 1.93 (m, 4H), 1.75 (s, 2H), 1.54 - 1.46 (m, 2.H), 1.39 - 1.18 (m, 281 1). 0.90 - 0.84 (m, 3H);!3C NMR (100 MHz, CDCI3) 3 166.37, 165.18, 147.98, 130.10, 129.94, 68.07, 66.87, 62.02, 59.66, 55.85, 53.69, 53.14, 49.21, 48.52, 32.75, 32.04, 29.91, 29.84, 29.80. 29.69, 29.68, 29.66, 29.45, 29.43, 2.8.10, 27.53. 27.36, 22.82, 14.57. 14.26, 14.15. HRMS (m / z): [M+H] + calcd for C35H64N3O5606.4845, found 606.4845; 0 ppm. xvii. DIETHYL 1-DODECYL-3-(2-(4-METHYLPIPERAZIN-1- YL)ETHYL)-1,2,3,6-TETRAHYDROPYRIYHDINE-4,5- DICARBOXYLATE (THP17)

[0309] 1H NM (4R00 MHz, CDCI3) 34.32 (q, J= 7.2, 7.2, 7.2 Hz, 2H), 4.07 (q, J=7.1 , 7.1 , 7.1 Hz, 2H), 4.01 (s, 2H), 3.46 (s, 2H), 3.13 (t, J= 6.9, 6.9 Hz, 2H), 2.27 (s, 3H), 1.48 (q, J--- 7.2, 7.0, 7.0 Hz, 2.H), 1.34 (t, J = 7.2, 7.2 Hz, 3H), 1.32 - 1.21 (m, 18H), 1.19 (t, J--- 7.1, 7.1 Hz, 3H), 0.88 - 0.81 (m, 3H);13C NMR (100 MHz, CDCI3) 8 166.37. 165.06, 147.94, 92.24, 68.75, 62.00, 59.57, 57.56, 55.05, 53.16, 52.89, 48.25, 48.18, 45.93, 32.00, 29.76, 29.73, 29.68, 29.44, 28.15, 27.49, 22.77, 14.52, 14.21. HRMS (m / z): [M+H] + calcd for C29H55N4O4523.4223, found 523.4223; 0 ppm.xviii. DIETHYL (Z)-3-(2-(4-METHYiJpiPERAzrN-l-YL)ETHYL)-l- (OCTADEC-9-EN-1-YL)-1,23,6-TETRAHYDROPYRIMIDINE- 4,5-DICARBOXYL ATE (THP18)

[0310] 1H NMR (400 MHz, CDCh) 84.34 (q, J = 7.2 Hz, 2H), 4.09 (q, . / 7. 1 Hz,2H), 4.02 (s, 2H), 3.47 (s, 2H), 3.14 (t, J= 6.9, Hz, 2H), 2.56 - 2.38 (m, 10H), 2.29 (s, 3H),1 .99 (dd, J= 13.5, 6.6 Hz, 4H), 1.50 (d, J= 7.2 Hz, 2H), 1 .36 (t, J= 7.2, Hz, 3H), 1 .28 (dd, J ---- 19.5, 5.6 Hz, 22H), 1.21 (t, J ----- 7.1, Hz, 3H), 0.87 0. . / 6.8, Hz, 3H);i3C NMR (100 MHz, CDCh) 8 166.42, 165.11, 147.99, 130.08, 129.94, 92.27, 68.80, 65.10. 62.06, 59.63, 57.61,56.74, 55.10, 53.23, 52.93, 48.27, 48.23, 45.98, 32.74, 32.04, 29.91 , 29.90, 29.83, 29.79,29.74, 29.71, 29.69, 29.65, 29.45, 29.43, 28.20, 27.54, 27.35, 22.81 , 14.57, 14.25, 14.13. HRMS (m / z): [M+H] + calcd for C35H65N4O4605.5005, found 605.5005; 0 ppm. xix. DIETHYL 1-HEXADECYL-3-(2-(4-METHYLPIPERAZIN-1-YL)ETHYL)-1,2,3,6-TETRAHYDROPYRIMIDINE-4,5-DICARBOXYL ATE (THP19)

[0311] 1H NMR (400 MHz, CDCh) 84.33 (q, J == 7.2. 7.2. 7.2 Hz. 2H), 4.09 (q, . / 7.1, 7.1, 7.1 Hz, 2.H), 4.02. (s, 2H), 3.47 (s, 2H), 3.14 (t, J= 6.9, 6.9 Hz, 2H), 2.59 - 2.37 (m, 12H), 2.28 (s, 3H), 1.50 (q, J= 7.2, 7.0, 7.0 Hz, 2H), 1.35 (t, J= 7.2, 7.2 Hz, 3H), 1.21 (t, J= 7.1, 7.1 Hz, 3H), 0.90 - 0.83 (m, 3H);13C NMR (100 MHz, CDCh) 3 166.42, 165. 11, 147.99, 92.24, 68.80, 62.05. 59.62, 57.65, 55.14, 53.31, 52.93, 48.28, 48.23. 46.04, 32.06,29.83, 29.79, 29.74, 29.49, 28.20, 2.7.54, 2.2.82, 14.57, 14.25, 14.13. HRMS (m / z): [M+H] + calcd for C33H53N4O4579.4849, found 579.4849; 0 ppm. xx. DIETHYL (Z)-3-(2-(1H-IMIDAZOL-4-YL)ETHYL)-1 -(OCTADEC-9-EN-1-YL)-1,2,3,6-TETRAHYDROPYRIMIDINE- 4,5-DICARBOXYLATE (THP20)

[0312] 1H NMR (400 MHz, CDCh) 3 7.58 (s, 1H), 6.81 (s, 1H), 5.39 - 5.32 (m, 2H), 4.28 (q, . / 7.2, 7.2, 7.2 Hz, 2.H), 4.10 (q, J == 7.1. 7.1, 7.1 Hz, 2H), 3.85 (s, 2H). 3.44 (s, 2H), 3.35 (t, J ------ 6.8, 6.8 Hz, 2H). 2.86 (t, J--- 6.8, 6.8 Hz, 2H), 2.49 (dd, J - 8.5. 6.5 Hz, 2H), 2.03 - 1.94 (m, 4H), 1.53 (t, J= 7.4, 7.4 Hz, 2H), 1.33 - 1.18 (m, 29H), 0.90 - 0.84 (m, 311);13C NMR (100 MHz, CDCh) 8 166.16, 165.16, 147.91 , 135.03, 130.08, 129.91, 68.33, 62.13,59.82. 53.62, 51.13, 49.08, 32.73, 32.02, 2.9.89, 29.82. 29.78, 29.64, 29.44. 29.39, 27.80,27.56, 27.34, 26.76, 22.80, 14.52, 14.24, 14.01. FIRMS (m / z): [M+H] + calcd for C33H57N4O4 573.4379, found 573.4380; 0 ppm. xxi. DIETHYL 3-(2-(1H-IMIDAZOL-4-YL)ETHYL)-1- HEXADECYL-1,2,3,6"TET1LIHYDROPYRIYIIDINE-4,5- DICARBOXYLATE (THP21)

[0313] 1H NMR (400 MHz, CDCI3) 5 7.59 (s, 1H). 6.81 (s, 1H), 4.28 (q, J - 7.2 Hz. 2H), 4.11 (q, J= 7.1 Hz, 2.H), 3.85 (s, 2H), 3.44 (s, 2H), 3.35 (t, J= 6.7 Hz, 2.H), 2.86 (t, J = 6.7 Hz, 2H), 2.53 - 2.46 (m, 2H), 1.59 - 1.49 (m, 2H), 1.32 - 1.20 (m, 33H), 0.87 (t, .7= 6.8 Hz, 3H);13C NMR (100 MHz, CDCI3) 8 166.11, 165.16, 147.88, 134.96, 68.34, 62.17, 59.87, 53.74, 51.1 1, 49.21, 32.07, 29.84, 29.81, 29.77, 2.9.68, 2.9.51, 27.78, 27.60, 22.84, 14.54, 14.27, 14.03. HRMS (m / z): [M+H] + calcd for C31H55N4O4547.4223, found 547.4223; 0 ppm. xxii. DIETHYL (Z)-1-(OCTADEC-9-EN-1-YL)-3-(2-(PYRROLIDIN- 1-YL)ETHYL)-1,2,3,6-TETRAHYDROPYRIMIDINE-4,5- DICARBOXYLATE (THP22)

[0314] 1H NMR (400 MHz, CDCh) 8 5.39 - 5.31 (m, 2H), 4.35 (q, .7 7.2 Hz, 2H), 4.10 (q, J == 7.1 Hz. 2H), 4.01 (s, 2H). 3.47 (s, 2H), 3.2.0 (s, 2H), 2.74 - 2.48 (m, 8H), 2.04 - 1 .93 (m, 4H), 1 .78 (s, 4H), 1 .55 - 1 .47 (m, 2H), 1 .39 - 1 .35 (m, 3H), 1.34 - 1 .24 (m, 22H), 1.21 (d, J= 7.1 Hz, 311), 0.90 - 0.85 (m, 3H);13C NMR (100 MHz, CDCh) 3 166.38, 165.17, 130.08, 129.96, 68.57, 62.08, 59.65, 54.31, 52.97, 48.47, 32.04, 29.91, 29.84, 29.79, 29.69, 29.66, 29.46, 29.43, 28.11, 27.52, 27.36, 23.63, 22.82, 14.57, 14.25, 14.13. HRMS (m / z): [M+H] + calcd for C34H52N3O4576.4740, found 576.4739; 0.2 ppm. xxiii. DIETHYL 1-HEXADECYL-3-(2-(PYRROLIDIN-1-YL)ETHYL)- 1,2,3,6-TETRAHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP23)

[0315] 1H NMR (400 MHz, CDCI3) 84.34 (q, J= 7.2, 7.2, 7.2 Hz, 211), 4.23 (q, J= 7.1, 7.1, 7.1 Hz, 2H), 4.09 (q. . / 7.1, 7.1, 7.1 Hz, 2H), 4.00 (s, 2H), 3.46 (s, 2H), 1.54 - 1.47 (m, 2.H), 1.36 (t. . / 7.2, 7.2 Hz, 3H), 1.32 - 1.2.4 (m, 26H), 1.23 - 1.18 (m, 3H), 0.89 - 0.84 (m, 3H);13C NMR (100 MHz, CDl3) δ 166.33, 165.18, 147.78, 68.59, 67.98, 62.10, 60.39, 59.65, 55.89, 55.20, 54.28, 54.05, 53.00, 49.59, 48.48, 32.04, 29.82, 29.77, 29.69, 29.47,28.06, 27.50, 23.67, 23.60, 22.81 , 14.54, 14.46, 14.23, 14.11. FIRMS (m / z): [M+H] + calcd for C32H60N3O4550.4583, found 550.4584; 0 ppm. xxiv. DIETHYL 1-HEXADECYL-3-((4-METHYLMORPHOLIN-2- YL)METHYL)-1,2,3,6-TETRAHYDROPYRIMIDINE-4,5- DICARBOXYLATE (THP24)

[0316] 1H NMR (400 MHz, CDCb,) 54.34 (qq, . / 7.1, 3.6 Hz, 2H), 4.14 - 4.00 (m, 4H), 3.84 (dd, J= 11.5, 1.9 Hz, 1H), 3.71 - 3.59 (m, 2H), 3.48 (s, 2.H), 3.14 - 3.02 (m, 2H), 2.65 (t, . / = 9.7 Hz, 2H), 2.52 (It, J = 7.3, 3.8 Hz, 2H), 2.29 (s, 3H), 2.12 - 2.04 (m, 1H), 1 .79 (s, 1H), 1.55 - 1.47 (m, 2H), 1.36 (t, . / 7.2 Hz, 3H), 1.23 (d, . / 13.4 Hz, 29H), 0.91 - 0.84 (m, 3H);l3C NMR (100 MHz, CDCh) 8 166.41 , 165.04, 147.97, 92.78, 75.50, 69.17, 66.58, 62.05, 59.62, 57.63, 54.65, 53.40, 52.84, 48.43, 46.34, 32.02, 29.80, 29.76, 29.75, 29.69, 29.46, 28.08, 27.50, 22.79, 14.53, 14.22, 14.09. FIRMS (m / z): [M+H] + calcd for C32H60N3O5 566.4532, found 566.4533; 0 ppm. xxv. DIETHYL (Z)-3-((4-METHYLMORPHOLIN-2-YL)METHYL)- 1-(OCTADEC-9-EN-1-YL)-1, 2,3,6- TETRAHYDROPYRIMIDINE-4,5-DICARBOXYLATE (THP25)

[0317] 1H NMR (400 MHz, CDCh) 8 5.41 - 5.31 (m, 2H). 4.34 (qd, . / 7.2, 7.2, 7.2, 2.9 Hz, 2H), 4,17 - 3.98 (m, 4H), 3.48 (s, 2H), 3.16 - 3.04 (m, 2H), 2.70 (s, 2H), 2.56 - 2.46 (m, 2H), 2.32 (s, 3H), 1.51 (d, J= 6.9 Hz, 2H), 1.41 - 1.17 (m, 28H), 0.88 (t, J= 6.8, 6.8 Hz, 3H);13C NMR (100 MHz, CDCI3) 6 166.32, 164.94, 147.90, 129.93, 129.83, 92.67, 75.40, 69.09, 66.46, 61.96, 59.53, 57.51, 54.54, 53.31, 52.74, 48.35, 46.22, 32.62, 31.92, 29.79, 2.9.72, 29.67, 29.58, 29.56, 29.54, 29.38, 29.33, 2.9.30, 27.98, 27.41, 27.23, 22.70, 14.45, 14.14, 14.00. FIRMS (m / z): [M+H] + calcd for C31H59N4O6567.4277, found 567.4277; 0 ppm. xxvi. DIETHYL 3-(2-(4-METiiYLPiPERAZiN-l -YL)ETHYL)-1- TETR / XDECYL-1,2,3,6-TETR / XHYDROPYRIMIDINE-4,5- DICARBOXYLATE (THP26)

[0318] 1H NMR (400 MHz, CDl3) 54.32 (q, . / 7.1 Hz, 2H), 4.07 (q, J == 7.1, Hz, 2H), 4.00 (s, 2H), 3.45 (s, 2H), 3.12 (t, J= 6.9, Hz, 3H), 2.66 - 2.34 (m, 12H), 2.28 (s, 3H), 1.49 (t, .7 = 7.3, Hz, 2H), 1.39 - 1.14 (m, 28FI), 0.85 (t, J= 6.7, Hz, 3H);13C NMR (100MHz, CDCh) 6 166,39, 165.08, 147,96, 92.23, 77.48, 77, 16, 76.84, 68.72, 62.03, 59.59, 57.53, 55.04, 55.01, 53.13, 52.89, 52.57, 48.23, 48.17, 45.89, 45.82, 32.00, 29.78, 29.76, 29.73, 29.68, 2,9.44, 28.13, 27.49, 22.77, 14.57, 14.51, 14.20, 14.07. HRMS (m / z): [M+H] + calcd for C31H39N4O4551 .4536, found 551 .4534; 0,36 ppm. xxvii. DIOCTYL 1-(2-(1H-IMTOAZOL-4-YL)E.THYL)-3- HEXA»ECYL-l,2,3,6-TETRAHYDROPYRIMroiNE-4,5- DiCARBOXYLATE (MTHP14 )

[0319] 1HNMR (400MHz, CDCh) 5 7.53 (d, J= 10,6 Hz, IH), 6.83 (d, J= 9.3 Hz, 1 H), 4.26 (i. . / 6.9 Hz, IH), 4.18 (t, J--- 6.8 Hz, IH), 4.09 (t, J--- 6.7 Hz, IH), 4.05 - 3.98 (m, 3H), 3.88 (s, IH), 3.81 - 3.72 (m, 2H), 3.63 - 3.54 (m, 2H), 2.96 (dd, J= 13.9, 7.6 Hz, 2.H), 2.87 - 2.80 (m, 2H), 1 .74 - 1 .65 (m, 2H), 1 .63 - 1 .48 (ra, 5H), 1 .23 (s, 45H), 0.89 - 0.84 (m, 9H). See FIG. 22. e. EXAMPLARY COMPOUNDS1003201 All analogs shown in Table 1 below were synthesized by following the above synthetic procedures.TABLE 1.f. MATERIALS AND METHODS i. MATERIALS

[0321] l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamme (DOPE), 1,2- Distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from Avanti Polar Lipids, DLin-MC3-DMA was purchased from Cayman Chemicals. Triton X-l 00 was purchased from Sigma. Fetal Bovine serum was purchased from Gibco. QuantiT RiboGreen RNA Assay Kit was purchased from Invitrogen, Clean cap FLuc-mRN A and CleanCap Cre mRNA was purchased from Trilink Biotechnology. HEK 293 and RAW 264.7 cell lines purchased from ATCC were cultured according to the ATCC guidelines. DMEM growth medium (Gibco, USA) containing sodium bicarbonate, without sodium pyruvate and HEPES,was supplemented with 10% fetal bovine serum (Gibco, USA) and 1 % penicillin / streptomycin (Thermo Fisher Scientific). ii. FORMULATION OF LIPID NANOPARTICLES

[0322] Tire organic phase was prepared by dissolving the corresponding ionizable Lipid, DOPE, cholesterol, and DMG-PEG2K in ethanol at a molar ratio of 35: 16:46.5:2.5, respectively. The aqueous phase was prepared by dissolving the corresponding mRNA in 10 mM citrate buffer at pH 3 (Teknova, Hollister, CA, USA). The ionizable lipid to mRNA weight ratio for all LNPs was 10: 1 .50 The two phases were loaded into separate glass syringes (Hamilton Company, Reno, NV) and LNPs were formed by chaotic mixing of the organic and aqueous phases at a 1 :3 volume ratio either using a pipette (hand mixing) or in a microfluidic device using Fusion 400 X (Chemyx Inc, USA)(Kauffman, K. J.; et al., (2015) Nano Lett. 15 (11), 7300-7306). The LNPs were subsequently dialyzed against IX PBS (Thermo Fisher Scientific, Walthrnan, MA, USA) in 20 kDa molecular weight cutoff dialysis cassettes (Thermo Fisher Scientific) for 4 hours. iii. CHARACTERIZATION OF LIPID NANOPARTICLES

[0323] Encapsulated mRNA concentration and encapsulation efficiency of the LNPs were measured using a Quant-iT RiboGreen assay (Thermo Fisher Scientific) as previously described. (Heyes, J.; et al., (2005) J. Controlled Release 107 (2), 276-287; Swingle, K. L.; et al., (2023) J. Am. Chem. Soc. 145 (8), 4691-4706) Each LNP was diluted 100-fold in two microcentrifuge tubes containing either 1 * TE buffer or 1% (v / v) Triton X-100 (Sigma, USA) in 1 < TE buffer. The Triton X-100 samples were mixed thoroughly and allowed to incubate for 5 min to achieve lysis of LNPs. LNPs in 1 z TE buffer, LNPs in 1% Triton X-100 buffer, and mRNA standards were placed in quadruplicate in black-walled 96-well plates. RiboGreen detection reagent was added to each well per manufacturerinstructions. The plate was shaken on a plate reader in the dark at 200 rpm for 5 min, and then fluorescence intensity was read on GloMax Explorer plate reader (Promega, USA) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Encapsulated mRNA concentration was estimated using a standard curve estimated from a univariate least-squared linear regression. Encapsulation efficiency was calculated as EE = 1 — — ™ , where RTE is the measured free RNA content inTE buffer, and R?x is the measured total RNA content in 0.1% Triton X-100 buffer. The hydrodynamic diameter and polydispersity index of each LNP were measured using a Mobius(Wyat Technology, Santa Barbara, CA, USA). Each LNP was diluted 100-fold in lx PBS and placed in a cuvette (Wyat Technology) inside the Mobius. The surface ^-potential of each LNP was measured using a Mobius (Wyatt Technology, Santa Barbara, CA, USA).Each LNP was diluted 100-fold in deionized water (Thermo Fisher Scientific) and placed into a capillary cell before measurements were collected by the instrument, ^-potential and size were reported as mean ± standard deviation (n = 3 technical replicates). iv. IN VIIROSTUDIES[00324 j Cells were plated at 18,000 cells per well in 100 pL of DMEM and left to adhere for 24 h in a 96 well plate. The media was removed and 75 pL of fresh media without penicillin — streptomycin was added. The cells were then treated with 150 ng of mRNA per 18,000 cells to evaluate the in vitro luciferase expression mediated by each LNP. DMEM alone was used as the negative control, and LNPs formulated with MC3 were used as the positive control. The LNP -treated cells were incubated at 37 °C for 24 h. After 24 h, 100 pL of luciferase assay substrate (Promega) was added to each well . The plate was shaken on a plate reader in the dark at 200 rpm for 10 min, and GloMax Explorer plate reader (Promega, USA) was used to quantify luminescence intensity. v. ANIMAL EXPERIMENTS

[0325] All animal use was in accordance with the guidelines and approval from theUniversi ty of Las Vegas, Nevada's Institutional Animal Care and Use Commi ttee (IACUC; protocol #01218). C57BL / 6J (Strain#000664) female and male mice (6~— -8 weeks old, approximately 20 g average weight) and(6-8 weeks old, approximately 20 g average weight) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Mice were injected with LNPs formulated with FLuc mRNA at a dose of 0.5 mg of mRNA per kg of body mass (mg / kg) via the lateral tail vein or intramuscularly. A solution of D-lucifenn (PerkinElmer) m i x PBS was prepared at a concentration of 30 mg / mL. After 6 h (in case of intramuscular injections) or 24 h (in case of intravenous injections), the mice were injected intraperitoneally with the D-luciferin solution (130 pL, 30 mg / mL). After 10 min, the mice were euthanized with CO, and the organs (liver, spleen, kidney, pancreas, heart, lung) were harvested and imaged using an in vivo imaging system (IVIS; PerkinElmer, Walthman, MA, USA). Luminescence flux was quantified with the Living Image Software (PerkinElmer). For Ail4 experiments, fluorescence wasquantified with the Living Image Software at an excitation / emission of 554 / 581 nm. Regions of interest (ROI) of constant size were placed around the corresponding images of each organ. Total luminescence flux and total radiant efficiency was reported as mean ± standard deviation. For the evaluation of toxicity, blood samples were collected via cardiac puncture 24 h after treatment; subsequently mice were sacrificed by CO, asphyxiation and organs were collected. Blood samples were allowed to coagulate for 20 mm at RT and were subsequently centrifuged at 2000 x g for 20 min at 4 °C to retrieve serum . Serum liver enzyme concentrations were measured by VRL Animal Health Diagnostics. High quality total RNA was isolated by Qiagen RNeasy Mini Kit (Qiagen) for three PBS samples and three THP samples. Sequencing libraries were prepared by TruSeq Stranded mRNA Library Prep Kit (Illumina) and then sent to GAA cores at UNLV for 75bp pair-end sequencing. The sequencing generated 20, 26 and 37 million pair-end (PE) short reads respectively for the three PBS samples and 18, 23, and 33 million PE reads respectively for the three THP samples. FASTQC was used to check the quality of these RNA-seq data and cutadapt67 to remove low-quality bases in two tails of each read. The short reads of each sample were aligned against the mouse reference genome mm39 using STAR v2.7. 10a68 with the gene annotation GENCODE vM34. After alignment, edgeR69 was used to detect differential gene expression between PBS and THP samples and visualized the results in volcano plot. vi. TISSUE IMMUNOHISTOCHEMISTRY (IHC) AND H&E STAJN

[0326] H&E and IHC on liver tissues from Ail 4 mice was performed on FFPE (formalin fixed paraffin embedded) sections. Tissues were post-fixed in 10% formalin 24-48 hours at room temperature (can stay up to a week in 10% formalin) and directly transferred to 70% ethanol. Histology was performed by HistoWiz Inc, (NY, USA) using a Standard Operating Procedure and fully automated workflow'. Samples were embedded in paraffin and sectioned at 4mm. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Bio- systems) with heat-mediated antigen retrieval using Epitope Retrieval Solution I (Leica Biosystems). Bond Polymer Refine Detection (Leica Biosystems) was used according to the manufacturer's protocol. After staining, sections were dehydrated and film coverslip- ped using a TissueTek-Prisma and Coverslipper (Sakura). Whole slide scanning (40x) was performed on an Aperio AT2. (Leica Biosystems).I MMIJNOFEUORESCE NCE

[0327] Unstained slides of Ai 14 mice liver tissue samples post treatment were obtained from HistoWiz Inc. (NY, USA). Sections of 5pm thickness were deparaffinized in xylene, rehydrated through a descending ethanol series, and subjected to antigen retrieval in citrate buffer (pH 6.0) at 95 °C for 20 minutes. After cooling to room temperature, sections were permeabilized with 0.1% Triton X-100 in PBS for 10 minutes and blocked with 5% normal goat serum in PBS for 1 hour. Direct staining was performed using DAPI (1 pg / mL in PBS) for 10 minutes to visualize nuclei, since tdTomato expression was already present in the liver tissues. After washing with PBS, slides were mounted using an anti-fade mounting medium and covered with a glass coverslip. Images were captured using a Zeiss LSM 980 fluorescence microscope (Oberkochen, Germany) with filters for tdTomato (red fluorescence) and DAPI (blue fluorescence). viii. STATISTICS

[0328] Statistical analysis of the results was performed by two-tailed unpairedStudent's t-test or One-Way ANOVA followed by Bonferroni post-hoc analysis to compare multiple replicate means using Prism 10 (GraphPad). Differences were considered significant when p < 0.05. ix. TNS ASSAY

[0329] LNP pKa was determined using the TNS binding assay. A 0.16mM stock solution of TNS (6-(p-Toluidino)-2-naphthalenesulfonic acid) reagent from Sigma Aldrich was prepared in DI water. The LNPs were diluted to 40 ng / mL, and then 10 p.L of the TNS stock solution was added to each well. The final volume in each well was 250 pL, which included 150 mM sodium chloride, 20 mM sodium phosphate, 20 mM ammonium acetate, and 25 rnM ammonium citrate. pH values ranging from 2 to 12 in 0.5 increments were tested. The samples were prepared in black 96-well plates and mixed using a plate shaker at 300 rpm for 5 minutes at room temperature in the dark. Subsequently, the plate was transferred to a GloMax Explorer plate reader (Promega), and the fluorescence was measured using excitation and emission wavelengths of 322 nrn and 431 nm, respectively, with bandwidths of 2.0 nm each and a gain of 60. Following this, plots of normalized fluorescence versus pH were generated, and the pKa value was determined as the pH at the inflection point of the titration curve.2. RESULTS

[0330] A facile, versatile, and catalyst-free MCR platform specifically designed to create tetrahydropyrimidine (THP) ionizable lipids in just 3 hours. The THP core structure has previously demonstrated various pharmacological activities, including calcium channel modulation, as well as antiviral and anticancer properties through receptor-mediated

[0331] Interactions (Malin, G.; Lapidot, A. (1996) J. Bacteria (2), 385-395; Darandale, S. N.; et al., (2013) Bioorg. Med. Chem. Lett. 23 (9), 2632-2635; Kobayashi, J.; et al., (1991) J. Org. Chem. 56 (14), 4574-4576; Messer, William S.; et al., (1997) J. Med. Chem. 40 (8), 1230-1246; Godhani, D. R.; et al., (2014) Med. Chem. Res. 23 (5), 2417-2425). THP MCR is a catalyst-free, facile, and versatile domino reaction that employs yne-diacid esters, amines, and formaldehyde (Zhang, M.; et al,, (2007) Org. Lett. 9 (21), 41 11-41 13). However, its potential for gene delivery has not yet been explored. Utilizing this innovative MCR method — previously uncharted for LNP formulation and mRNA delivery — 26 new lipids with unique structures were developed. These lipids were designed with biodegradable ester groups and varying carbon tail lengths to improve biocompatibility and functionality. The head group of these lipids features a tetrahydropyrimidine ring substituted with a carboxylic acid ester, providing a robust platform for further chemical modifications. The design of these compounds was guided by principles of medicinal chemistry, aiming to balance the hydrophobic and hydrophilic properties necessary for effective LNP formation and mRNA encapsulation. The efficiency of ionizable THP lipids in encapsulating mRNA and producing proteins was assessed, both in vitro and in vivo. Through strategic alteration of the electronic and steric properties of the substituents, a wide range of physicochemical and biological properties were explored. Despite the structural diversity, it was observed that THP lipids featuring electronic functional groups and single lipid tail did not exhibit substantial efficacy in mRN A delivery in vivo. This finding emphasizes the importance of fine-tuning the electronic properties of ionizable lipids, as well as lipophilicity of lipid tails, to achieve effective mRNA encapsulation and release. One particular compound, THP1 , has emerged as a promising biocompatible lipid capable of delivering mRNA intramuscularly for up to 5 days, and intravenously in a dose-dependent manner with highefficiency, highlighting the importance of strategic functional group placement and lipid architecture optimization for efficient mRNA delivery. THP1 exhibits a balanced amphiphilic nature, which aids in creating stable LNPs that effectively deliver mRNA to target cells with minimal toxicity. This underscores the significance of innovative chemical design in overcoming the challenges ofmRNA therapeutics. In summary', the advancement in mRNA delivery' using MCRs, particularly the development of THP-based ionizable lipids, broadens the toolkit of ionizable lipids and paves the way for future optimization of mRNA deliveiy' technologies. This provides valuable insights for developing next-generation LNPs for vaccine delivery and CRISPR / Cas9-mediated gene editing in the liver with potential for clinical translation. a. SYNTHESIS AND EVALUATION OF BIOCOMPATIBLE TETRAHYDROPYRIMIDINE LIPIDS FOR MRNA DELIVERY[00332 j The development of biocompatible nanomaterials that exhibit low toxicity and high transfection efficiency is crucial for the clinical success of mRNA therapeutics (Syama, K.; et al., (2022) Sei. Rep. 12 (1), 18071 ; Athinarayanan, J.; et al., (2022) ACS Omega 7 (23), 19270-19279; Duran-Lobato, M.; et al., (2016) Drug Dev. Ind. Pharm. 42 (2), 190-198). Achieving this necessitates a chemical structure with ionizable nitrogen atoms, appropriate lengths of aliphatic carbon chains, and biodegradable functional groups. In this study, 26 THP ionizable lipids were synthesized in just 3 hours (FIG, 1 A and FIG. IB) and their effectiveness in delivering mRNA was assessed. The designed THP lipid molecules contain ionizable nitrogen atoms within their pharmacologically significant six-membered heterocyclic (THP) cores, hydrolyzable diester functional groups, and various aliphatic carbon tails paired with different heterocyclic moieties.

[0333] A multicomponent reaction was employed to synthesize THP-based lipid molecules, aiming to discover novel mRNA deliveiy vehicles. This THP MCR reaction utilized but-2-ynedioic acid diethy 1 ester as a Michael acceptor, formaldehyde, and two identical or different primary' amines as building blocks for the heterocyclic THP ring. For the synthesis of dialkylated THP molecules where R^-NH, and R4-NH2 are identical, R%NH2 was initially reacted with but-2-ynedioic acid diethyl ester. Formaldehyde was then added, resulting in the production of dialkylated THP molecules with aliphatic chains ranging from C8 to C 18, achieving moderate to good yields (65 - 80%). The structure of the THP molecule was precisely controlled by the sequential addition of primary amines. When using a heterocyclic amine, such as imidazole amine, in combination with different aliphatic carbon tails (R3-NH, and R4-NH2 are different), R4-NH2 was initially reacted witli but-2-ynedioic acid diethyl ester, followed by the addition of R3-NH2. This procedure yielded the desired products -with 45-60% efficiency. Notably, reactions involving morpholine amine and N- methyl piperazine amine resulted in higher yields of THP lipids, ranging from 64 - 83%. Onthe other hand, reactions with histamine and pyrrolidine amine yielded lower yields of 15 - 30%. The synthesis strategy facilitated the formation of a diverse set of THP ionizable lipids with different structural features, enabling a comprehensive evaluation of their potential as an mRNA delivery system. h. FORMULATION OF THP LIPID NANOPARTICI.ES (LNPS) ANDEVALUATION IN VITRO

[0334] To evaluate the effectiveness of THP ionizable lipids in delivering mRNA efficiently, all 26 THP ionizable lipids were incorporated into LNPs. These ionizable lipids were mixed with l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1 ,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) in 35: 16:46,5:2.5 ratio with firefly luciferase (FLuc) mRNA to form THP LNPs for initial in vitro screening (FIG. 2A). DLin-MC3-DMA was also formulated alongside these LNPs as a positive control for the study using l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC) (Eygeris, Y.; et al., (2020). Nano Lett. 20 (6), 4543-4549), FLuc mRNA is utilized here for direct, quantification and imaging because it produces a non-secretory protein. The nanoparticle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency for each LNP formulation are detailed in FIG, 2B and FIG, 2C. The particle size analysis revealed that the formulated LNPs had diameters ranging from 80 to 200 nm with polydispersity indices below 0.2, indicating a uniform particle size distribution suitable for cellular uptake and endosomal escape. The zeta potential measurements ranged from 2 to 6 mV, suggesting sufficient colloidal stability, which is crucial for maintaining the integrity and delivery’ efficiency of the nanoparticles. Encapsulation efficiencies varied between 20% and 75%, highlighting the influence of the lipid structure on the encapsulation process. Higher encapsulation efficiencies were observed in LNPs with longer aliphatic chains and more hydrophobic moieties. This is likely due to increased lipid-mRN A interactions and enhanced stability of the nanoparticle core. On the other hand, lower encapsulation efficiencies in certain formulations could be attributed to suboptimal interactions between the lipid components and the mRN A, underscoring the importance of fine-tuning lipid compositions for optimal performance.

[0335] Next, the transfection efficiency of THP LNPs was evaluated in vitro using HEK 293 (FIG. 6) and RAW 264.7 (FIG. 7) cells. The in vitro transfection experiments showed that THP1 and THP3 LNPs resulted in significant protein expression in both HEK293 and RAW 264.7 cells. Particularly, THP1 and THP3 have double alkyl tails and exhibited comparable in vitro protein production to MC3, the control. This indicates that the double alkyl tails of THP 1 and THP3 facilitate beter endosomal escape and mRNA release into the cytoplasm. These results emphasize the potential of THP-based ionizable lipids in mRNA delivery, highlighting specific structural features that enhance transfection efficiency and nanoparticle stability. c. In vivo SCREENING OF THP LNPS FOR MRNA DELIVERY

[0336] Following the in vitro results, the in vivo transfection efficacy of the THP LNPs was evaluated. The same formulation for intramuscular delivery' was used as in the in vitro studies. Both methods provide insights into cellular uptake and endocytosis, considering intramuscular injection as a form of local delivery'. A thorough batch screening method was utilized to quickly screen all the THP LNPs. The nanoparticles -were categorized into five distinct groups based on their chemical structures and administered to C57BL / 6 mice via intramuscular injections at a dose of 0.5 mg / kg. These groupings, detailed in the supplementary' information (FIG. 8A-E), allow' for a systematic assessment of the influence of structural variations on transfection efficiency. The choice of intramuscular administration was based on its relevance for various therapeutic applications, including vaccines and protein replacement therapies. Byquantifying the luminescence intensity following mRNA translation to protein, the efficiency- of mRN A delivery and expression was measured. In vivo results revealed a clear differentiation in transfection efficacy among the batches. Batch 1 demonstrated significant transfection, as indicated by a strong luminescence signal, while no luminescence was observed in the other batches (FIG. 2D). FIG. 2D shows IVIS images at 6h post injection and graphical representation of total flux of FLuc mRNA encapsulated with all THPs, batched based on chemical structures detailed in Figure S3. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled THP nanoparticles (n===2 biologically independent mice, initial screening). This initial screening highlighted the potential of specific structural motifs within Batch 1, which are long alkyl tail amine functionalized and included THP1, THP2, THP3, THP4, and THP11. Next, LNPs from Batch 1 were individually- administered and found that THP1 had the highest transfection efficiency (FIG. 2E). FIG. 2E shows individual intramuscular injections of all the THPs from Group 1. Representative IVIS images at 6h post injection and graphical representation of total flux of THP 1, THP2, THP3, THP4, and THP11 LN Ps at a dose of 0.5 mg / kg (n=2 biologically independent mice, initial screening). This superior performance was consistent with its favorable physiochemical properties, such as optimal particlesize, zeta potential, and encapsulationefficiency observed during in vitro characterization, and notably may be due to the double alkyl tail in the structure. d. OPTIMIZATION OF HELFER LIPIDS FOR IMPROVED MRNA DELIVERY

[0337] Recognizing the pivotal role phospholipids play in nucleic acid delivery, lipid nanoparticles utilizing the lead ionizable lipid, THP 1, were optimized by investigating various phospholipid components to achieve higher mRNA delivery in vivo. Phospholipids with phosphatidylcholine (PC) headgroups, such as l-stearoyl-2-oleoyl-sn-glycero-3- phosphocholine (SOPC), typically form cylindrical structures that stabilize lipid bilayers. While lipids such as DOPE and 1 -palmitoy l -2 -oleoyI-sn-glycero-3-phosphoethanolamine (POPE), with phosphatidylethanolamine (PE) headgroups, tend to adopt a conical shape that can transition into a hexagonal conformation (Gan, Z.; et al., (2020). Bioeng. Trani. Med. 5 (3), el 0161; Alvarez-Benedicto, E.; et al., (2022) Biomater Sei. 10 (2), 549-559). This hexagonal structure is associated with membrane fusion properties, which can facilitate the release of mRNA into the cytoplasm. Without wishing to be bound by theory, based on these structural and functional distinctions, it was hypothesized that varying the phospholipid component could improve the effectiveness of delivering RNA.

[0338] To enhance the transfection efficacy following intramuscular injections, DOPE was replaced with five different phospholipids 1 -stearoyl -2 -oleoyl-sn-glycero-3- phosphoethanolamme (SOPE), POPE, SOPC, l,2-dioleoyl-3-trimethylammonium propane (DOTAP) and l,2-di-O-phytanyl-sn-glycero-3-phosphoethanolamine (4Me) (FIG. 9), and tested in vivo intramuscularly at a dose of 0.5 mg / kg. These phospholipids differ in tail length, number of unsaturations in the tail, and charge on the nitrogen headgroup. Among the different phospholipids tested, the THP1 formulated with POPE exhibited the highest transfection efficiency, significantly surpassing the positive control, DLin-MC3-DMA. Tire average total flux observed in vivo with the initial formulation of THP 1 with DOPE was 2.7x 106, compared to 5.2xl07with THP1 POPE, while the positive control yielded 3.2x10° (FIG. 2F). FIG. 2F shows IVIS Images at 6h post injection and graphical representation of total flux of THP1 formulated with phospholipids SOPE, POPE, SOPC, DOTAP, and 4Me injected intramuscularly in C57BL / 6 mice at a dose of 0.5 mg / kg (n:::2 biologically independent mice, initial screening). This represents a 19-fold increase in mRNA transfection efficiency with THP1 POPE compared to the parent formulation with DOPE, and a 16-fold increase compared to DLin-MC3-DMA. Without wishing to be bound by theory.this substantial difference supported the hypothesis that the structural properties of POPE could enhance mRNA delivery by promoting better membrane fusion and mRNA release).

[0339] To validate these findings and further explore the potential of POPE in enhancing mRNA deliver}', all 26 THP lipids were reformulated with POPE. This comprehensive batch analysis was aimed at determining whether the enhanced transfection observed with THP1 could be generalized to other THP lipids. The formulations were pooled following the same batch process as before and administered intramuscularly to mice at a dose of 0.5 mg / kg to compare transfection efficiency (FIG. 2G). FIG. 2G shows Representative IVIS images at 6h post injection and graphical representation of total flux of all the THPs formulated with POPE, batched based on chemical structures detailed in FIG. 8. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled THP nanoparticles (n=2 biologically independent mice, initial screening). The findings show' that Batch 1, which was previously found to be effective, demonstrated a significant increase in transfection with POPE. This confirms that optimizing phospholipids plays a crucial role in improving delivery'. To determine the most effective lipid, THPs from Batch 1 were individually formulated w'ith POPE and conducted a comprehensive comparison. The individual analysis showed that THP I with POPE consistently had the highest transfection efficiency across all formulations. Compared to the positive control DLin-MC3-DMA, THP1 with POPE-formulated LNPs achieved a two-fold increase in luminescence signal, demonstrating its superior capability in mRNA delivery and expression (FIG. 2H). FIG. 2H shows representative IVIS images at 6h post injection and graphical representation of total flux of all the THPs from Group 1 formulated with POPE and injected intramuscularly in C57BL / 6 mice at a dose of 0.5 mg / kg (n=2 biologically independent mice, initial screening). This significant improvement highlights the critical impact of phospholipid selection on the performance of LNP formulations. The in vivo evaluation of THP LNPs for mRNA delivery successfully identified THP1 as a highly effective ionizable lipid for intramuscular mRNA administration . The systematic approach — from structural grouping and initial batch screening to individual lipid assessment ----- aimed to systematically dissect the contributions of lipid structure to transfection efficacy. As a result, the structural advantages of THP 1 were effectively translated into superior transfection performance through phospholipid optimization. This optimization not only emphasizes the importance of phospholipid structure in nucleic acid delivery but also illustrates the importance of balancing electronic properties with lipid tail structure in the design of delivery systems and singles out THP1 POPE as a promising candidate for further development.e. V ALIDATION AND EVALUATION OF INTRAMUSCULAR VERSUSINTRAVENOUS ADMINISTRATION OF THP1 FOR MRNA DELIVERY

[0340] To further optimize the LNP formulations and explore their scalability for clinical applications, two different methods of LNP preparation for the lead compound THPI were compared: hand mixing (using pipette) and microfluidic mixing. Initial formulation preparation exclusively used hand mixing. While this straightforward pipette mixing technique is convenient for bench-scale experiments due to its simplicity and flexibility in handling small volumes, it has limitations in reproducibility and scalability (Ripoil, M.; et al., (2022) Set. Rep. 12 (1), 9483; Strelkova Petersen, D. M.; et al., (2023). Eur. J. Pharm. Biopharm. 192, 126-135). Microfluidic mixing devices, on the other hand, offer precise control over mixing parameters, ensuring consistent particle size and encapsulation efficiency, which are crucial for clinical translation and large-scale manufacturing (Nakamura, T.; et al., (2020) Mol. Pharm. 17 (3), 944-953; Maeki, M.; et al., (202.2.) J. Controlled Release 344, 80-96). The size, PDI, zeta potential, and encapsulation efficiency of the resulting particles were examined to understand the influence of the preparation method on these crucial parameters. Microfluidic mixing involves the precise control of fluid flow rates to ensure rapid and uniform mixing of lipid and aqueous phases, resulting m nanoparticles with consistent properties. Conversely, hand mixing relies on manual techniques, potentially introducingv affability due to less controlled mixing conditions. Using microfluidic mixing, LNPs with smaller and more uniform particle sizes (80 nm to 150 nm) were observed compared to hand-mixed LNPs (100 nm to 200 nm) (FIG. 3A). FIG. 3A show's comparison of size, PDI, zeta potential measurements, and mRNA encapsulation efficiency of THP1 POPE, THPI DOPE, and D-Lm-MC3-DMA formulated using hand mixing and microfluidic mixing. The PDI values were significantly lower for microfluidic LNPs, indicating a narrower size distribution and better homogeneity. Additionally, the zeta potential of microfluidic LNPs was consistently higher, between 4 to 6 mV, compared to 2 to 6 mV for hand-mixed LNPs, suggesting improved stability. It was also noted that microfluidic LNPs exhibited superior encapsulation efficiency, reaching up to 75%, whereas hand-mixed LNPs achieved a maximum of 65%. To evaluate the impact of microfluidic mixing on transfection efficiency, THPI POPE and DLin-MC3-DMA LNPs were intramuscularly administered m C57BL / 6 mice, lire results demonstrated that LNPs formulated using microfluidic mixing exhibited significantly higher transfection efficiency compared to hand-mixed LNPs. Specifically, THPI POPE formulated with microfluidicmixing showed a luminescence signal (average total flux of 1 .3 x 10”) that was markedly higher than both the hand-mixed counterpart and the positive control, DLin-MC3-DMA (average total flux of 5.0x106) (FIG. 3B). FIG. 3B shows representative IVIS images at 6h post injection and graphical representation of total flux of THPl POPE formulated using microfluidic mixing and injectedintramuscularly in C57BL / 6 mice at a dose of 0.5 mg / kg (n=4 biologically independent mice, ±SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, two-tailed unpaired Student's t-test analysis). Notably, THPl POPE demonstrated 26-fold increase in transfection when compared to MC3. This improved performance can be attributed to the more consistent and stable particle characteristics achieved through microfluidic mixing, which likely facilitated enhanced cellular uptake and mRNA release. These results highlight the potential of the THPl L.NP formulation for clinical use for vaccine applications, as it offers a scalable and reproducible method for achieving high mRNA delivery efficiency.

[0341] To better understand the in vivo decay of mRNA and the kinetics of THPl LNPs, THPl POPE and MC3 LNPs were administered intramuscularly into C57BL / 6 mice. It was observed that the bioluminescence intensity of THPl LNPs was higher than that of MC3 LNPs on day 5 after injection, and the decay of FLuc mRNA w!as significantly faster in MC3 LNPs compared to THPl LNPs. Additionally, THPl showed a 116-fold increase in mRNA expression compared to DLin-MC3-DMA on day 5. These results indicated that the duration of protein expression in vivo is significantly longer in THPl LNPs compared to MC'3 LNPs. Furthermore, the THPl POPE LNPs formulation showed sustained protein expression in vivo for up to 5 days when delivered intramuscularly (FIG. 3C). FIG. 3C shows in vivo kinetics of FLuc expression following intramuscular injection. THPl LNPs formulated with Luc mRNA were injected intramuscularly into mice at a dose of 0.5 mg / kg (n==:3biologically independent mice, ± SD). The luciferase expression was visualized at 12 h, 24 h, 48 h, 72 h, and 120 h after injection by IVIS.

[0342] Next, the distribution and transfection efficiency of THPl LNPs were assessed through intravenous injection. THPl with both DOPE and POPE with DLin-MC3-DMA was compared, and the biodistribution studies showed that both THP 1 DOPE and THP 1 POPE were able to efficiently deliver mRNA to the liver (FIG. 4A). FIG. 4A shows representative IVIS images at 24h post injection and graphical representation of total flux of THPl DOPE, THPl POPE and D- Lin-MC3-DMA LNPs injected intravenously at a dose of 0.5 mg / kg (n::::3 biologically independent mice, -t SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). THPl POPPI LNPs showedmuch higher transfection efficiency compared to DLin-MC3-DMA, with an average total flux of 1.6x109 for THP1 POPE LNPs and 1 .3x107 for DLin-MC3-DMA. Tins corresponds to a remarkable 123-fold increase in signal intensity for THP1 POPE LNPs over the positive control. This can be attributed to enhanced stability in circulation and their ability to interact with target cells in the liver. Next, a dose-response study was conducted to assess if the protein expression levels could be regulated, different doses of THP1 POPE containing FLuc mRNA (0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg) were injected intravenously and observed a dose-dependent increase in luciferase expression, validating the feasibility of manipulating endogenous protein expression (FIG. 4B). FIG. 4B show's representative IVIS images at 24h post injection and graphical representation of total flux of different doses (0.25, 0.5, and 1 mg / kg) of THP1 POPE LNPs. THP1 POPE LNPs were intravenously injected in C57BL / 6 mice (n=3 biologically independent mice, ± SD). Assessing the safety profile of LNPs is critical for clinical translation. Body weight changes was monitored over 21 days postinjection and conducted hematological examinations to determine toxicity. THP1 POPE LNPs exhibited no significant body weight loss (FIG. 10). Moreover, hepatic parameters, including ALT, AST, and alkaline phosphatase, remained within normal ranges with a more favorable safety profile than mice treated with MC3 LNPs (FIG. 4C). FIG. 4C show's serum levels of liver enzymes, aspartate mmotransferase (AST), alanine aminotransferase (ALT) and Alkaline Phosphatase after dosingwith PBS, THP1 POPE and D-Lin-MC3-DMA (n = 3, ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, one-w'ay ANOVA with Bonferroni post- hoc analysis). Additionally, transcriptom ics was used to evaluate further the effect of THP1 on gene expression and toxicity at the molecular level. High-quality total RNA was isolated and conducted 75bp pair-end sequencing. The volcano plot displaying the differential gene expression is presented in FIG. 4D, with the x-axis showing the logarithm of fold change and the y-axis showing the False Discovery' Rate (FDR) in a logarithmic scale (FIG. 4D). Tire results clearly demonstrate that none of the genes show significant differences between PBS and THP using FDR < 0.05, further validating no substantial toxic effect of THP1.

[0343] lire apparent pKa has been reported to influence the in vivo tissue specificity of theLNP (Shobaki, N.; et al., (2018) Int. J. Nanomedicine 13, 8395-8410; Patel, P.; et al., (2021) Trends Pharmacol. Sei. 42 (6), 448-460). Negatively charged LNPs tend to target the spleen, whereas positively charged LNPs target the lungs (Kularatne, R. N.; et al., (2022) Pharmaceuticals 15 (7), 897). LNPs with intermediate charges can target the liver, spleen, and lungs. Although charge-mediated targeting has not been thoroughly examined for intramuscular administration, studies indicate that LNPs injected intramuscularly can enterthe vasculature and express in liver hepatocytes, potentially due to passive ApoE-mediated targeting (Sakurai, Y.; et al., (2023) J. Controlled Release 2023. 353, 125-133; Paunovska, K.; et al., (2.022) Nano Lett. 22 (24), 10025-10033; Sebastiani, F.; et al., (2021) ACS Nano 15 (4), 6709-6722). To understand how the pKa affects the in vivo behavior of THP1, the pKa of THP1 POPE compared to MC3 LNPs was evaluated. Both LNPs exhibited apparent pKa values comfortably within the well-established range for liver-targeted LNPs (FIG. HA and FIG. 11B). FIG. 11A and FIG. 11B show pKa measurement of THP1 DOPE and MC3 nanoparticles using TNS fluorescence assay. These results align with prior research on the relative pKa of LNPs and their tissue-specific activity. Additionally, these findings highlight the complexity of factors, including pKa and electric charge, that govern tissue-specific mRNA delivery’ activity through intricate mechanisms. f. THP 1 POPE LNPs ENABLED IMPROVED IN VIVO MRNA DELIVERYAND GENE EDITING.

[0344] After evaluating the in vivo transfection efficiency’ of THP 1 LNPs, their potential for tissue-specific gene editing was investigated. To achieve this, THP1 POPE LNPs were used to examine Cre recombinase-mediated editing in Ail4 mice. The Ail4 transgenic mouse model contains a LoxP-stop-LoxP cassette that inhibits tdTomato expression in all tissues. Upon removal of this stop cassette, tdTomato fluorescence is activated, allowing for the detection of gene-edited cells. Specifically, the Ail4 mice feature a Lox-Stop-Lox-tdTomato construct downstream of a CAG promoter (Xue, L.; et al., (2024) Nat. Commun. 15 (1), 1884; Tuma, J.; et al., (2023) Biochemistry 62 (24), 3533-3547; Sago, C,; et al ., (2021) Blood 138, 2.931), Thus, if Cre mRNA is successfully delivered and translated into Cre protein, the target cells will express tdTomato fluorescence (FIG. 5A). FIG. 5A shows schematic illustration of Cre mRNA delivery and Cre-mediatedgenetic deletion of the stop cassette to activate tdTomato expression in a cre-loxP mouse model (Ail4). LNPs formulated with Cre mRNA were injected intravenously m Ail4 mice, and isolated livers were imaged using the IVIS. In this study, THP1 LNPs were employed to deliver Cre-recombinase mRNA (Cre mRNA), which then expresses Cre protein to excise the stop cassette. Two days post-delivery, highly efficient and liver-selective gene editing were observed, as evidenced by intense tdTomato fluorescence throughout the liver (FIG. 5B). FIG. 5B shows representative tdTomato expression in liver and graphical representation of total flux of THP1 POPE and D-Lin-MC3-DMA LNPs containing Cre mRNA, and PBS treatedcontrol injected intravenously to Ail4 mice at a dose of 0.5 mg / kg (n=3 biologically independent mice, ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). The average total fluorescence for THP 1 was 7.7xlOn, compared to 5.1xl0ufor MC3, indicating a 1.5-fold increase in tdTomato expression with THP1 LNPs compared to MC3 LNPs. Immunohistochemistry and immunofluorescence further validated the expression of tdTomato in hepatocytes of THP1- injected liver samples (FIG. 5C, panels (i) and (ii)). H&E staining revealed no significant liver tissue damage caused by THP1 LNPs (FIG. 5C, panel (iii)). FIG. 5C shows (i) Immunohistochemistry of tdtomato in Ail4 liver: Representative images showing immunohistochemical staining of tdTomato in the liver from Ail4 mice treated with THP1 POPE and D-Lin-MC3-DMA LNPs encapsulating Cre mRNA, and with PBS (n=3 biologically independent mice, images captured at 20x magnification, ± SD). (ii) Immunofluorescence analysis of tdtomato in Ail4 liver: Representative immunofiuorescent images of liver sections from Ail4 mice treated with THP1 POPE and D-Lin-MC3-DMA LNPs encapsulating Cre mRNA, and with PBS, with DAPT staining to highlight nuclei. Images were captured at 20x magnification from three biologically independent mice, (iii) Histological examination of LNP treated Ail 4 livers: Representative histology images of liver sections post-treatment with THP1 POPE and D-Lin-MC3-DMA encapsulating Cre mRNA, and PBS (control) via intravenous injection in Ail 4 mice. Hematoxylin and Eosin (H&E) staining was performed, with images taken at 20x magnification (n = 3 biologically independent mice). Without washing to be bound by theory, these findings demonstrate that THP1 is a highly efficient ionizable lipid and a robust deliver}’ vehicle with promising potential for treating various liver diseases in the future.

[0345] To ensure reproducibility across different synthesis batches, THP1 LNPs from multiple batches were intravenously injected into C57BL / 6 mice and evaluated their mRNA delivery' efficiency. Consistent transfection efficacy across batches affirmed the robustness and reliability of THP 1POPE LNPs (FIG. 12), which is crucial for advancing toward clinical trials and commercialization. FIG. 12 shows the robustness study' for THP1 to check for variation in transfection with different batches of synthesized THP1. Representati ve IVIS images at 24h post injection of LNPs and graphical representation of the total flux of THP 1 POPE formulated using two different batches of THP 1. The formulation was injected intravenously in C57BL / 6 mice at a dose of 0.5 mg / kg (n=3 biologically independent mice, ± SD). In addition to the initial studies with female mice, THP1 LNPs was also injected into male mice and observed similar results, confinning the consistency and reliability of the findings across different genders (FIG. 13). FIG. 13 shows representative IVIS images andgraphical representation of the total flux 24 h after intravenous injection of FLuc mRNA- loaded THP1 POPE LNPs in male mice. The formulation was injected intravenously in C57BL / 6 mice at a dose of 0.5 mg / kg (n=3 biologically independent mice, ± SD), Ensuring the stability of LNPs is pivotal fortheir clinical applicability, especially during storage and transport. The stability of THP 1 POPE LNPs was evaluated by storing them at 4 °C and -20 °C for one month before assessing their mRNA delivery efficacy. Surprisingly, LNPs maintained robust FLuc expression even after 30 days of storage, highlighting their resilience and potential for extended shelf life (FIG. 14A and FIG. 14B). FIG. 14A shows the Z- average size and zeta potential of THP1 POPE LNPs under different conditions. FIG. 14B shows the representative 1VIS images and graphical representation of total flux 24 h after intravenous injection of FLuc mRNA-loaded THP1 POPE LNPs (0.5 mg / kg, n = 2),

[0346] In conclusion, the comprehensive assessment of THP1 POPE LNPs underscores their strong efficiency in delivering mRNA, their safety profile, stability under challenging conditions, and most importantly, their potential for targeted gene editing. These findings position THP1 POPE LNPs as versatile and promising candidates for advancing therapeutic RNA deliver}' strategies, paving the way for their application in clinical trials and future biomedical applications. g. CONCLUSION

[0347] In summary, a facile, catalyst-free, and versatile THP MCR was employed to synthesize a diverse library of novel THP ionizable lipids in just 3 hours, each meticulously tailored for optimal performance in LNP formulations. Through a systematic exploration of these THP ionizable lipids for mRNA delivery, both therapeutic efficacy and safety in nucleic acid-based therapies were enhanced. In initial in vitro screenings, THP1 outperformed the conventional DLin-MC3-DMA in transfection efficiency across HER and RAW 264.7 cell lines. Encouraged by these findings, the library' was evaluated in vivo and optimized the formulations by' varying helperphospholipids. Notably, incorporating POPE significantly' enhanced the transfection efficiency of the mRNA pay load for intramuscular delivery' with sustained protein expression for up to 5 days. Compared to DLin-MC3-DMA, THP1 POPE demonstrated a 116-fold higher protein expression on day 5. Next, it was found that THP1 POPES can deliver mRNA to the liver in a dose dependent manner more efficiently than MC3 and has remarkable stability and safety profiles, sustaining robust luciferase expression without eliciting notable systemic toxicity. Furthermore, these LNPs exhibited targeted geneediting capabilities in the liver of a tdTomato transgenic mouse model, underscoring their precision in therapeutic delivery. These findings underscore the transformative potential of THP ionizable lipids in advancing mRNA therapeutics. Future efforts will focus on refining these formulations for clinical translation, aiming to address critical challenges in nucleic acid delivery and expand applications in gene therapy, gene editing, and vaccine development.3. SYNTHESIS AND EVLAUTION OF MTHP-BASED IONIZABLE LIPIDS

[0348] Additional data was generated through a systematic synthesis and evaluation process involving a combinatorial library' of 108 modified tetrahydropyridine (mTHP)-based ionizable lipids. Initially, six diester intermediates were synthesized by esterifying acetylene dicarboxylic acid with six different alcohols under reflux conditions in toluene. Each diester was then subjected to a multicomponent reaction with three different long -chain primary amines (R1 ), six varied amine head groups (R2), form aldehyde, and a catalytic amount of acetic acid under sonication in ethanol for 8 hours, yielding a diverse set of ionizable lipids.

[0349] These ionizable lipids were formulated into lipid nanoparticles (LNPs) by mixing them with helper lipids (DOPE, cholesterol, and DMG-PEG 2000) and firefly' luciferase (Flue) mRNA at defined molar ratios. The physical characteristics of the LNPs, including particle size and polydispersity, were measured using dynamic light scattering (DLS).

[0350] In vitro transfection efficiency' was assessed by delivering the mTHP LNPs carrying Flue mRNA into HEK 293 cells, followed by quantification of luciferase protein expression.

[0351] For m vivo evaluation, LNPs were grouped based on diester core, alkyl chain length, and amine head group identity. These formulations were intravenously administered into C57BL / 6 mice at 0.5 mg / kg dose. The biodistribution and transfection efficiency were monitored by measuring bioluminescence from luciferase expression using IVIS imaging 18 hours post-injection. Comparative analyses against tire clinically used ionizable lipid MC3 provided benchmarks for performance assessment.

[0352] Without wishing to be bound by theory?, this comprehensive approach allowed identification of key structural features influencing mRNA delivery efficiency and organ targeting.4. SUMMARY OF RESULTS

[0353] A combinatorial library of 108 modified tetrahydropyridine (mTHP)-based ionizable lipids was synthesized using a two-step method. This involved six diester intermediates along with 3 different alkyl chain amines and 6 different amine head groups.These lipids were made into lipid nanoparticles (LNPs) and tested for mRNA delivery effectiveness in vitro and in vivo. In vitro tests showed that the mTHP LNPs had uniform particle sizes of about 100 to 160 nm with low polydispersity, making them suitable for cellular uptake. Among these, mTHP 14, which has tuple alkyl tails and an imidazole head group, displayed transfection efficiency similar to the standard MC3 lipid in HEK 293 cells.This suggests it effectively escapes endosomes and releases mRNA. In vivo studies in mice showed that LNPs grouped by diester core (DI to D6) had different biodistribution patterns. Diesters D I and 04 led to much higher liver expression of luciferase compared to control MC3 LNPs. Further analysis found that longer alkyl chains (Cl 6) improved liver targeting, likely by enhancing membrane interactions. The type of amine head groups (R2) also affected biodistribution, with amine A2 providing better liver delivery. Two lead candidates, mTHP 14 (D1 C16A2) and mTHP68 (D4C16A2), demonstrated beter hepatic delivery’ efficiency than MC3 in vivo. These results highlight the significant impact of diester core, alkyl chain length, and amine head group on LNP performance. They also underscore the promise of these new ionizable lipids for liver-targeted mRNA therapies.J. REFERENCES

[0354] (1) Kariko, K.; Whitehead, K.; Meet, R. van der. What Does the Success of mRNA Vaccines Tell Us about the Future of Biological Therapeutics? Cell Syst. 2021 , 12 (8), 757-758.

[0355] (2) Mukai, H.; Ogawa, K,; Kato, N.; Kawakami, S. Recent Advances in LipidNanoparticles forDelivery of Nucleic Acid, mRNA, and Gene Editing-Based Therapeutics. Drug Metab. Pharmacokinet. 2022, 44, 100450.

[0356] (3) Qin, M.; Li, Y.; Bloomer, H.; Xu, Q. Developing Biodegradable LipidNanoparticles for Intracellular mRNA Delivery and Genome Editing. Acc. Chem. Res. 2021, 54 (21), 4001-401 1.

[0357] (4) Holt, C. E.; Bullock, S. L. Subcellular mRNA Localization in Animal Cells and Why It Matters. Science 2009, 326 (5957), 1212-1216.

[0358] (5) Olszewska, M.; Bujarski, J. J.; Kurpisz, M. P-Bodies and Their Functions during mRNA Cell Cycle: Mini-Review. Cell Biochem. Fund. 2012, 30 (3), 177-182.

[0359] (6) Arribas-Layton, M.; Wu, D Lykke-Andersen, J.; Song, H. Structural andFunctional Control of the Eukaryotic mRNA Decapping Machinery. Biochim. Biophys. Ada BBA - Gene Regul. Meeh. 2013, 1829 (6), 580-589.[00360 j (7) Ross, J. mRNA Stability in Mammalian Cells. MicrobioL Rev. 1995, 59 (3), 423-450.

[0361] (8) Lutz, J.: Lazzaro, S.; Habbeddine, M.; Schmidt, K. E.; Baumhof. P.; Mui,B. L.; Tam, Y. K.; Madden, T. D.; Hope, M. J.; Heidenreich, R.; Fotin-Mleczek, M.Unmodified mRNA inLNPs Constitutes a Competitive Technology for Prophylactic Vaccines. Npj Vaccines 2017, 2 (1), 1-9.

[0362] (9) Geng, ('.: Zhou, K.; Yam Y.; Li. C.; Ni, B.; Liu, J.; Wang, ¥.: Zhang, X.;Wang, D.; Lv, L.; Zhou, Y.; Feng, A.; Wang, Y.; Li, C. A Preparation Method for mRNA- LNPs with Improved Properties. J. Controlled Release 2023, 364, 632-643.

[0363] (10) Schoenmaker, L.; Witzigmann, D.; Kulkarni, J. A.; Verbeke, R.; Kersten,G.; Jiskoot, W.; Crommelin, D. J. A. niRNA-Lipid Nanoparticle COVID-19 Vaccines: Structure and Stability. Ini. J. Pharm. 2021, 601, 12.0586.

[0364] (11 ) Kiaie, S. H.; Majidi Zolbanin, N.; Ahmadi, A.; Bagherifar, R.; Valizadeh,FI.; Kashanchi, F.; Jafari, R. Recent Advances in mRNA-LNP Therapeutics: Immunological and Pharmacological Aspects. J Nanobiotechnology 2022, 20 (1), 276.

[0365] (12) Cui, L,; Pereira, S.; Sonzini, S.; Pelt, S, van; M. Romanelli, S,; Liang, L.;Ulkoski, D.; R. Krishnamurthy, V.; Brannigan, E.; Brankin, C.; S. Desai, A. Development of a High-Throughput Platform for Screening Lipid Nanoparticles for mRNA Delivery.Nanoscale 2022, 14 (4), 1480-1491.

[0366] (13) Jiirgens, D. C.; DeBloch, L.; Porras-Gonzalez, D.; WinkeJjann, J.;Zielinski, S.; Munschauer, M.; Horner, A. L.; Burgstaller, G.; Winkeljann, B.; Merkel, O. M. Lab-Scale siRNA and mRNA LNP Manufacturing by Various Microfluidic Mixing Techniques — an Evaluation of Particle Properties and Efficiency. OpenNano 2023, 12, 100161.

[0367] (14) Eygeris, Y .; Gupta, M.; Kim, J.; Sahay, G. Chemistry of LipidNanoparticles for RNA Delivery. Acc. Chem. Res. 2022, 55 (1), 2-12.

[0368] (15) Safety and Efficacy of the BNT162b2 mRNA Covid- 19 Vaccine I NewEngland Journal of Medicine . https: / / www.nejm.org / doi / full / 10.1056 / nejmoa2034577 (accessed 2024-07-20).

[0369] (16) Advances in COVJD-19 mRNA vaccine development I SignalTransduction and Targeted Therapy. ht^s: / / www.nature.com / articles / s41392-022 -00950-y (accessed 2024-07-20),

[0370] (17) Ndeupen, S.; Qin, Z.; Jacobsen, S.; Bouteau, A,; Estanbouli, H.; Igyarto,B. Z. The mRNA-LNP Platform's Lipid Nanoparticle Component Used in Preclinical Vaccine Studies Is Highly Inflammatory. iScience 2021, 24 (12).

[0371] (18) A, Kulkami, J.; Witzigmann, D.; Leung, J.; C. Tam, Y. Y.; R, Cullis, P.On the Role of Helper Lipids in Lipid Nanoparticle Formulations of siRNA. Nanoscale 2019, I I (45), 21733-21739.

[0372] (19) Zhang. L.: More, K. R.; Ojha, A.; Jackson, C. B.: Quinlan, B. D.; Li, H.:He, W Farzan, M.; Pardi, N.; Choe, H, Effect of mRNA-LNP Components of TwoGlobally -Marketed COVID-19 Vaccines on Efficacy and Stability. Npj Vaccines 2023, 8 (1), 1-14.

[0373] (20) Hald Albertsen, C.: Kulkami, J. A.; Witzigmann, D.; Lind, M.; Petersson,K.; Simonsen, J. B. The Role of Lipid Components in Lipid Nanoparticles for Vaccines and Gene Therapy. Adv. Drug Deliv. Rev. 2022, 188, 1 14416.

[0374] (21) Zhang, Y.; Sun, C.; Wang, C.; Jankovic, K. E.; Dong, Y. Lipids and LipidDerivatives for NA Delivery. Chem. Rev. 2021, 12.1 (2.0), 12181-12277.

[0375] (22) Ball, R. L.; Bajaj, P.; Whitehead, K. A. Achieving Long-Term Stability ofLipid Nanoparticles: Examining the Effect of pH, Temperature, and Lyophilization. Int. J. Nanomedicine 2017, 12, 305-315.

[0376] (23) Koitabashi, K.; Nagumo, H.; Nakao, M.; Machida, T.; Yoshida, K.;Sakai-Kato, K. Acidic pH-Induced Changes in Lipid Nanoparticle Membrane Packing, Biochi m. Biophys. Acta BBA - Biomembr. 2021, 1863 (8), 183627.

[0377] (24) Lam, K.; Leung, A.; Martin, A.; Wood, M.; Schreiner, P.; Palmer, L.;Daly, O.; Zhao, W.; McClintock, K.; Heyes, J. Unsaturated, Trialkyl Ionizable Lipids Are Versatile Lipid- Nanoparticle Components for Therapeutic and Vaccine Applications, Adv. Mater 2023, 35 (15), 2209624.

[0378] (25) Schlich, M.; Palomba, R.; Costabile, G.; Mizrahy, S.; Pannuzzo, M.; Peer,D.; Decuzzi, P. Cytosolic Delivery of Nucleic Acids: The Case of Ionizable Lipid Nanoparticles. Bioeng. Transl. Med. 2021, 6 (2), e 102.13.

[0379] (26) Billingsley, M. M.; Singh, N.; Ravikumar, P.; Zhang, R.; June, C. H.;Mitchell, M. J. Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering, Nano Lett. 2020, 20 (3), 1578-1589.

[0380] (27) Kulkami, J. A.; Darjuan, M. M.; Mercer. J. E.; Chers. S.; van der Meel,R.; Thewalt, J. L.; Tam, Y. Y. C.; Callis, P. R. On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA. ACS Nano 2018, 12 (5), 4787-4795.

[0381] (28) De, A.; Ko, Y. T. Why mRNA -Ionizable LNPs Formulations Are soShort-Lived: Causes and Way-Out Expert Opin. Drug Deltv. 2023, 20 (2), 175-187.

[0382] (29) Lamoot, A.; Lamm ens, J.; Lombaerde, E. D.; Zhong, Z.; Gontsarik, M.;Chen, Y.; Beer, T. R. M. D.; Geest, B. G. D. Successful Batch and Continuous Lyophilization of mRNA LNP Formulations Depend on Cryoprotectants and Ionizable Lipids. Biomater Set. 2023, 11 (12), 4327-4334.

[0383] (30) Carrasco, M. J.; Alishetty, S.; Alameh, M.-G.; Said, H.; Wright, L.;Paige, M.; Soliman, 0.; Weissman, D.; Cleveland, T. E.; Grishaev, A.; Buschmann, M. D. Ionization and Structural Properties of mRNA Lipid Nanoparticles Influence Expression in Intramuscular and Intravascular Administration. Commun. Biol. 2021, 4 (1), 1-15.

[0384] (31) Meanwell, M. W O'Sullivan, C.; Howard, P.; Fyles, T. M. Branched-Chain and Dendritic Lipids for Nanoparticles. Can. J Chem. 2017, 95 (2), 120-129.

[0385] (32) Han, X.; Alameh, M.-G.; Gong, N.; Xue, L.; Ghattas, M.; Bojja, G.; Xu,J.; Zhao, G.; Warzecha, C. C.; Padilla, M. S.; El-Mayta, R.; Dwivedi, G.; Xu, Y .; Vaughan, A. E.; Wilson, J. M.; Weissman, D.; Mitchell, M. J. Fast and Facile Synthesis of Amidine- Incorporated Degradable Lipids for Versatile mRNA Delivery in Vivo. Nat. Chem. 2024, 1- 11.

[0386] (33) He, Z.; Liu, Z.; Chen, Y. Chemical Design Strategy of Ionizable Lipids for In Vivo mRNA Delivery. ChemMedChem 2024 e202400199.

[0387] (34) Farbiak, L.; Cheng, Q.; Wei, T.; Alvarez-Benedicto, E.; Johnson, L. T.;Lee, S.; Siegwart, D. J. All-In-One Dendrimer-Based Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo. Adv. Mater. 2021, 33 (30), 2006619.

[0388] (35) Dong, W.; Li, Z.; Hou, T.; Shen, Y.; Guo, Z.; Su, Y.-T.; Chen, Z.; Pan,H.; Jiang, W2; Wang,Y. Multicomponent Synthesis of Imidazole-Based Ionizable Lipids for Highly Efficient and Spleen-Selective Messenger RNA Delivery. J. Am. Chem. Soc. 2024, 146 (22), 15085-15095.

[0389] (36) De, A.; Ko, Y, T. Single Pot Organic Solvent-Free ThermocyclingTechnology for siRNA-ionizable LNPs: A Proof-of-Concept Approach for Alternative to Microfluidics. Drug Deltv. 2022, 29 (1), 2644-2657.

[0390] (37) Malin, G.; Lapidot, A. Induction of Synthesis of TetrahydropyrimidineDerivatives in Streptomyces Strains and Their Effect on Escherichia Coli in Response to Osmotic and Heat Stress. J Bacteria. 1996, 178 (2), 385-395.

[0391] (38) Darandale, S. N.; Pansare, D. N.; Mulla, N. A.; Shinde, D. B. GreenSynthesis of Tetrahydropyrimidine Analogues and Evaluation of Their Antimicrobial Activity. Bioorg. Med. Chem. Lett. 2013, 23 (9), 2632-2635.

[0392] (39) Kobayashi, J.; Kanda, F.; Ishibashi, M.; Shigemori, H. Manzacidins A-C,Novel Tetrahydropyrimidine Alkaloids from the Okinawan Marine Sponge Hymeniacidon Sp. J. Org. Chem. 1991, 56 (14), 4574-4576.

[0393] (40) Messer, William S.; Abuh, Y. F.: Liu, Y.; Periyasamy, S.; Near. D. O.;Edgar, M. A. N.; El-Assadi, A. A.; Sbeih; Dunbar, P. G.; Rokmch, S.; Rho, T.; Fang, Z.; Ojo, B.; Zhang, H.;Huz1, J. J.; Nagy, P. I. Synthesis and Biological Characterization of 1 ,4,5, 6- Tetrahydropyrimidine and 2-Amino-3,4,5,6-Tetrahydropyridine Derivatives as Selective Ml Agonists. J. Med. Chem. 1997, 40 (8), 1230-1246.

[0394] (41 ) Godhani, D. R.; Dobariya, P. B.; Jogel, A . A .; Sanghani, A. M.; Mehta, J.P. An Efficient Synthesis, Characterization, and Antimicrobial Screening of Tetrahydropyrimidine Derivatives. Med. Chem. Res. 2014, 23 (5), 2417-2425.

[0395] (42) Zhang, M.; Jiang, H.; Liu, H.; Zhu, Q. Convenient One-Pot Synthesis ofMultisubstituted Tetrahydropyrimidines via Catalyst-Free Multicomponent Reactions. Org. Lett. 2007, 9 (21), 4111-4113.

[0396] (43) Syania, K.; Jakubek, Z. J.; Chen, S.; Zaifman, I; Tam, Y. Y. C.; Zou, S.Development of Lipid Nanoparticles and Liposomes Reference Materials (II): Cytotoxic Profiles. Sci. Rep. 2022, 12 (1), 18071.

[0397] (44) Athinarayanan, J.; Periasamy, V. S.; Alshatwi, A. A. Unveiling theBiocompatible Properties of Date Palm Tree (Phoenix Dactylifera L.) Biomass-Derived Lignin Nanoparticles. -ACS' Omega 2022, 7 (2.3), 192.70-19279.

[0398] (45) Duran-Lobato, M.; Martin-Banderas, L.; Lopes, R.; Gonsalves, L. M. D.;Fernandez-Arevalo, M.; Almeida, A. J. Lipid Nanoparticles as an Emerging Platform for Cannabinoid Delivery: Physicochemical Optimization and Biocompatibility. Drug Dev. Ind. Pharm. 2016, 42 (2), 190-198.

[0399] (46) Eygeris, Y.; Patel, S.; Jozic, A.; Sahay, G. Deconvoluting LipidNanoparticle Structure for Messenger RNA Delivery. Nano Lett. 2020, 20 (6), 4543-4549.

[0400] (47) Gan, Z.; Lokugamage, M. P.; Hatit, M. Z. C.; Longhrey, D.; Paunovska,K.; Sato, M.; Cristian, A.; Dahlman, J. E. Nanoparticles Containing ConstrainedPhospholipids Deliver mRNA to Liver Immune Cells in Vivo without Targeting Ligands. Bioeng. Tram. Med. 2020, 5 (3), e 10161 .

[0401] (48) Alvarez-Benedicto, E.; Farbiak, L.; Ramirez, M, M,; Wang, X,; T.Johnson, L.; Mian, O.; D. Guerrero, E.; J. Siegwart, D. Optimization of Phospholipid Chemistry for Improved Lipid Nanoparticle (LNP) Delivery of Messenger RNA (mRNA). Biomater Sci. 2022, 10 (2), 549-559.

[0402] (49) Pattipeiluhu, R.; Zeng, Y.; Hendrix, M. M. R. M.; Voets, I. K.; Kros, A.;Sharp, T. H. Liquid Crystalline Inverted Lipid Phases Encapsulating siRNA Enhance Lipid Nanoparticle Mediated Transfection. Nat. Commun. 2024. 15 (1), 1303.

[0403] (50) Yu, H.; Iscaro, J.; Dyett, B.; Zhang, Y.; Seibt, S.; Martinez, N.; White, J.;Drummond, C. J.; Bozinovski, S.; Zhai, J. Inverse Cubic and Hexagonal Mesophase Evolution within Ionizable Lipid Nanoparticles Correlates with mRNA Transfection in Macrophages. J. Am. Chem. Soc. 2023, 145 (45), 24765-24774.

[0404] (51) Ripoil, M.; Martin, E.: Enot, M.; Robbe, O.; Rapisarda, C.; Nicolai, M.~C.; Dehot, A.; Labeling, P.; Authelin, J.-R.; Nakach, M.; Wils, P. Optimal Self-Assembly of Lipid Nanoparticles (LNP) in a Ring Micromixer. Sci. Rep. 2022, 12 (I), 9483.

[0405] (52) Strelkova Petersen, D. M.; Chaudhary, N.; Anal, M. L.; Weiss, R. M.;Whitehead, K. A. Hie Mixing Method Used to Formulate Lipid Nanoparticles Affects mRNA Delivery Efficacy and Organ Tropism, Eur. J. Pharm. Biopharm. 2023, 192, 126-135. |00406] (53) Nakamura, T.; Kawai, M.; Sato, Y.; Maeki, M.; Tokeshi, M.; Harashirna,H. The Effect of Size and Charge of Lipid Nanoparticles Prepared by Microfluidic Mixing on Their Lymph Node Transitivity and Distribution. Mol. Pharm.. 2020, 17 (3), 944-953.

[0407] (54) Maeki, M.; Uno, S.; Niwa, A.; Okada, Y Tokeshi, M. MicrofluidicTechnologies and Devices for Lipid Nanoparticle-Based RNA Delivery. J. Controlled Release 2022, 344, 80-96.

[0408] (55) Shobaki, N.; Sato, Y.; Harashirna, H. Mixing Lipids to Manipulate theIonization Status of Lipid Nanoparticles for Specific Tissue Targeting. Int. J. Nanomedicine 2018, 13, 8395-8410.

[0409] (56) Patel, P.; Ibrahim, N. M.; Cheng, K. The Importance of Apparent pKa in the Development of Nanoparticles Encapsulating siRNA and mRNA. Trends Pharmacol. Sci. 2021, 42 (6), 448-460.

[0410] (57) Kularatne, R. N.; Crist, R. M.; Stern, S. T. Tire Future of Tissue-TargetedLipid Nanoparticle-Mediated Nucleic Acid Delivery. Pharmaceuticals 2022, 15 (7), 897.

[0411] (58) Sakurai, Y.; Yoshikawa, K.; Aral, K.; Kazaoka, A.; Aoki, S.; Ito, K.;Nakai, Y.; Tange, K.; Furihata, T.; Tanaka, H.; Akita, H. siRNA Delivery to Lymphatic Endothelial Cells viaApoE-Mediated Uptake by Lipid Nanoparticles. J. Controlled Release 2023, 353, 125-133.|00412[ (59) Paunovska, K.; Da Silva Sanchez, A. J.; Lokugamage, M. P.; Loughrey,D.; Echeverri, E. S.; Cristian, A.; Hath, M. Z. C.; Santangelo, P. J.; Zhao, K.; Dahlman, J. E. The Extent to Which Lipid Nanoparticles Require Apolipoprotein E and Low-Density Lipoprotein Receptor for Delivery Changes with Ionizable Lipid Structure. Nano Lett, 2022, 22 (24), 10025-10033.

[0413] (60) Sebastiani, F.; Yanez Arteta, M.; Lerche, M.; Porcar, L.; Lang, C.; Bragg,R, A,; Elmore, C. S.; Krishnamurtby, V, R.; Russell, R. A.; Darwish, T.; Pichler, H.; Waldie,S.; Moulin, M.; Haertlein, M.; Forsyth, V. T.; Lindfors, L.; Cardenas, M. Apolipoprotein E Binding Drives Structural and Compositional Rearrangement of mRNA-Containing Lipid Nanoparticles. A CSNano 2021, 15 (4), 6709-6722.

[0414] (61) Xue, L.; Hamilton, A. G.; Zhao, G.; Xiao, Z.; El-Mayta, R.; Han, X.;Gong, N.; Xiong, X.; Xu, J.; Figueroa-Espada, C. G.; Shepherd, S. J.; Mukalel, A. J.;Alameh, M.-G.; Cui, J.; Wang, K.; Vaughan, A. E.; Weissman, D.; Mitchell, M. J. High- Throughput Barcoding of Nanoparticles Identifies Cationic, Degradable Lipid-like Materials for mRNA Delivery to the Lungs in Female Preclinica! Models. Nat. Commitn. 2024, 15 (1), 1884.

[0415] (62) Tuma, J.; Chen, Y.-J.; Collins, M. G.; Paul, A.; Li, J.; Han. H.; Sharma,R.; Murthy, N.; Lee, H, Y, Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection. Biochemistry 2023, 62 (24), 3533-3547.

[0416] (63) Sago, C.; Campbell, E.; Lutz, B.; Patwardhan, N.; Hamilton, G.; Wong,C.; Lee, D.; Keating, S.; Murray, K.; Singh, M.; Ciaramella, G. Screening of Chemically Distinct LipidNanoparticles In Vivo Using DNA Barcoding Technology Towards Effectively Delivering Messenger RN A to Hematopoietic Stem and Progenitor Cells. Blood 2021, 138, 2931.

[0417] (64) Kauffman, K. J.; Dorkin, J. R.; Yang, J. H.; Heartlein, M. W.; DeRosa,F.; Mir, F. F Fenton, O. S.; Anderson, D. G. Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs. Nano Lett. 2015, 15 (11), 7300-7306.

[0418] (65) Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. Cationic LipidSaturation Influences Intracellular Delivery of Encapsulated Nucleic Acids. J. Controlled Release 2005, 107 (2), 276-287.

[0419] (66) Swingle, K. L.; Safford, H. C.; Geisler, H. C.; Hamilton, A. G.; Thatte, A.S.; Billingsley, M. M.; Joseph, R. A.; Mrksich, K.; Padilla, M. S.; Ghalsasi, A. A.; Alameh, M.-G.; Weissman, D.; Mitchell, M. J. Ionizable Lipid Nanoparticles for In Vivo mRNA Delivery' to the Placenta during Pregnancy. J Am. Chem. Soc. 2023, 145 (8), 4691-4706.

[0420] (67) Martin, M. Cutadapt Removes Adapter Sequences from High-ThroughputSequencing Reads. EMBnetjournal Vol 17 No 1 Gener Seq. Data Anal. 2011.

[0421] (68) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.;Batut, P.; Chaisson, M.; Gingeras, T. R, STAR: Ultrafast Universal RNA-Seq Aligner, BioInformatics 2013, 29 (1), 15-21.

[0422] (69) Robinson, M. D.; McCarthy, D. J.; Smyth, G. K. edgeR: A BioconductorPackage for Differential Expression Analysis of Digital Gene Expression Data.Bioinformatics 2010, 26 (1), 139-140.

[0423] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in tire art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

CLAIMSWhat is claimed is:1 . A compound having a structure represented by a formula:wherein each of R1and R2is independently selected from C2-C21 alkyl and C2-C21 alkenyl; wherein R3is selected from a C8-C20 alkyl, a C8-C20 alkenyl, a -(Cl-C20 alkyl)NR10aR10b’, -(C1-C20 alkylene)Cy3, and -(C2-C20 alkenylene)Cy!; wherein each of R10aand R10bis independently selected from C1-C4 alkyl and Cl- C4 hydroxy alkyl: wherein Cy1is selected from a 5- to 7-membered N-linked heterocy clyl and a 5- membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1-C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino; wherein R4is selected from a C8-C20 alkyl, a C8-C2.0 alkenyl, a-(C1-C20 alkyl)N R11aR11b, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2; wherein each of R11aand R11bis independently selected from C1-C4 alkyl and Cl- C4 hydroxyalkyl; and wherein Cy2is selected from a 5- to 7-membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from halogen, -NO2, -CN, -OH, -SH, -NH2, C1 -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 haloalkyl, C1-C4 cyanoalkyl, C1 -C4 hydroxyalkyl, C1-C4 haloalkoxy, C1-C4 alkoxy, C1-C4 thioalkyl, C1-C4 alkylthiol, C1-C4 aminoalkyl, C1-C4 alkylamino, and (C1-C4)(C1-C4) dialkylamino, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein RJis selected from a C8-C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy1, and -(C2-C20 alkenylene)Cy1and R4is selected from a C8- C20 alkyl, a C8-C20 alkenyl, -(C1-C20 alkylene)Cy2, and -(C2-C20 alkenylene)Cy2.

3. The compound of claim 1 or claim 2, wherein each of R!and R2is independently a C2-C21 alkyl.

4. The compound of claim 1 or claim 2, wherein each of R1and R2is independently a C2-C8 alkyl.

5. The compound of claim 1 or claim 2, wherein each of R1and R2is ethyl.

6. The compound of any one of claims 1 to 5, wherein R3is a C8-C20 alkyl.

7. The compound of any one of claims 1 to 5, wherein R3is a C8 alkyl.

8. The compound of any one of claims 1 to 5, wherein R3is a C8-C20 alkenyl.

9. The compound of claim 8, wherein the C8-C20 alkenyl has a structure represented by a formula:wherein each of r and q is independently an integer selected from 2 to 8,10. The compound of any one of claims 1 to 9, wherein R3is selected from -(C1-C20 alk ylene )Cy1and -(C2-C20 alkenylene)Cy1.

11. The compound of claim 10, wherein Cy1is selected from pyrrolidinyl, morpholinyl, piperazinyl, and imidazolyl, and is unsubstituted.

12. The compound of any one of claims 1 to 11, wherein R3is selected from -(C1-C5 alk ylene )Cy1and -(Cl-C5 alkenylene)Cy1.

13. The compound of any one of claims 1 to 12, wherein R4is a C8-C20 alkyl.

14. The compound of any one of claims 1 to 12, wherein R4is a C8-C20 alkenyl.

15. The compound of claim 14, w herein the C8-C20 alkenyl has a structure represented by a formula:wherein each of s and t is independently an integer selected from 2 to 8.

16. The compound of any one of claims 1 to 12, wherein R4is selected from -(Cl -C20 alkylene)Cy2and -(C2-C20 alkenylene)Cy2.

17. The compound of claim 16, wherein Cy2is selected from pyrrolidinyl, morpholinyl, piperazinyl, and imidazolyl, and is substituted with 0 or 1 methyl group.

18. The compound of any one of claims 1 to 12, wherein R4is selected from —(Cl -C5 alkylene)Cy2and -(C1-C5 alkenylene)Cy2.

19. The compound of any one of claims 1 to 18, wherein Cy1is selected from a 5- to 7- membered N-linked heterocyclyl and a 5-membered N-linked heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from Cl -C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl.

20. The compound of any one of claims 1 to 18, wherein Cy1is selected from a 5- to 7- membered N-linked heterocyclyl and a 5 -membered N-linked heteroaryl, and is substituted with 0 or 1 methyl group.21 . The compound of any one of claims 1 to 20, wherein Cy2is selected from a 5- to 7- membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0, 1, 2, or 3 groups independently selected from C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C1-C4 hydroxyalkyl, and C1-C4 aminoalkyl.

22. The compound of any one of claims 1 to 20, wherein Cy2is selected from a 5- to 7- membered heterocyclyl, and a 5-membered heteroaryl, and is substituted with 0 or 1 methyl group.

23. The compound of claim 1 , wherein the compound has a structure represented by a formula:wherein each of R 3 and R4is a C8-C20 alkyl, or a pharmaceutically acceptable salt thereof.

24. The compound of claim 1 , wherein the compound has a structure represented by a formula:wherein R4is selected from -(C1-C20 alkylene)Cy2and -(C2-C20 alkenylene)Cy2, or a pharmaceutically acceptable salt thereof.

25. The compound of claim 24, wherein R4is selected from -(C1-C5 alkylene)Cy2and - (C1-C5 alkenylene)Cyz.

26. The compound of claim 1, wherein the compound is selected from:or a pharmaceutically acceptable salt thereof.The compound of claim 1, wherein the compound is:or a pharmaceutically acceptable salt thereof.

28. A lipid nanoparticle (LNP) formulation comprising a plurality of nanoparticles, wherein each nanoparticle comprises a phospholipid and the compound of any one of claims 1 to 27.

29. The LNP formulation of claim 28, wherein the phospholipid is selected from 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l-palmitoyl-2-oleoyi-sn-glycero-3- phosphoethanolamme (POPE), l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), l -stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), l,2-dioIeoyl-3- trimethylammonium propane (DOTAP), and l,2-di-O-phytanyl-sn-glycero-3- phosphoethanolamine (4 Me).

30. The LNP formulation of claim 28, wherein the phospholipid is POPE.

31. The LNP formulation of any one of claims 28 to 30, wherein the phospholipid is present in an amount of from about 10 wt% to about 20 wt%.

32. Tire LNP formulation of any one of claims 28 to 30, wherein the phospholipid is present in an amount of about 16 wt%.

33. The LNP formulation of any one of claims 28 to 32, further comprising cholesterol.

34. The LNP formulation of claim 33, wherein cholesterol is present in an amount of from about 40 wt% to about 50 w4%.

35. The LNP formulation of claim 33, wherein cholesterol is present in an amount of about 47 wt%.

36. The LNP formulation of any one of claims 28 to 35, further comprising a PEGylated lipid.

37. The LNP formulation of claim 36, wherein the PEGylated lipid is selected from 1,2- dimyristoyl-rac-glycero-3 -methoxypoly ethylene glycol (DMG-PEG), 1 ,2-distearoy 1-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) (DSPE-PEG), 1 ,2- di stearoyl-sn-Glycero-3 -Phosphoethanolamine with conjugated methoxyl poly (ethylene glycol) (mPEG-DSPE), 1 ,2-dmiyristoyl-sn-glycero~3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000], and l,2-dioleoyl-sn-glycero-3-phosphoethanolamine- N-[amino(polyethylene glycol)] (DOPE), or pharmaceutically acceptable salts thereof.

38. The LNP formulation of claim 36, wherein the PEGylated lipid is DMG-PEG.

39. The LNP formulation of any one of claims 28 to 38, wherein each nanoparticle has a diameter of from about 80 nm to about 200 nm.

40. The LNP formulation of any one of claims 28 to 39, wherein each nanoparticle comprises the phospholipid, the compound of claim 1 , cholesterol, and a PEGylated lipid.

41. The LNP formulation of claim 40, further comprising a messenger ribonucleic acid (mRNA) that encodes a protein or peptide, wherein the mRNA is encapsulated within each nanoparticle.

42. The LNP formulation of claim 41, wherein the protein or peptide is a viral antigen.

43. The LNP formulation of claim 42, wherein the viral antigen is from a virus selected from human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), chicken pox, infectious mononucleosis, mumps, measles, rubella, shingles, ebola, viral gastroenteritis, viral hepatitis, viral meningitis, human metapneumo virus, human parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus, viral pneumonia, Chikungunya vims(CHIKV), Venezuelan equine encephalitis (VEEV), dengue (DENV), influenza, West Nile virus (WNV), human coronavirus, and zika (ZIKV).

44. The LNP formulation of claim 41, wherein the protein or peptide is a tumor antigen.

45. The LNP formulation of claim 44, wherein the tumor antigen is selected from CD19, BCMA, CD36, CD71, CD41a, CD61, CD4, and CD7.

46. The LNP formulation of claim 41 , wherein the protein or peptide is a growth factor.

47. The LNP formulation of claim 46, wherein the growth factor is selected from an epidermal growth factor (EGF), a transforming growth factor (TGF), a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGF), a bone morphogenic protein (BMP), a hepatocyte growth factor (HGF), an insulin-like growth factor (IGF), a platelet derived growth factor (PDGF), and a keratinocyte growth factor (KGF).

48. The LNP formulation of claim 41, wherein the protein or peptide is a gene-editing tool.

49. The LNP formulation of claim 48, wherein the gene-editing tool is selected from a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), Cas9, Cas9 nickase, and Casl2a.

50. A pharmaceutical composition comprising the LNP formulation of any one of claims 28 to 49 and a pharmaceutically acceptable carrier.

51. The pharmaceutical composition of claim 50, wherein the plurality of nanoparticles has a polydispersity index of less than about 0.2.

52. The pharmaceutical composition of claim 50 or claim 51, wherein the plurality of nanoparticles has a zeta potential of from about 2 mV to about 6 mV.

53. The pharmaceutical composition of any one of claims 50 to 52, wherein the composition is formulated for intravenous administration, pulmonary’ administration, or intramuscular administration.

54. A method for delivering an mRNA into a cell, the method comprising contacting the cell wdth the pharmaceutical composition of any one of claims 50 to 53.

55. The method of claim 54, wherein the cell exhibits aberrant expression or activity of the protein or peptide that is encoded by the mRNA prior to the contacting step.

56. The method of claim 55, wherein the aberrant expression or activity comprises expression of a non-functional variant of the protein or peptide.

57. The method of claim 55, wherein the aberrant expression or activity is associated with a genetic disease or disorder.

58. The method of claim 57, wherein the genetic disease or disorder is selected from cystic fibrosis, citrin deficiency, hemophilia, phenylketonuria, classic galactosemia, arginase 1 deficiency, Crigler-Najjar syndrome, alpha-1 antitrypsin deficiency, ornithine transcarbamylase deficiency, progressive familial intrahepatic cholestasis type 3, familial hypercholesterolemia, thylmalonic academia, Fabry disease, glycogen storage disease, acute intermittent porphyria, argininosuccinic aciduria, surfactant protein B deficiency, and propionic acidemia.

59. The method of any one of claims 55 to 58, wherein, upon contacting the cell the pharmaceutical composition, the mRNA is expressed in the cell to produce a functional variant of the protein or peptide.

60. The method of any one of claims 56 to 59, wherein the functional variant of the protein or the peptide is produced in an amount that is greater than an amount of the functional variant of the protein or peptide generated in the absence of the contacting step.

61. The method of any one of claims 54 to 60, wherein said contacting is in vivo.

62. The method of any one of claims 54 to 60, wherein the cell is in a tissue or organ of a subject.

63. The method of claim 62, wherein the tissue or organ is a functionally compromised tissue or organ.

64. The method of claim 62 or claim 63, wherein contacting comprises administering the pharmaceutical composition to the subject.

65. The method of any one of claims 54 to 64, further comprising repeating the contacting step.

66. The method of any one of claims 54 to 65, wherein contacting comprises contacting a plurality of cells with the pharmaceutical composition.

67. Tire method of claim 66, wherein, upon contacting the plurality of cells, the mRNA is expressed in at least 40% of the plurality of cells to produce a functional variant of the protein or peptide that is encoded by the mRNA.

68. A method for delivering an mRNA into a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 50 to 53.

69. The method of claim 68, wherein the subject is a mammal.

70. The method of claim 69, wherein the mammal is a human.

71. The method of any one of claims 68 to 70, wherein, prior to the administering step, the subject has been diagnosed as having a viral infection, cancer, or a genetic disease or disorder.

72. The method of claim 71 , wherein the viral infection is due to a virus selected from human immunodeficiency virus (HIV), human papillomavirus (HPV), herpes simplex virus (HSV), human cytomegalovirus (HCMV), chicken pox, infectious mononucleosis, mumps, measles, rubella, shingles, ebola, viral gastroenteritis, viral hepatitis, viral meningitis, human metapneumovirus, human parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus, viral pneumonia, Chikungunya vims (CHIKV), Venezuelan equine encephalitis (VEEV), dengue (DENV), influenza, West Nile vims (WNV), human coronavirus, and zika (ZIKV).The nanoparticle of claim 41, wherein the protein or peptide is a tumor antigen.

73. The method of claim 71, cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma).

74. The method of claim 71, wherein the genetic disease or disorder is selected from cystic fibrosis, citrin deficiency, hemophilia, phenylketonuria, classic galactosemia, arginase 1 deficiency, Crigler-Najjar syndrome, alpha- 1 antitrypsin deficiency, ornithine transcarbamylase deficiency, progressive familial intrahepatic cholestasis type 3, familial hypercholesterolemia, thylmalonic academia, Fabry disease, glycogen storage disease, acute intermittent porphyria, argininosuccinic aciduria, surfactant protein B deficiency, and propionic acidemia.

75. The method of any one of claims 68 to 74, wherein the subject has been diagnosed with a need for treatment of a viral infection, cancer, or a genetic disease or disorder prior to the administering step.

76. The method of any one of claims 68 to 75, further comprising the step of identifying a subject in need of treatment of a viral infection, cancer, or a genetic disease or disorder.

77. The method of any one of claims 68 to 76, further comprising administering to the subject an effective amount of at least one agent selected from an antiviral agent and a chemotherapeutic agent.

78. The method of claim 77, wherein the composition and the agent are administered simultaneously.

79. The method of claim 77, wherein the composition and the agent are administered sequentially.

80. The method of claim 77, wherein the antiviral agent is selected from acemannan, acyclovir, acyclovir sodium, adamantanamine, adefovir, adenine arabinoside, alovudine, alvircept sudotox, amantadine hydrochloride, aranotin, arildone, atevirdme mesylate, avridine, cidofovir, cipamfylline, cytarabine hydrochloride, BMS 806, C31G, carrageenan, cellulose sulfate, cyclodextrins, dapivinne, delavirdine mesylate, desciclovir, dextrin 2- sulfate, didanosine, disoxaril, dolutegravir, edoxudine, enviradene, envirozime, etravirine, famciclovir, famotme hydrochloride, fiacitabine, fialuridine, fosarilate, foscamet sodium, fosfonet sodium, FTC, ganciclovir, ganciclovir sodium, GSK 1265744, 9-2 -hydroxy-ethoxy methylguanine, ibalizumab, idoxuridine, interferon, 5-iodo-2'-deoxyundine, IQP-0528, kethoxal, lamivudine, lobucavir, maraviroc, memotine pirodavir, penciclovir, raltegravir, ribavirin, rimantadine hydrochloride, rilpivirine (TMC-278), saquinavir mesylate, SCH-C,SCH-D, somantadine hydrochloride, sorivudine, statolon, stavudine, T20, tilorone hydrochloride, TMC120, TMC125, triflnridine, trifluorothymidine, tenofovir, tenofovir alefenamide, tenofovir disoproxyl fumarate, prodrugs of tenofovir, UC-781 , UK-427, UK- 857, valacyclovir, valacyclovir hydrochloride, vidarabine, vidarabine phosphate, vidarabine sodium phosphate, viroxime, zalcitabene, zidovudine, and zinviroxime.81 . The method of claim 77, wherein the chemotherapeutic agent is selected from an alkylating agent, an antimetabolite agent, an antineoplastic antibiotic agent, a mitotic inhibitor agent, and a mTor inhibitor agent.

82. The method of claim 81, wherein the antineoplastic antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorabicin, dactinomycin, epirubicin, idambicin, plicamycin, mitomycin, pentostatm, and valrubicin, or a pharmaceutically acceptable salt thereof.

83. The method of claim 81 , wherein the antimetabolite agent is selected from gemcitabine, 5 -fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof.

84. The method of claim 81, wherein the alkylating agent is selected from carboplatm, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharmaceutically acceptable salt thereof.

85. The method of claim 81, wherein the mitotic inhibitor agent is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or a pharmaceutically acceptable salt thereof.

86. The method of claim 81, wherein the mTor inhibitor agent is selected from everolimus, siroliumus, and temsirolimus, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.

87. The method of any one of claims 68 to 86, wherein administering is via intravenous administration, pulmonary administration, or intramuscular administration.

88. The method of any one of claims 68 to 88, wherein the effective amount is a therapeutically effective amount.

89. Tire method of any one of claims 68 to 88, wherein the effective amount is a prophylactically effective amount.

90. A kit comprising the pharmaceutical composition of any one of claims 50 to 53, and one or more selected from:(a) instructions for delivering an mRNA; and(b) an agent selected from an antiviral agent and a chemotherapeutic agent.

91. The kit of claim 90, wherein the composition and the agent are co-packaged.

Images