Imidazole-based synthetic lipidoids for in vivo mRNA delivery to immune cells

Abstract

Disclosed are lipidoid nanoparticles (LNPs) comprising a lipid compound comprising an imidazole head and the lipidoid compound disclosed herein for efficient nucleic acid delivery to T cells.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 983,997, filed on March 2, 2020.

[0002] Government support This invention was made with government support under Grant Nos. R01 EB027170 - 01 and UG3 TR002636 - 01 awarded by the National Institutes of Health. The government has certain rights in this invention.

Background Art

[0003] Manipulating T lymphocytes has great potential in the advancement of cancer, viral infection, inflammation, and autoimmune therapies. For example, chimeric antigen receptor T cells (CART) have become one of the FDA - approved lymphoma and leukemia therapeutics in recent years. In current clinical strategies, intracellular delivery of therapeutic molecules into primary T lymphocytes depends on viral delivery systems or physical methods such as electroporation. However, this requires ex vivo enrichment of T lymphocytes, resulting in complex procedures and high costs. Therefore, the development of in vivo T cell engineering that provides time - efficient and low - cost treatments is essential. mRNA is a newly emerging approach for cell engineering due to the ease of its synthesis, rapid and transient protein expression, and minimal risk of mutagenesis. Nanomaterials, including polymers and lipid nanoparticles, have been studied for mRNA delivery to various types of cells. However, delivery of mRNA to T lymphocytes remains a technical challenge due to the limited endocytosis and protein translation of T lymphocytes. Therefore, the development of a better delivery system for enhanced in vivo T cell engineering is desired.

Summary of the Invention

Means for Solving the Problems

[0004] In certain embodiments, compounds of formula I and pharmaceutically acceptable salts thereof are disclosed herein: [Chemistry] [wherein, R 5 is -L-W -R 脂質 , hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; wherein only one and only one of R 5 is -L-W -R 脂質 ; L is a divalent linker; W is NR 20 , O, or S; R 脂質 are independently substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkenyl, substituted or unsubstituted C 1-20 alkynyl, substituted or unsubstituted C 1-20 heteroalkyl, substituted or unsubstituted C 1-20 heteroalkenyl, or substituted or unsubstituted C 1-20 heteroalkynyl; R 20 is R 脂質 , H, C 1-6 alkyl, C 1-6 alkenyl, or C 1-6 alkynyl].

[0005] In certain embodiments, W is NR 20 or S. In certain embodiments, W is S. In certain embodiments, W is NR 20 .

[0006] In certain embodiments, R 20 is R 脂質 . Brief Description of the Drawings

[0007] [Figure 1] Figures 1A-1C show the optimization of lipidoid formulations, delivery time, and delivery concentration. Figure 1A shows the luminescence expression of primary human CD8+ T lymphocytes treated with Fluc mRNA loaded 93-O17S formulated with different ratios of excipients. Only the formulated lipidoids showed successful delivery activity. UT: Untreated. Data are presented as mean ± SD, n = 3. Figures 1B and 1C show the luminescence expression of primary human CD8+ T lymphocytes treated with Fluc mRNA loaded 93-O17S in a time-dependent (Figure 1B) and dose-dependent (Figure 1C) manner. UT: Untreated. Data are presented as mean ± SD, n = 2. [Figure 2] Figure 2 is a schematic diagram from rough screening to detailed screening. Imidazole-containing lipidoids were selected from rough screening. A library containing imidazole and imidazole analogs was constructed for detailed screening. The lipidoids selected from the screening were used for in vivo bioluminescence and gene recombination. [Figure 3] Figures 3A-3C show the rough screening of lipidoids for mRNA delivery to primary T lymphocytes in vitro. Figure 3A is the synthetic route of lipidoids. Figure 3B shows the chemical structures of the amine head and carbon tail for lipidoid synthesis. Figure 3C shows the results of the rough screening of different lipidoid libraries in primary human CD8+ T lymphocytes using Fluc mRNA. "F" indicates lipidoids formulated with cholesterol, DOPE, and DSPE-PEG at a weight ratio of 16:4:1:1. "NF" indicates unformulated lipidoids. LF2000: Lipofectamine 2000. mRNA: mRNA alone not loaded into nanoparticles. Data are presented as the mean ± SD of two separate experiments performed in triplicate each. [Figure 4]Figures 4A-4B show the detailed screening of imidazole and imidazole analog head-containing lipidoids. Figure 4A shows the chemical structures of the analog heads of amine head 93. Amines 9310-9315 have different branches and different spacer lengths on the spacer; amines 9321-9324 have branches on 2-imidazole; amines 9331-9334 have branches on 1-imidazole and a spacer on 2-imidazole; amines 9341-9352 have imidazole analogs in place of imidazole. The red structures show a positive effect on delivery, and the blue structures show a negative effect on delivery. Figure 4B shows the results of the detailed screening of imidazole analog heads in primary human CD8+ T lymphocytes using FLuc mRNA. UT: Untreated. Data are presented as mean ± SD, n = 3. [Figure 5] Figures 5A-5B show the detailed screening of lipidoid tails. Figure 5A shows the chemical structures of lipidoids with different tails. Figure 5B shows the results of the detailed screening of lipidoid tails in primary human CD8+ T lymphocytes using FLuc mRNA. Data are presented as mean ± SD, n = 3. *p < 0.05. **p < 0.005. [Figure 6] Figure 6 is a graph showing the flow cytometry histograms of primary human CD8+ T lymphocytes after treatment with different concentrations of 93-O17S or 9322-O17S loaded with EGFP mRNA. UT: Untreated. [Figure 7A] Figures 7A-7B are bar graphs of the pKa and hemolysis analysis of lipidoid nanoparticles. Figure 7A is the pKa analysis of lipidoids in a detailed amine head 93 analog library and tail library. [Figure 7B] Figure 7B is the hemolysis analysis of lipidoids in a detailed amine head 93 analog library and tail library. [Figure 8]Figures 8A - 8C are bioluminescence images by IVIS using the selected lipidoid. Figures 8A - 8B are representative bioluminescence images of the whole mouse (Figure 8A) and organs (Figure 8B) after intravenous injection of FLuc mRNA - loaded lipidoid. Figure 8C shows the detection of luminescence expression from mice intravenously injected with FLuc mRNA - loaded lipidoid after homogenizing and lysing tissues and cells. The luminescence intensity was normalized to the total protein amount. PBS: PBS was injected into the mice. The data are presented as mean ± SD, n = 3. [Figure 9] Figures 9A - 9C are bioluminescence images by IVIS using an invalid lipidoid. Figure 9A is a representative bioluminescence image of the whole mouse by IVIS after intravenous injection of FLuc mRNA - loaded lipidoid. Figure 9B is a representative bioluminescence image of organs by IVIS after intravenous injection of FLuc mRNA - loaded lipidoid 9313 - O18S - S. Figure 9C shows in vivo delivery of Cre recombinase mRNA to Ai14 mice by intravenous administration. tdTomato expression in the spleen was detected by confocal microscopy 10 days after injection. T cells were labeled with CD3ε antibody. [Figure 10] Figures 10A - 10C show in vivo delivery of Cre recombinase mRNA to Ai14 mice by intravenous administration. In Figures 10A and 10B, tdTomato expression in the spleen was detected by confocal microscopy 10 days after injection. T cells were labeled with CD3ε antibody (Figure 10A) and CD8a antibody (Figure 10B). Figure 10C is flow cytometry analysis of the spleen 10 days after injection. tdTomato expression in CD4 + T cells, CD8 + T cells, B cells (CD45R), macrophages (F4 / 80), and dendritic cells (CD11c) was quantified. The data are presented as mean ± SD of 3 mice performed in duplicate respectively. *p < 0.05. **p < 0.005. ***p < 0.001. [Figure 11]Figure 11 shows the in vivo delivery of Cre recombinase mRNA to macrophages. tdTomato expression in the spleen was detected by confocal microscopy 10 days after injection. Macrophages were labeled with an F4 / 80 antibody. [Figure 12] Figure 12 is a graph showing a flow cytometry dot plot of splenocytes after in vivo delivery of Cre recombinase mRNA to Ai14 mice by intravenous administration. tdTomato expression in CD4+ T cells and CD8+ T cells was analyzed 10 days after delivery. **DETAILED DESCRIPTION OF THE INVENTION**

[0008] In certain embodiments, compounds of formula I and pharmaceutically acceptable salts thereof are disclosed herein: **[Chemical Formula]** [wherein, R 5 is -L-W -R 脂質 , hydrogen, halogen, amino, hydroxyl, alkoxy, cyano, nitro, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; wherein only one and only one of R 5 is -L-W -R 脂質 ; L is a divalent linker; W is NR 20 , O, or S; R 脂質 is independently substituted or unsubstituted C 1-20 alkyl, substituted or unsubstituted C 1-20 alkenyl, substituted or unsubstituted C 1-20 alkynyl, substituted or unsubstituted C 1-20 heteroalkyl, substituted or unsubstituted C 1-20 heteroalkenyl, or substituted or unsubstituted C 1-20 heteroalkynyl; R 20 is R脂質 、 H, C 1-6 alkyl, C 1-6 alkenyl, or C 1-6 is alkynyl].

[0009] In certain embodiments, W is NR 20 or S. In certain embodiments, W is S. In certain embodiments, W is NR 20 .

[0010] In certain embodiments, R 20 is R 脂質 .

[0011] In certain embodiments, R 脂質 is represented by Formula II: [Chemical formula] [wherein, R 1 and R 2 are independently H, methyl, OH, NHR 30 , or SH; R 3 and R 4 are both H; or, R 3 and R 4 together form an oxo (=O) group; Z is O, NR 30 , or S; X and Y are independently CH2, NR 30 , O, S, or Se; m is an integer selected from 1 to 3; n is an integer selected from 1 to 14; p is 0 or 1; q is an integer selected from 1 to 10; t is 0 or 1; R 30 is H, C 1-6 alkyl, C 1-6 alkenyl, or C 1-6 is alkynyl].

[0012] In certain embodiments, R 3 and R 4 are both H. In certain embodiments, R 3 and R 4 together form an oxo (=O) group.

[0013] In certain embodiments, p is 0. In certain embodiments, p is 1.

[0014] In certain embodiments, Z is O or NR 30 . In certain embodiments, Z is O. In certain embodiments, Z is NR 30 .

[0015] In certain embodiments, the compound is a compound of Formula III:

Chemical Structure

[0016] In some embodiments, R 1 and R 2 are both H. In certain embodiments, R 1 is H and R 2 is methyl. In certain embodiments, R 1 is H and R 2 is OH.

[0017] In certain embodiments, X and Y are independently CH2 or O. In certain embodiments, X and Y are both CH2. In certain embodiments, X and Y are independently CH2 or O and X and Y are not the same.

[0018] In certain embodiments, X and Y are independently CH2 or S. In certain embodiments, X and Y are both S. In certain embodiments, X and Y are independently CH2 or S and X and Y are not the same.

[0019] In certain embodiments, m is 1 or 2. In certain embodiments, m is 1.

[0020] In certain embodiments, n is an integer selected from 4 to 12. In certain embodiments, n is an integer selected from 6 to 10.

[0021] In certain embodiments, q is an integer selected from 2 to 8. In certain embodiments, q is an integer selected from 4 to 8.

[0022] In certain embodiments, t is 0. In certain embodiments, t is 1.

[0023] In certain embodiments, L is a substituted or unsubstituted C 1-6 alkylene, a substituted or unsubstituted C 1-6 alkenylene, or a substituted or unsubstituted C 1-6 alkynylene, a substituted or unsubstituted C 1-6 heteroalkylene, a substituted or unsubstituted C 1-6 heteroalkenylene, or a substituted or unsubstituted C 1-6 heteroalkynylene.

[0024] In certain embodiments, L is a substituted or unsubstituted C 1-6 alkylene. In certain embodiments, L is an unsubstituted C 1-6 alkylene. In certain embodiments, L is a C 1-6 alkylene substituted with C 1-6 alkyl.

[0025] In certain embodiments, L is

Chemical formula

[0026] In certain embodiments, R 5 is C 1-6 alkyl, C 2-6Alkenyl, C 2-6 is alkynyl. In certain embodiments, R 5 is C 1-6 alkyl.

[0027] In certain embodiments,

Chemical formula

Chemical formula

[0028] In certain embodiments,

Chemical formula

Chemical formula

[0029] In certain embodiments, each instance of R 脂質 is independently selected from the group consisting of

Chemical formula

[0030] In certain embodiments, each instance of R 脂質 is independently selected from the group consisting of

Chemical formula

[0031] In certain embodiments,

Chemical formula

Chemical formula

[0032] In another aspect, lipidoid nanoparticles comprising the compounds disclosed herein are provided.

[0033] In certain embodiments, the lipidoid nanoparticles further comprise cholesterol.

[0034] In certain embodiments, the lipidoid nanoparticles further comprise DOPE or PEG2K-DEPC.

[0035] In certain embodiments, the lipidoid nanoparticles further comprise divalent nickel, and the compound chelates with divalent nickel.

[0036] In certain embodiments, the lipidoid nanoparticles further comprise a protein or a nucleic acid.

[0037] In certain embodiments, the protein or nucleic acid is GFP-Cre or CRISPR / Cas9. In certain embodiments, the protein or nucleic acid is GFP-Cre. In certain embodiments, the protein or nucleic acid is CRISPR / Cas9.

[0038] In certain embodiments, divalent nickel binds to the protein or nucleic acid via non-covalent interaction.

[0039] In certain embodiments, the lipidoid nanoparticles further comprise a small molecule.

[0040] In certain aspects, the small molecule is an antifungal agent or a chemotherapeutic agent.

[0041] In certain embodiments, the small molecule is selected from the group consisting of bortezomib, imatinib, gefitinib, erlotinib, afatinib, osimertinib, dacomitinib, daunorubicin hydrochloride, cytarabine, fluorouracil, irinotecan hydrochloride, vincristine sulfate, methotrexate, paclitaxel, vincristine sulfate, epirubicin, docetaxel, cyclophosphamide, carboplatin, lenalidomide, ibrutinib, abiraterone acetate, enzalutamide, pemetrexed, palbociclib, nilotinib, everolimus, ruxolitinib, epirubicin, pirarubicin, idarubicin, valrubicin, amrubicin, bleomycin, phleomycin, dactinomycin, mitomycin, streptozotocin, pentostatin, mitosanes mitomycin C, enediynes calicheamycin, glycosides rebeccamycin, macrolide lactones epotihilones, ixabepilone, pentostatin, salinosporamide A, vinblastine, vincristine, etoposide, teniposide, vinorelbine, docetaxel, camptothecin, hycamtin, pedrin, theopederins, annamides, trabectedin, aplidine, and ecteinascidin 743 (ET743).

[0042] In one aspect, the small molecule is amphotericin B or doxorubicin.

[0043] In certain embodiments, the lipidoid nanoparticles have a particle size of from about 25 nm to about 1000 nm. In certain embodiments, the lipidoid nanoparticles have a particle size of from about 50 nm to about 750 nm.

[0044] In yet another aspect, there is provided a pharmaceutical composition comprising the lipidoid nanoparticles disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0045] Definitions Unless defined otherwise herein, scientific and technical terms used in this application shall have the meanings commonly understood by one of ordinary skill in the art. Generally, the nomenclature used in connection with, and the laboratory procedures of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics, and protein and nucleic acid chemistry described herein, as well as those techniques, are those well known and commonly employed in the art.

[0046] The methods and techniques of the present disclosure, unless otherwise indicated, generally are carried out according to conventional methods described in various general and more specific references cited and discussed throughout this specification that are well known in the art. See, for example, “Principles of Neural Science”, McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics”, Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.”, W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.”, W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.”, Sinauer Associates, Inc., Sunderland, MA (2000).

[0047] Chemical terms used herein, unless otherwise defined herein, are used according to their conventional usage in the art as exemplified by “The McGraw-Hill Dictionary of Chemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, C.A. (1985).

[0048] As used herein, the term “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, “optionally substituted alkyl” means that the alkyl may or may not be substituted, and also refers to instances where the alkyl is not substituted.

[0049] It is understood that the substituents and substitution patterns on the compounds of the present invention can be selected by those skilled in the art such that they can result in chemically stable compounds that can be readily synthesized from readily available starting materials by techniques known in the art and the methods described below. It is understood that when the substituent itself is substituted with a plurality of groups, these plurality of groups may be on the same carbon or on different carbons as long as a stable structure is obtained.

[0050] As used herein, the term "optionally substituted" refers to replacing one to six hydrogen radicals in a given structure with radicals of specific substituents including, but not limited to: hydroxyl, hydroxyalkyl, alkoxy, halogen, alkyl, nitro, silyl, acyl, acyloxy, aryl, cycloalkyl, heterocyclyl, amino, aminoalkyl, cyano, haloalkyl, haloalkoxy, -OCO-CH2-O-alkyl, -OP(O)(O-alkyl)2, or -CH2-OP(O)(O-alkyl)2. Preferably, "optionally substituted" refers to replacing one to four hydrogen radicals in a given structure with the above substituents. More preferably, one to three hydrogen radicals are substituted by the above substituents. It is understood that the substituents may be further substituted.

[0051] Articles such as "a," "an," and "the" can mean one or more unless indicated otherwise or clear from context. A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or clear from context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments in which one or more or all of the group members are present in, used in, or otherwise relevant to a given product or process.

[0052] As used herein, the term "alkyl" refers to a saturated aliphatic group, C1 to C 10 Straight chain alkyl group or C1-C 10 This includes, but is not limited to, branched alkyl groups. Preferably, the "alkyl" group refers to a C1-C6 straight chain alkyl group or a C1-C6 branched chain alkyl group. Most preferably, the "alkyl" group refers to a C1-C4 straight chain alkyl group or a C1-C4 branched chain alkyl group. Examples of "alkyl" include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, n-butyl, sec-butyl, tert-butyl, 1-pentyl, 2-pentyl, 3-pentyl, neopentyl, 1-hexyl, 2-hexyl, 3-hexyl, 1-heptyl, 2-heptyl, 3-heptyl, 4-heptyl, 1-octyl, 2-octyl, 3-octyl, or 4-octyl. The "alkyl" group may be substituted.

[0053] The term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0054] The term "acylamino" is recognized in the art and refers to an amino group substituted with an acyl group, and can be represented, for example, by the formula hydrocarbyl C(O)NH-.

[0055] The term "acyloxy" is recognized in the art and refers to a group represented by the general formula hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0056] The term "alkoxy" refers to an alkyl group having an oxygen atom attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0057] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group and can be represented by the general formula alkyl-O-alkyl.

[0058] The term "alkyl" refers to a saturated aliphatic group including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substituted alkyl groups. In a preferred embodiment, a straight-chain or branched-chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C for straight-chain and C for branched-chain), more preferably 20 or fewer carbon atoms. 1-30 、branch-chain for C 3-30 )

[0059] Furthermore, the term "alkyl" as used throughout this specification, the examples, and the claims is intended to include both unsubstituted alkyl groups and substituted alkyl groups, the latter referring to an alkyl moiety having a substituent that replaces a hydrogen on one or more carbons of the hydrocarbon backbone and including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl.

[0060] 「C x-y 」or「C x ~C yThe term "」", when used with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, means a group containing x to y carbons in the chain. C0 alkyl represents hydrogen when the group is in a terminal position and a bond when it is internal. For example, C 1-6 The alkyl group contains 1 to 6 carbon atoms in the chain.

[0061] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0062] The term "alkylthio", as used herein, refers to a thiol group substituted with an alkyl group and can be represented by the general formula alkylS-.

[0063] The term "amide", as used herein, refers to the following groups:

Chemical formula

[0064] The terms "amine" and "amino" are recognized in the art and refer to both unsubstituted and substituted amines and their salts, and refer to groups that can be represented, for example, by the following:

Chemical formula

[0065] The term "aminoalkyl", as used herein, refers to an alkyl group substituted with an amino group.

[0066] The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group.

[0067] The term "aryl" as used herein includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is aromatic. For example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0068] The term "carbamate" is recognized in the art and refers to the following group:

Chemical formula

[0069] The term "carbocyclic alkyl", as used herein, refers to an alkyl group substituted with a carbocyclic (carbocyclic) group.

[0070] The term "carbocyclic ring" includes monocyclic rings of 5 to 7 members and bicyclic rings of 8 to 12 members. Each ring of the bicyclic carbocyclic ring can be selected from saturated, unsaturated, and aromatic rings. The carbocyclic ring includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused carbocyclic ring" refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with another ring. Each ring of the fused carbocyclic ring can be selected from saturated, unsaturated, and aromatic rings. In an exemplary embodiment, an aromatic ring, such as phenyl, can be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of the carbocyclic ring as long as the valency permits. Exemplary "carbocyclic rings" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocyclic rings include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. The "carbocyclic ring" can be substituted at any one or more positions that can have hydrogen atoms.

[0071] The term "carbocyclic alkyl", as used herein, refers to an alkyl group substituted with a carbocyclic group.

[0072] The term "carbonate" is recognized in the art and refers to the group -OCO2-.

[0073] The term "carboxy", as used herein, refers to the group represented by the formula -CO2H.

[0074] The term "ester", as used herein, refers to the group -C(O)OR 9 where R 9 represents a hydrocarbyl group.

[0075] As used herein, the term "ether" refers to a hydrocarbyl group bonded to another hydrocarbyl group through oxygen. Thus, an ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. The ether may be symmetric or asymmetric. Examples of ethers include, but are not limited to, heterocyclic-O-heterocyclic and aryl-O-heterocyclic. Ethers include "alkoxyalkyl" groups, which can be represented by the general formula alkyl-O-alkyl.

[0076] As used herein, the terms "halo" and "halogen" mean halogen and include chloro, fluoro, bromo and iodo.

[0077] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a hetaryl group.

[0078] The terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, the ring structure of which contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is heteroaromatic, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0079] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

[0080] As used herein, the term "heterocyclic alkyl" refers to an alkyl group substituted with a heterocyclic group.

[0081] The terms "heterocyclyl", "heterocyclic" and "heterocyclic ring" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3- to 10-membered rings, more preferably 3- to 7-membered rings, the ring structure of which contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic ring" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings and at least one of the rings is a heterocyclic ring, for example, the other cyclic rings can be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, and the like.

[0082] As used herein, the term "hydrocarbyl" refers to a group bonded through a carbon atom that has no =O or =S substituents, typically has at least one carbon-hydrogen bond and predominantly a carbon skeleton, and may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and even trifluoromethyl are considered hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has an =O substituent on the linking carbon) and ethoxy (which is linked through oxygen rather than carbon) are not. Examples of hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, alkynyl, and combinations thereof.

[0083] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0084] When the term "lower" is used in combination with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, it means a group containing up to 10, preferably up to 6 atoms in the substituent. "Lower alkyl" refers to an alkyl group containing, for example, up to 10, preferably up to 6 carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituents as defined herein, whether they appear alone or in combination with other substituents such as in the description of hydroxyalkyl and aralkyl (wherein, for example, when counting the carbon atoms in the alkyl substituent, the atoms in the aryl group are not counted), are each lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy.

[0085] The terms "policyclic", "polycyclic" and "polycyclic ring" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings". Each of the polycyclic rings may or may not be substituted. In certain embodiments, each ring of the polycyclic ring contains from 3 to 10, preferably from 5 to 7 atoms in the ring.

[0086] The term "sulfate" is recognized in the art and refers to the group -OSO3H or a pharmaceutically acceptable salt thereof.

[0087] The term "sulfonamide" is recognized in the art and is represented by the following general formula:

Chemical formula

[0088] The term "sulfoxide" is recognized in the art and refers to the group -S(O)-.

[0089] The term "sulfonate" is recognized in the art and refers to the group SO3H or a pharmaceutically acceptable salt thereof.

[0090] The term "sulfone" is recognized in the art and refers to the group -S(O)2-.

[0091] The term "substituted" refers to a moiety having a substituent that replaces hydrogen on one or more carbons of the backbone. It is understood that "substitution" or "substituted with" includes the implicit condition that such substitution follows the valences of the substituted atoms and substituents and results in a stable compound that does not undergo spontaneous transformation, such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is intended to include all acceptable substituents of an organic compound. In a broad aspect, acceptable substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. Acceptable substituents may be one or more and may be the same or different for a suitable organic compound. For the purposes of the present invention, a heteroatom such as nitrogen may have any acceptable substituent of the organic compounds described herein that satisfies the valence of the hydrogen substituent and / or the heteroatom. Substituents can include any of the substituents described herein, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thiomethanoate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. One of ordinary skill in the art will understand that a substituted moiety on a hydrocarbon chain may itself be substituted where appropriate.

[0092] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0093] As used herein, the term "thioester" refers to the group -C(O)SR 9 or -SC(O)R 9 wherein R 9 represents a hydrocarbyl.

[0094] As used herein, the term "thioether" corresponds to an ether in which oxygen is replaced by sulfur.

[0095] The term "urea" is recognized in the art and can be represented by the following general formula:

Chemical formula

[0096] As used herein, the term "modulate" includes inhibition or suppression of a function or activity (such as cell proliferation), as well as enhancement of a function or activity.

[0097] The phrase "pharmaceutically acceptable" is recognized in the art. In certain embodiments, this term refers to compositions, excipients, adjuvants, polymers, and other materials and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0098] As used herein, the term "salt" is used to refer to an acid addition salt or a base addition salt.

[0099] Many of the compounds useful in the methods and compositions of the present disclosure have at least one stereocenter in their structure. This stereocenter may be present in the R or S configuration, and the R and S notations are used in accordance with the rules described in Pure Appl. Chem. (1976), 45, 11-30. The present disclosure contemplates all stereoisomeric forms, such as enantiomeric and diastereomeric forms of the compounds, salts, prodrugs, or mixtures thereof (including all possible mixtures of stereoisomers). See, for example, WO 01 / 062726.

[0100] Furthermore, certain compounds containing an alkenyl group may exist as Z (together) or E (opposite) isomers. In each case, the present disclosure includes both mixtures and the separate individual isomers.

[0101] Some of the compounds may also exist in tautomeric forms. Such forms are not explicitly shown in the formulas described herein but are intended to be included within the scope of the present disclosure.

[0102] "Pharmaceutically acceptable" means approved or approvable by a federal or state government regulatory agency or the corresponding agency of a country other than the United States or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, more specifically in humans.

[0103] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and can be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include: (1) acid addition salts formed using inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid; or organic acids such as, for example, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, mucic acid; or (2) salts formed when the acidic proton present in the parent compound is replaced by a metal ion such as, for example, an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or when coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. The salts further include, by way of mere example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc.; and, when the compound contains a basic functional group, salts of non-toxic organic or inorganic acids, such as, for example, hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc.

[0104] The term "pharmaceutically acceptable cation" refers to an acceptable cationic counterion of an acidic functional group. Such cations are exemplified by sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium cations, etc. (see, e.g., Berge, et al., J. Pharm. Sci. 66 (1):1-79 (January 77)).

[0105] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient or carrier with which the compounds of the present invention are administered.

[0106] "Pharmaceutically acceptable metabolically cleavable group" refers to a group that is cleaved in vivo to yield the parent molecule of the structural formulas shown herein. Examples of metabolically cleavable groups include -COR, -COOR, -CONRR, and -CH2OR groups, where R is independently selected, at each occurrence, from alkyl, trialkylsilyl, carbocyclic aryl, or carbocyclic aryl substituted with one or more of alkyl, halogen, hydroxy or alkoxy. Specific examples of representative metabolically cleavable groups include acetyl, methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.

[0107] "Prodrug" refers to a compound (including derivatives of the compounds of the present invention) having a cleavable group and becoming a pharmaceutically active compound of the present invention in vivo by solvolysis or under physiological conditions. Such examples include, but are not limited to, choline ester derivatives, N-alkylmorpholine esters, etc. Other derivatives of the compounds of the present invention are active in both their acid and acid derivative forms, but in the acid-sensitive form, often provide advantages in solubility, tissue compatibility, or delayed release in mammalian organisms (see Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well-known to those skilled in the art, such as esters prepared by the reaction of a parent acid with a suitable alcohol, or amides prepared by the reaction of a parent acid compound with a substituted or unsubstituted amine, or acid anhydrides, or mixed anhydrides. Simple aliphatic or aromatic esters, amides, and anhydrides derived from acidic groups pendant to the compounds of the present invention are specific prodrugs. In some cases, it is desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters or (alkoxycarbonyl)oxy)alkyl esters. In particular, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, aryl, C7-C 12 substituted aryl, and C7-C 12 arylalkyl esters.

[0108] "Solvate" usually refers to the form of a compound that associates with a solvent or water (also called "hydrate") by solvolysis reaction. This physical association involves hydrogen bonding. Conventional solvents include water, ethanol, acetic acid, etc. The compounds of the present invention can be prepared, for example, in crystalline form and can be solvated or hydrated. Suitable solvates include pharmaceutically acceptable solvates such as hydrates, and further include both stoichiometric solvates and non-stoichiometric solvates. In certain examples, a solvate can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "Solvate" encompasses both the solution phase and isolable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

[0109] "Subjects" for which administration is contemplated include humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, toddlers, juveniles) or adult subjects (e.g., young adults, middle-aged adults or the elderly)) and / or non-human animals, such as mammals including primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs, but are not limited thereto. In certain embodiments, the subject is a human. In one aspect, the subject is a non-human animal. The terms "human", "patient", and "subject" are used interchangeably herein.

[0110] "Effective amount" means an amount of a compound that, when administered to a subject for treating or preventing a disease, is sufficient to effect such treatment or prevention. The "effective amount" can vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject being treated. "Therapeutically effective amount" refers to an effective amount for therapeutic treatment. "Prophylactically effective amount" refers to an effective amount for prophylactic treatment.

[0111] "Preventing" or "prevention" or "preventive treatment" refers to reducing the risk of acquiring or developing a disease or disorder (i.e., not yet being exposed to an agent that causes the disease or, in a subject predisposed to the disease prior to its onset, not developing at least one of the clinical symptoms of the disease).

[0112] The term "Prophylaxis" is related to "prevention" and refers to means or procedures whose purpose is to prevent rather than to treat or cure a disease. Non-limiting examples of prophylactic means can include administration of a vaccine; administration of low molecular weight heparin to a hospitalized patient at risk of thrombosis due to immobility; and administration of an antimalarial agent such as chloroquine prior to travel to an area where malaria is endemic or where there is a high risk of contracting malaria.

[0113] "Treating" or "treatment" or "therapeutic treatment" of any disease or disorder, in one embodiment, refers to improving the disease or disorder (i.e., halting the disease or reducing the signs, degree or severity of at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to improving at least one physical parameter, which may not be distinguishable by the subject. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of distinguishable symptoms) or physiologically (e.g., stabilization of physical parameters) or both. In a further aspect, "treating" or "treatment" relates to slowing the progression of the disease.

[0114] As used herein, the term "isotope variant" refers to a compound that contains an unnatural proportion of isotopes in one or more of the atoms that make up such a compound. For example, an "isotope variant" of a compound can be, for example, deuterium ( 2 H or D), carbon-13 ( 13 C), nitrogen-15 ( 15It can contain one or more non-radioactive isotopes such as N). In such a compound with isotope substitution, when present, the following atoms can vary, for example, such that any hydrogen can be " 2 H / D", such that any carbon can be 13 C, or such that any nitrogen can be 15 N. It will be understood that the presence and arrangement of such atoms can be determined within the skill of those in the art. Similarly, the present invention can include the preparation of isotope variants having radioactive isotopes, for example, when the resulting compounds can be used in tissue distribution studies of drugs and / or substrates. The radioactive isotopes tritium, that is 3 H, and carbon-14, that is 14 C, are particularly useful for this purpose in view of the ease of their incorporation and the ease of detection means. Further, 11 C, 18 F, 15 O, 13 N and other compounds substituted with positron-emitting isotopes can also be prepared, and they are useful for positron emission tomography (PET) studies to examine the occupancy of substrate receptors. All isotope variants of the compounds provided herein are intended to be encompassed within the scope of the present invention, whether or not they are radioactive substances.

[0115] It should also be understood that compounds having the same molecular formula but different in the nature or order of bonding of their atoms, or the arrangement of those atoms in space, are called "isomers". Isomers that differ in the arrangement of their atoms in space are called "stereoisomers"

[0116] Stereoisomers that are not mirror images of each other are called "diastereomers", and those that are mirror images that cannot be superimposed on each other are called "enantiomers (mirror image isomers)". When a compound has an asymmetric center, for example, when bonded to four different groups, a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and are described by the Cahn and Prelog R- and S-configuration rules or by the manner in which the molecule rotates the plane of polarization and are designated as dextrorotatory or levorotatory (i.e., the (+)- or (-)-isomers, respectively). A chiral compound can exist as either the individual enantiomers or a mixture thereof. A mixture containing equal ratios of enantiomers is called a "racemic mixture".

[0117] "Tautomers" are interchangeable forms of a particular compound structure and refer to compounds that differ in the displacement of hydrogen atoms and electrons. Thus, the two structures can be in equilibrium by the movement of electrons and atoms (usually H). For example, enol and ketone are tautomers because they are rapidly interconverted by treatment with either an acid or a base. Another example of tautomerism is the aci- and nitro-forms of phenylnitromethane, which are similarly formed by treatment with an acid or a base. Tautomeric forms can be relevant to achieving the optimal chemical reactivity and biological activity of the compound of interest.

[0118] As used herein, a pure enantiomeric compound is substantially free of other enantiomers or stereoisomers of that compound (i.e., is enantiomerically pure). In other words, the "S" form of a compound is substantially free of the "R" form of the compound and is thus enantiomerically pure with respect to the "R" form. The terms "enantiomerically pure" or "pure enantiomer" mean that the compound contains greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, or greater than 99.9% by weight of the enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0119] As used herein, unless otherwise indicated, the term "enantiomerically pure R-compound" refers to at least about 95% by weight of the R-compound and at most about 5% by weight of the S-compound, at least about 99% by weight of the R-compound and at most about 1% by weight of the S-compound, or at least about 99.9% by weight of the R-compound and at most about 0.1% by weight of the S-compound. In certain embodiments, the weight is based on the total weight of the compound.

[0120] As used herein, unless otherwise indicated, the term "enantiomerically pure S-compound" or "S-compound" refers to at least about 95% by weight of the S-compound and at most about 5% by weight of the R-compound, at least about 99% by weight of the S-compound and at most about 1% by weight of the R-compound, or at least about 99.9% by weight of the S-compound and at most about 0.1% by weight of the R-compound. In certain embodiments, the weight is based on the total weight of the compound.

[0121] In the compositions provided herein, an enantiomerically pure compound or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof may be present together with other active or inactive ingredients. For example, a pharmaceutical composition containing an enantiomerically pure R compound can contain, for example, about 90% excipient and about 10% enantiomerically pure R compound. In certain embodiments, the enantiomerically pure R compound in such a composition can contain, for example, at least about 95% by weight of the R compound and up to about 5% by weight of the S compound, based on the total weight of the compound. For example, a pharmaceutical composition containing an enantiomerically pure S - compound can contain, for example, about 90% excipient and about 10% enantiomerically pure S - compound. In certain embodiments, the enantiomerically pure S - compound in such a composition can contain, for example, at least about 95% by weight of the S - compound and up to about 5% by weight of the R - compound, based on the total weight of the compound. In certain embodiments, the active ingredient can be formulated without or with little excipient or carrier.

[0122] The compounds of the present invention can have one or more chiral centers; thus, such compounds can be prepared as individual (R)- or (S)-stereoisomers or as mixtures thereof.

[0123] Unless otherwise indicated, the description or naming of a particular compound herein and in the claims is intended to include both the individual enantiomers and mixtures thereof (racemic or otherwise). Methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art.

[0124] One of ordinary skill in organic synthesis will recognize that the maximum number of heteroatoms contained in a stable chemically - achievable heterocyclic ring is determined by the ring size, degree of unsaturation, and valence of the heteroatoms, whether it is aromatic or non - aromatic. Generally, a heterocyclic ring can have from 1 to 4 heteroatoms, provided that the heteroaromatic ring is chemically achievable and stable.

Example

[0125] The following examples are described so that the invention described in this specification can be more fully understood. The examples described in this application are provided to illustrate the compounds, compositions, materials, devices, and methods provided herein and should in no way be construed as limiting their scope.

[0126] Materials and Methods Lipidoid synthesis Chemicals for lipidoid synthesis were purchased from Sigma-Aldrich and used as received. In a glass screw-top vial lined with Teflon (registered trademark), an aliphatic amine head and either an acrylate tail (O17O, O17S, O17Se, O18S-S, O16S-S, and N16S-S) or an epoxide tail (EC18 and EC16) were mixed at a molar ratio of 1 to 2.4 at 70 °C for 48 hours. The crude product was purified using a Teledyne Isco chromatography system.

[0127] Nanoparticle formulation All lipidoids, cholesterol, DOPE, and DSPE-PEG were dissolved in an ethanol solution prior to nanoparticle preparation. For the lipidoid formulation, a 16:4:1:1 (lipidoid:cholesterol:DOPE:DSPE-PEG) w / w ratio was selected (Figure 1A). The ethanol solution was added dropwise to three volumes of 25 mM sodium acetate buffer (pH 5.2). The formulated nanoparticles were dialyzed in a 3.5K MWCO Slide-A-Lyzer dialysis device (Thermo Fisher) for at least 2 hours.

[0128] Isolation of human primary CD8+ T cells Human peripheral blood mononuclear cells (PBMCs) were purchased from Research Blood Components, LLC. Lymphocytes were isolated by density gradient centrifugation using Lympholyte-H (Cedarlane) and washed with PBS. Red blood cells were lysed using RBC lysis buffer (Multi-species) (eBioscience) and washed with PBS. CD8+ T cells were isolated using the CD8+ T cell isolation kit, Human (Miltenyi Biotec) according to the manufacturer's protocol. The purified CD8+ T cells were characterized by flow cytometry using a CD3+ antibody.

[0129] In vitro luciferase assay for lipidoid screening Human primary CD8+ T cells were seeded at 25,000 cells / well in 250 μL of serum-free RPMI medium containing 10 ng / mL of recombinant human IL-2 (BD Biosciences), ImmunoCult Human CD3 / CD28 T cell Activator (STEMCELL Technologies), and 100 U / mL of Pen-Strep (Gibco) in a 48-well plate. FLuc mRNA was delivered to the T cells immediately after seeding. For each well, 0.5 μg of CleanCap FLuc mRNA (TriLink Biotechnologies) and 5 μg of lipidoid were mixed in 50 μL of 25 mM sodium acetate buffer (pH 5.2) and incubated at room temperature for 15 minutes for encapsulation. The mRNA / lipidoid complex was then added to the cell culture medium at final concentrations of 1.7 μg / mL and 17 μg / mL, respectively. The T cells were incubated at 37 °C for 6 hours and then lysed, and luciferase expression was measured using the Firefly Luciferase Assay Kit 2.0 (Biotium) and a SYNERGY H1 microplate reader (BioTek). The graph represents the average luminescence of triplicate experiments, and the error bars represent ±SD. The optimal delivery time and lipidoid concentration were found to be approximately 6 hours and 30 μg / mL, respectively (Figures 1B - 1C).

[0130] In vivo FLuc mRNA Delivery and Bioluminescence Lipidoid / mRNA complexes were prepared as described above. 15 μg of CleanCap FLuc mRNA (5 moU) (TriLink Biotechnologies) and 150 μg of lipidoid were used per mouse in a total volume of 150 μL of PBS. The lipidoid / mRNA complexes were injected intravenously into BALB / c mice. Six hours after injection, 3 mg of D-luciferin was injected intraperitoneally, and bioluminescence was measured using an IVIS Spectrum CT Biophotonic Imager (PerkinElmer). After bioluminescence imaging, the mice were sacrificed, and luminescence from each organ was also measured using the IVIS. Subsequently, the delivery efficacy of FLuc mRNA to T cells was quantified by a luciferase assay. Single cell suspensions were recovered from the spleen by passing splenocytes through a 70 μm cell strainer followed by lysing red blood cells using RBC lysis buffer (Multi-species) (eBioscience). The cells were washed once with PBS, and CD8+ T cells were recovered using CD8a MicroBeads, mouse (Miltenyi Biotec) according to the manufacturer's protocol. Splenocytes and other organs were lysed using Firefly Luciferase Lysis Buffer (Biotium), and luminescence intensity was measured using a Firefly Luciferase Assay Kit 2.0 (Biotium) and a SYNERGYHI microplate reader (BioTek). The total protein amount was quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific). Luciferase intensity was normalized to the total protein amount in the cells or tissues.

[0131] in vivo Cre mRNA Delivery and Confocal Microscopy The lipidoid / mRNA complex was prepared as described above. 15 μg of CleanCap Cre mRNA (5moU) (TriLink Biotechnologies) and 150 μg of lipidoid were used per mouse in a total volume of 150 μL of PBS. The lipidoid / mRNA complex was intravenously injected into Ai14 mice twice every 5 days. 10 days after the first injection, the mice were sacrificed and the spleens were harvested. For confocal microscopy imaging, the spleens were cryosectioned to a thickness of 15 μm. The sections were washed with PBS, fixed with acetone, and then stained with fluorescent antibodies against CD3ε (APC), CD8a (APC), or F480 (APC). The fluorescence signals from tdTomato and the antibodies were observed using an SP8 confocal microscope (Leica). For flow cytometry analysis, splenocytes were passed through a 100 μm cell strainer, and then single cell suspensions were collected from the spleens by lysing red blood cells using RBC lysis buffer (Multi-species) (eBioscience). The cells were washed once with PBS and stained with fluorescent antibodies against CD4 (APC) and CD8a (APC) in Flow Cytometry Staining Buffer (eBioscience). The fluorescence signals were measured using an LSR-II flow cytometer (BD Biosciences).

[0132] Results and Discussion Rough Screening of Lipidoids for mRNA Delivery to Human Primary CD8+ T Cells Cationic lipid-like materials (lipidoids) are synthesized by a combinatorial approach of hydrophilic amine heads and hydrophobic carbon tails by Michael addition reaction (Figures 3A and 3B).

[0133] Lipidoids showing relatively good efficacy for nucleic acid and protein delivery were selected and subjected to a rough screening for luciferase mRNA delivery to primary T lymphocytes in vitro (Figure 3C). Lipidoids were named using a naming rule in the form of R-OnX, R-NnX, or R-ECn. In all cases, "R" indicates the amine number shown in Figure 3B (left column), "n" indicates the number of carbons in the hydrophobic tail before atomic substitution, "X" indicates the content of heteroatoms (O, S, Se, or disulfide) in the tail, and "EC" indicates that the lipidoid tail was synthesized from an epoxide. For the first-stage rough screening, both formulated and non-formulated lipidoids were tested using three excipients, namely cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-[poly(ethylene glycol)-2000] (DSPE-PEG). None of the selected lipidoids showed effective delivery to T lymphocytes when used alone, but some lipidoids were found to show effective delivery when formulated with the above excipients (Figure 3C). Among various libraries, the library with chalcogen (O, S, Se)-containing tails showed the most effective delivery (Figure 3C). Interestingly, lipidoids with amine head 93 (l-(3-aminopropyl)imidazole) always showed effective delivery in all tail variants (93-O17O, 93-O17S, and 93-O17Se) (Figure 3C). Treatment with mRNA alone showed no luminescence expression, indicating that mRNA itself cannot enter cells without an effective lipidoid. Furthermore, even Lipofectamine 2000 (LF2000), the most commonly used commercial lipid reagent, showed no transfection effect at all, revealing a major challenge in T cell transfection by non-viral particles. According to the results of the rough screening, lipidoids with an imidazole group (amine head 93) were found to have great potential in T lymphocyte transfection.

[0134] Synthesis and detailed screening of lipidoids containing imidazole and imidazole analogs Based on the results of the rough screening, it was determined that imidazole is an important structure of lipidoids for mRNA delivery to primary T lymphocytes. New lipidoids containing imidazole or imidazole analogs as shown in Figure 4A were then synthesized.

[0135] The imidazole or imidazole analog-containing amine heads were classified into four groups: i) the carbon spacer between imidazole and the amine group was modified with several carbon branches and different lengths (R: 9310 - 9315); ii) carbon branches with different structures were added to the 2-imidazole position (R: 9321 - 9324); iii) the carbon spacer at the 1-imidazole position was moved to the 2-imidazole position and replaced with carbon chains having various lengths and branches (R: 9331 - 9334); iv) the imidazole ring was replaced with several similar structures (R: 9341 - 9352). Three aliphatic tail variants (O17O, O17S, and O16S-S) were used in the construction of a new library by Michael addition reaction.

[0136] This new library was screened in detail for luciferase mRNA delivery to primary T lymphocytes in vitro as shown in Figure 3b, and the correlation between the amine head structure and delivery efficacy was investigated. Generally, amine heads 9313, 9322, 9331, and 93 always showed high luminescence expression in the lipidoid library. Furthermore, some trends were found in the correlation between the chemical structure of the amine head and delivery efficacy. First, considering the linker branching structure, the inventors found that linkers with a single branch (e.g., 9312 and 9313) showed better efficacy than either linear linkers (e.g., 93, 9310, and 9315) or complex branched linkers (e.g., 9314 and 9316). The length of the linker also seemed to dramatically affect delivery efficacy: 4-carbon 9315 was not as effective as 3-carbon 93. Similarly, the 9311 head, which is structurally similar to 9313 but has one less carbon, showed no delivery effect, while 9313 was highly effective. Second, it was found that branching at the 2-imidazole position did not affect delivery potency (e.g., 9322, 9323, and 9324). Third, the inventors noted that within the group having a linker at the 2-imidazole position (e.g., 9331, 9332, 9333, and 9334), all amine heads had some positive signal, indicating that the linker position could be ortho-substituted and that branching on the imidazole itself did not affect the delivery effect. Finally, amine heads having imidazole ring analogs (e.g., 9341, 9351, 9352) showed no delivery efficacy, demonstrating that the imidazole ring is an important structure in T cell delivery of mRNA. In addition to the amine head structure, the carbon tail structure also affected delivery efficacy. Among the effective amine heads (i.e., 9313, 9322, 9331, and 93), lipidoids having O17O tails and O17S tails showed delivery efficacies that were 5-fold and 6-fold higher, respectively, than those having O16S-S tails (Figure 4B).From these results, it was found that the lipidoids synthesized from amine heads 9313, 9322, 9331, and 9332 were effective for mRNA delivery to T lymphocytes, and these amine heads were used for carbon tail screening.

[0137] Detailed screening of carbon tail structures and delivery efficacy As shown in Figure 4B, the tails of the lipidoids were other important factors in delivery efficacy. To further investigate the effect of tail structures on T cell transfection, a detailed lipidoid library with eight different carbon tails (Figure 5A) was synthesized using amine heads 9313, 9322, 9331, and 9332, which had high transfection efficacy in previous screening.

[0138] As shown in Figure 4B, the lipidoids with O17O, O17S, and O18S-S tails functioned more consistently and efficiently than other tails. Interestingly, for all lipidoids using the O17C tail composed entirely of carbon without heteroatoms, the delivery efficacy was significantly reduced. Furthermore, the length of the carbon chain also affected the delivery efficacy. The 18-carbon tail length (O18S-S) with two carbons replaced by S-S functioned significantly better than the 16-tail length (O16S-S). Additionally, the tail with an ester bond (O16S-S) functioned significantly better than the tail with an amide bond (N16S-S). The tail made from epoxide (ECn series) did not show effective delivery. To elucidate the percentage of positive T cells successfully transfected, EGFP mRNA was delivered to CD8+ T cells in vitro using 93-O17S and 9322-O17S, and GFP expression was quantified by flow cytometry. The delivery efficacy to CD8+ T cells reached 7.1% with 93-O17S and 11.1% with 9322-O17S (Figure 6).

[0139] Possible mechanisms for the structural - related differences in mRNA delivery to T cells may be related to various behaviors of LNPs, such as the apparent pKa value and membrane - disrupting ability. In this study, to further elucidate the possible mechanisms of different delivery effects among these lipidoids, the apparent pKa value and phospholipid bilayer membrane - disrupting ability were further analyzed. There was no significant difference in pKa between effective lipidoids and ineffective lipidoids, indicating that pKa may not be the main factor determining delivery efficacy (Figure 7A).

[0140] However, as shown in Figure 7B, most of the successful lipidoids showed higher membrane - disrupting ability than those that were not successful, especially for those lipidoids with the head of group ii).

[0141] In vivo mRNA delivery and biodistribution of nanoparticles In vitro screening has demonstrated that lipidoids found from structure - based screening can efficiently deliver mRNA to primary T cells. However, the in vivo delivery effect would be more important for in situ programming of T cells in the human body. Furthermore, due to the complex in vivo conditions, the in vivo delivery effect may hardly match the in vitro results. To investigate whether the lipidoids identified by in vitro screening function in vivo, the effect of lipidoid delivery was also evaluated by intravenous injection of the complex of FLuc mRNA and lipidoids into BALB / c mice. Six hours after injection, bioluminescence was detected in live mice, and then the mice were sacrificed and specific bioluminescence in the heart, liver, spleen, lung, and kidney was observed. All bioluminescence imaging was performed using an in vivo imaging system (IVIS). Amines 93 and 9322 with O17O and O17S tails showed strong and specific luminescence expression in the spleen (Figures 8A and 8B).

[0142] In mice delivered with 9322-O17S, the luminescence intensity in the spleen was 1.6-fold higher than that in the liver. On the other hand, amine heads 9313, 9331, and 9332 combined with O17O, O17S, or O18S-S tail showed no signal in any of the organs (Figs. 8A-8C and Figs. 9A-9B).

[0143] Mice were sacrificed, splenocytes were collected, and CD8+ T cells were purified using magnetic beads. Luminescence expression from either splenocytes or any of the organs was quantified. Luminescence signals from other types of cells in the liver and spleen were also present, but the inventors confirmed effective delivery to CD8+ T cells using 93-O17S, 9322-O17O, and 9322-O17S (Fig. 8C). The luminescence intensity also corresponded to the IVIS results (Figs. 8B and 8C).

[0144] Lipidoids enhanced Cre recombinase-mediated gene recombination of splenic T cells in vivo In vivo delivery of gene editing molecules, including mRNA, plasmid, and RNP, to T cells has great potential for applications in T cell engineering. The efficacy of lipidoids for in vivo gene recombination was demonstrated by intravenous delivery of a complex of Cre recombinase mRNA and lipidoid to Ai14 mice (The Jackson Laboratory). The Ai14 mice have a genetically integrated stop codon (STOP codon) adjacent to a loxP site located immediately upstream of the red fluorescent protein (tdTomato) gene 22. The STOP codon can be removed via the activity of Cre protein, which excises the DNA found between the loxP sites. Thus, the red fluorescent signal is mediated by the successful transfection of Cre mRNA, followed by the expression of Cre protein. Cre mRNA / lipidoid complexes were injected twice at 5-day intervals, followed by analysis of mouse spleens with a confocal microscope. Strong tdTomato expression in the spleen was observed in mice injected with either 93-O17S or 9322-O17S (Figures 10A and 10B), but no tdTomato signal was observed in mice injected with 9313-O18S-S or PBS alone (Figure 9C).

[0145] Spleen tissues were labeled with either CD3ε or F4 / 80 antibody to analyze the localization of the tdTomato signal by T lymphocytes or macrophages, respectively. Additionally, CD8a antibody was used to specifically label CD8+ T lymphocytes. The yellow signal in the fluorescence images indicates the co-localization of the red tdTomato signal and the green-tagged cell-type antibody (indicated by white arrows) (Figures 10A - 10B and Figures 8A - 8C).

[0146] A single-cell suspension was recovered from mouse spleens, and tdTomato expression was quantified by flow cytometry. Both 93-O17S and 9322-O17S showed significant expression of the tdTomato signal compared to the untreated control. 93-O17S reached delivery efficacy to approximately 8.2% of CD4+ T cells and approximately 6.5% of CD8+ T cells in vivo (Figures 10C and 10A - 10C).

[0147] Incorporation by Reference All U.S. and PCT patent publications and U.S. patents referred to herein are hereby incorporated by reference in their entirety, as if each individual patent publication or patent were specifically and individually indicated to be incorporated by reference. In case of conflict, this application, including any definitions herein, will control.

[0148] Other Embodiments One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but as set forth in the appended claims. One of ordinary skill in the art will understand that various changes and modifications can be made to this description without departing from the spirit or scope of the invention as defined in the following claims. Finally, the preferred embodiments of the present invention are described item by item. [Embodiment 1] A compound of formula I, or a pharmaceutically acceptable salt thereof:

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Claims

1. A compound of formula III, or a pharmaceutically acceptable salt thereof: 【Chemical 1】 [In the formula, The partial structure A-N is 【Chemistry 2】 having a structure selected from the group consisting of each R 1 and R 2 is independently H, methyl, OH, NHR 30 , or SH; each Y is independently CH 2 , NR 30 , O, S, or Se; each X is independently NR 30 , O, S, or Se; m is an integer selected from 1 to 3; n is an integer selected from 1 to 14; q is an integer selected from 1 to 10; t is 0 or 1; R 30 is H, C 1-6 alkyl, C 1-6 alkenyl, or C 1-6 alkynyl.

2. R １ and R ２ is independently H, methyl, or OH.

3. R １ and R ２ and R are both H.

4. The compound of claim 1, wherein R 1 is H and R 2 is methyl.

5. The compound of claim 1, wherein R 1 is H and R 2 is OH.

6. The compound according to any one of claims 1 to 5, wherein Y is CH 2 .

7. The compound according to claim 1, wherein Y is S.

8. The compound according to claim 1, wherein X is O.

9. The compound according to claim 1, wherein X is S.

10. A compound described in any one of claims 1 to 9, wherein n is 4 to 12.

11. The compound according to claim 1, wherein q is 2 to 8.

12. m is 1 or 2; n is an integer selected from 4 to 12, and The compound according to any one of claims 1 to 9, wherein q is an integer selected from 2 to 8.

13. The partial structure A-N is 【Chemistry 3】 The compound of any one of claims 1 to 12, having the structure:

14. 【Chemical 4】 Each occurrence of independently 【Chemistry 5】 The compound according to any one of claims 1 to 13, selected from the group consisting of:

15. 【Chemical 6】 Each occurrence of independently 【Chemistry 7】 The compound according to any one of claims 1 to 13, selected from the group consisting of:

16. Lipidoid nanoparticles comprising a compound according to any one of claims 1 to 15.

17. The lipidoid nanoparticle of claim 16 further comprising a protein or a nucleic acid.

18. 18. The lipidoid nanoparticle of claim 16 or 17, further comprising a phospholipid or cholesterol.

19. 19. The lipidoid nanoparticle of any one of claims 16 to 18, further comprising cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG), wherein the weight ratio of said compounds to cholesterol to DOPE to DSPE-PEG is about 16:4:1:

1.

20. The lipidoid nanoparticle of any one of claims 16 to 19, further comprising a nucleic acid, said nucleic acid comprising mRNA.

21. The lipidoid nanoparticle of claim 20 , wherein the nucleic acid is an mRNA encoding Cre recombinase or an mRNA encoding luciferase.

22. The lipidoid nanoparticle of any one of claims 16 to 21, further comprising a small molecule, wherein the small molecule is an antifungal agent or a chemotherapeutic agent.

23. 23. A pharmaceutical composition for modifying cells, comprising: a) a compound according to any one of claims 1 to 15, or a lipidoid nanoparticle according to any one of claims 16 to 22, and b) a pharmaceutically acceptable carrier or excipient.

24. 24. The pharmaceutical composition of claim 23, for intravenous administration.

25. 25. The pharmaceutical composition of claim 23 or 24, which is capable of targeting lymphatic tissue of a subject.

26. The pharmaceutical composition of any one of claims 23 to 25, wherein the cells comprise lymphocytes.

27. Use of lipidoid nanoparticles according to any one of claims 16 to 21 in the preparation of a medicament for modifying lymphocytes in vivo.

28. 28. The use of claim 27, wherein the lymphocytes comprise T cells.

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