Lipid conjugates prepared from a scaffold moiety

Abstract

The present application relates to a lipid conjugate of the formula M-X1-L, wherein M is a molecule of interest, e.g., a drug moiety; X1 is a linker, e.g., an ester, ether, or carbamate; and L is a lipid scaffold represented by formula (IId): -L1-[L2(H)(X2R)]n-L3-[L4(H)(X2R)]p-L5-L6, and wherein L comprises 5-40 carbon atoms and 0-20 carbon-carbon double bonds. The lipid conjugate can be formulated in a drug delivery vehicle, e.g., a lipid nanoparticle (LNP).

Description

Technical Field

[0001] This article provides lipid conjugates, lipid conjugate formulations, and precursor molecules for preparing such conjugates.

[0002] background

[0003] Many drugs have the potential to cure cancer, autoimmune diseases, and other ailments, but their therapeutic effects are often unattainable because they cannot reach the site of the disease. For example, when drugs are administered intravenously, typically only a small amount (e.g., usually less than 0.1%) of the drug actually reaches its target. The rest of the drug distributes throughout the body, leading to reduced therapeutic efficacy and adverse side effects.

[0004] Drug delivery systems, including lipid nanoparticles (LNPs) and polymer-based carriers, have the potential to overcome this problem. The aim of such systems is to encapsulate drugs and specifically target them to body sites requiring treatment, such as tumors or areas of inflammation. This effect can be achieved by utilizing the leaky vascular system and impaired lymphatic drainage often found at these disease sites. Regardless of the mechanism, by targeting drugs to specific sites, greater efficacy and lower toxicity can be achieved.

[0005] Nevertheless, only a small fraction of known drugs can be bound to many known drug delivery vehicles. In the case of bilayered LNPs, loading is primarily limited to multi-step, active loading techniques and typically requires the drug to possess an amino group. According to one active loading technique, a transmembrane pH gradient is established, making the interior of the LNP acidic while the external pH is adjusted according to physiological conditions. Uncharged amine-containing drugs, incubated with these LNPs, diffuse into the vesicles and become charged within the LNP due to the protonation of the amine. The charged drug no longer crosses the bilayer and is bound within the LNP. However, many widely prescribed and important drugs do not possess an amino group and cannot be simply encapsulated and retained in LNPs using this method. Therefore, the therapeutic benefits of many potentially effective drugs remain largely unrealized.

[0006] One approach to making a wider range of drugs suitable for incorporation into drug delivery carriers is to conjugate them to lipid moieties. Many drug delivery carriers contain hydrophobic components, and the lipid moieties on the conjugates can enhance the incorporation of drugs into such components. One known strategy involves conjugating the terminal C1 carboxyl end of a fatty acid to a hydroxyl or amino group of a drug. For example, fatty acids such as squalene, stearic acid, oleic acid, palmitic acid, DHA, linoleic acid, octadecanoic acid, lauric acid, and α-tocopherol have been linked to certain drugs to produce drug-lipid conjugates (as reviewed by Irby et al., “Lipid-Drug Conjugate for Enhancing Drug Delivery,” Mol. Pharm. 14(5): 1325-1338, 2017). Drugs can also be linked to lipid moieties via linkers that act as spacers between the drug and the lipids. Linkers for such purposes are known in the art and described, for example, in U.S. Patent No. 5,149,794, which is incorporated herein by reference.

[0007] The ability to control drug release from a delivery vehicle is a crucial factor in achieving optimal therapeutic efficacy. It is well known that hydrophobic compounds retain more binding to membranes or other hydrophobic components of the delivery vehicle than their less hydrophobic counterparts. Therefore, the overall hydrophobicity of a drug-lipid conjugate affects its ability to be released from the drug delivery vehicle after administration. In clinical applications where a longer circulation life in the bloodstream is required for the drug-lipid conjugate to reach the disease site, such as a distant tumor, it is important that the drug remain stably bound to the delivery vehicle for as long as possible. Other clinical applications, such as those requiring local delivery, may require faster release. However, from a practical standpoint, precisely tailoring the hydrophobicity of a given molecule is often challenging.

[0008] The inventors have identified a simple and widely applicable strategy for imparting desired physical properties to drug-conjugates, thereby enabling the clinical application of many potentially effective drugs. This strategy can also be applied to a variety of other molecules of interest besides drugs. Examples include hydrophilic polymers, genetic material, peptides and proteins, such as antibodies, and other molecules of interest.

[0009] The compositions and methods disclosed herein seek to solve this problem and / or provide useful alternatives to those previously described.

[0010] Overview

[0011] The embodiments described herein provide a scaffold molecule referred to as "L", which forms the carbon backbone of the lipid portion of a lipid conjugate, from which one or more groups may be conjugated. The carbon backbone of L has 5-40 or 5-30 carbon atoms and optionally has one or more cis or trans C=C double bonds. L is modular, meaning that it can function as a molecular scaffold from which various combinations of hydrocarbon groups (R and / or R') and molecules of interest (M) (including but not limited to pharmaceutical portions (D) or polymers (optionally via linkers)) can be linked along their carbon backbones via their respective functional groups.

[0012] In one embodiment, the method of the present invention described herein enables more precise control over the hydrophobicity of the molecule of interest, such as a prodrug. Without limitation, the molecule of interest can be designed to have a desired octanol / water LogP value by selecting a suitable hydrocarbon R conjugated to the scaffold L.

[0013] The ability to more precisely tailor the hydrophobicity of molecules of interest (e.g., drugs or other molecules of interest) offers several benefits. In some non-limiting embodiments, the inventors have shown that lipid conjugates with hydrophobic properties can be designed such that 100% encapsulation efficiency can be achieved when loaded into a given delivery carrier. Furthermore, the retention of lipid conjugates in the delivery carrier after administration to a patient can be more precisely controlled. For example, it has been found that the predicted LogP values ​​of certain lipid conjugates described herein are generally correlated with their ability to be retained in a drug delivery carrier. Therefore, by adjusting the LogP value of the prodrug, for example by selecting a suitable R group as described herein, more precise control of drug release can be achieved.

[0014] Typically, the lipid moiety of the molecule of interest governs the overall hydrophobicity of the conjugate. Therefore, a wide range of molecules can be selected for incorporation into prodrugs. This includes drugs, polymers, and other molecules of interest.

[0015] This document also describes novel pharmaceuticals and drug delivery compositions comprising lipid conjugates. These conjugates may be incorporated into pharmaceutical compositions comprising pharmaceutically acceptable salts and / or excipients, or into drug delivery carriers forming components of the pharmaceutical composition. Alternatively, the conjugates may be incorporated into consumer products, including but not limited to food, nutritional supplements, cosmetics, or cleaning products.

[0016] As described herein, this disclosure is also based on the finding that LNP formulations incorporating lipid conjugates exhibit spherical, electron-dense regions at the membrane. In such embodiments, the lipid nanoparticles comprise a bilayer, a lipid conjugate, and a hydrophobic oil phase composed of the lipid conjugate. In one embodiment, the lipid nanoparticles are liposomes. In another embodiment, the lipid conjugate has a structure of formula I, Ia, II, or IIa as illustrated herein.

[0017] In some embodiments, this document provides a lipid conjugate comprising a branched lipid moiety having a backbone L, the backbone serving as a scaffold for attaching one or more hydrocarbon R chains thereto, the lipid moiety having a structure of formula IId:

[0018] Formula IId:

[0019]

[0020] The main chain of the L lipid scaffold is represented by L1+L2+L3+L4+L5+L6, and L contains 5-40 carbon atoms and 0-2 cis or trans C=C double bonds;

[0021] Wherein L1 is a carbon chain with 3-30 carbon atoms, and optionally L1 has one or more cis or trans C=C double bonds or 0-2 cis or trans C=C double bonds;

[0022] L2 and L4 are each carbon atoms;

[0023] L3 consists of 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds;

[0024] L5 consists of 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds;

[0025] L6 is -CH3, =CH2, or H;

[0026] Each R is independently a linear or branched hydrocarbon chain having 0-30 carbon atoms and 0-2 cis or trans C=C double bonds, wherein if one or more of R are branched, each branching point includes an X2 functional group.

[0027] Where n is 0-8 and p is 0-8, and n+p is ≥1 or 1-8;

[0028] Each X2 is independently an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thiocarbonyl carbamate, guanine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, aminophosphate, phosphate, phosphonate, phosphate diester, phosphate phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, carbon-based functional group including alkanes, alkenes or alkynes, methylene (CH2) or urea;

[0029] Or where X2 is a link containing at least one hydrogen bond; and

[0030] The conjugates described therein are not ionizable lipids.

[0031] In some embodiments, X2 is independently a biodegradable group after administration to a patient. X2 may be independently linked to a carbamate, ether, or ester.

[0032] In yet another embodiment, L is linked at L1 to the molecule of interest M in the conjugate via X1 to form M-X1-L, wherein X1 is an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thiocarbonyl carbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, aminophosphate, phosphate ester, phosphonate, phosphate diester, phosphate phosphonomethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, carbon-based functional group including alkanes, alkenes, or alkynes, methylene (CH2), or urea; or wherein L is linked to the molecule of interest via a hydrogen bond between L and M of the lipid conjugate. In some embodiments, X1 is an ester, ether, or carbamate.

[0033] In another embodiment, the second L is linked to the molecule of interest via X1. Optionally, the second L has the structure of formula IId.

[0034] In one implementation, L1 has 3-30 carbon atoms or 4-30 carbon atoms.

[0035] In yet another embodiment, L is linked to the molecule of interest M via a hydrogen bond between L and M of the lipid conjugate, and wherein L-X1-M has the structure of formula V:

[0036] Formula V:

[0037]

[0038] E1, E2, E3, E4 and E5 are electronegative atoms independently selected from O, N and P;

[0039] E1, E2, and E3 are hydrogen bond acceptors, while E4 and E5 are hydrogen bond donors;

[0040] Dashed lines depict hydrogen bonds and solid lines depict covalent bonds;

[0041] Where L represents the lipid scaffold of the lipid portion;

[0042] n is 0 or 1; o is 0 or 1, and p is 0 or 1; and n + o + p ≥ 2;

[0043] q is 1-10, 2-10, or 4-10;

[0044] L represents the lipid scaffold of the lipid portion;

[0045] M is the molecule of interest; and

[0046] E1 and E3 optionally contain substituents connected thereto, the substituents being independently selected from alkyl, aryl, alkylene, or H.

[0047] In one embodiment, at least one R is branched and each branching point of R is independently selected from esters, ethers, or carbamates.

[0048] In another alternative embodiment, the lipid portion is non-cylindrical and is funnel-shaped or truncated conical in the direction from L1 to L6.

[0049] According to one implementation scheme, X2 is not a disulfide or thioether group.

[0050] According to another alternative embodiment, the lipid moiety is derived from a lipid having one or more reactive groups selected from hydroxyl, amino, and / or amide, the reactive groups being bonded to their internal carbon atoms to act as a scaffold carbon chain in the lipid moiety, and at least one other hydrocarbon chain in the hydrocarbon structure being derived from an acyl lipid, and wherein the X1 link is formed by the reaction of the reactive groups on the scaffold carbon chain with the carboxylic acid of the acyl chain.

[0051] This article further provides a lipid conjugate comprising a branched lipid moiety having a backbone L, the backbone L serving as a scaffold for attaching one or more R hydrocarbon chains thereto, the lipid moiety having a structure of formula IIe:

[0052] Formula IIe:

[0053]

[0054] Where L is composed of [CH2] m –L2–L3–L4–[CH2] q –CH3 indicates that the total number of carbon atoms in L is 5-30;

[0055] L2 and L4 are carbon atoms;

[0056] Where m is 0-20; n is 1-4; p is 0-4; and n+p is 1-4.

[0057] L3 consists of 0-10 carbon atoms and has 0-2 cis or trans C=C arrangements;

[0058] X2 is independently selected from ether, ester and carbamate groups;

[0059] Each R is independent of:

[0060] (a) A linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms, wherein each R is fused with one of its respective X2 at any carbon atom in the hydrocarbon chain; or

[0061] (b) A branched structure of formula IIb with a support represented by L':

[0062] Formula IIb:

[0063]

[0064] Where L' is composed of [CH2] r –L2–G3–L4–[CH2] u –CH3 indicates that the total number of carbon atoms in L is 3-30;

[0065] Where r is 0-20, 2-20, 3-20 or 4-20;

[0066] s is 0-4, t is 0-4; and s+t is either >1 or 1-4;

[0067] u is 1-20;

[0068] G3 consists of 0-10 carbon atoms and has 0-2 cis or trans C=C arrangements;

[0069] Each R' in formula IIb is independently a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms;

[0070] The total number of R' hydrocarbon chains in formula IIb is 1-16;

[0071] In the lipid moiety, each of the R and R' hydrocarbon chains is optionally substituted with a heteroatom, provided that no more than eight heteroatoms are substituted in the R and R' hydrocarbon chains, and the predicted or experimental logP of the conjugate is greater than 5; and

[0072] The lipid-conjugates mentioned therein are not ionizable lipids.

[0073] According to any of the above embodiments, the scaffold lipid L is derived from hydroxy lipids.

[0074] In yet another embodiment, the lipid conjugate has the structure of any of the lipid conjugates shown in FIG1.

[0075] According to another aspect, pharmaceutical compositions comprising the conjugates as described above are provided. For example, the conjugates can be formulated into nanoparticles, such as lipid nanoparticles. According to another embodiment, the nanoparticles comprise one or more bilayers.

[0076] Methods for treating cancer or infection are also provided, which include applying the conjugate as described above.

[0077] According to another embodiment, a prodrug having the structure of Formula I is provided:

[0078] Formula I:

[0079] M-X1-[L]-X2-R

[0080] in

[0081] M represents the drug component D;

[0082] X1 is a chemical connection that covalently links D to any carbon atom on L;

[0083] L is a scaffold carbon chain having 5-40 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0084] X2 is a chemical connection that covalently links R to any carbon atom on L; and

[0085] R is a linear or branched hydrocarbon having 1-40 carbon atoms and optionally one or more cis or trans C=C double bonds.

[0086] X1 and X2 are independently selected from functional groups or linker groups.

[0087] According to another embodiment, a prodrug having the structure of formula Ia as described above is provided:

[0088] Formula Ia:

[0089]

[0090] in

[0091] L1-L2 are scaffold carbon chains L with 5-40 carbon atoms;

[0092] L1 is a carbon chain having 5-40 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0093] L2 is a carbon chain having L minus L1 carbon atoms and optionally having one or more cis or trans C=C double bonds; and X2 causes R to be covalently attached to L2 at any carbon atom on L2.

[0094] In some embodiments, the prodrug has a value of at least 5 logP.

[0095] In an optional embodiment, the prodrug further comprises a second side R hydrocarbon chain having 1-40 carbon atoms, which is chemically linked to L via X2 covalent bonding.

[0096] The prodrug may also contain a third side chain R having 1-40 carbon atoms, which is covalently bonded to L via an X2 chemical linker.

[0097] The prodrug may include an R′ side chain that is connected to the first R via an X2 linker. The prodrug may also include an additional R′ side chain that is connected to another R via an X2 linker.

[0098] The X1 and X2 connections can be independently selected from connections containing one or more functional groups selected from esters, amides, amidines, hydrazones, disulfides, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphates, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas.

[0099] The X1 and X2 links of the prodrug may contain at least one group that is biodegradable after administration to a patient.

[0100] In one embodiment, the prodrug X1 is a linker and optionally biodegradable.

[0101] The (M-X1) part of equation I or Ia can have the following equation IV:

[0102] Formula IV:

[0103] M-[X4-M1-X5] X1

[0104] X4 and X5 are independently selected from esters, amides, amidines, hydrazones, disulfides, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acylalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2) or ureas; and M1 is an optional spacer group attached to the functional groups X4 and X5 and has 0-12 carbon atoms; or M1 is optionally CH2, CH2CH2, N-alkyl, N-acyl, O, or S.

[0105] In some embodiments, R is -CMe3, -Me, or a linear carbon chain having 2-40 carbon atoms and optionally 1-6 cis or trans double bonds.

[0106] Drug component D can be derived from anticancer drugs or immunomodulators.

[0107] Drug fraction D can be derived from docetaxel, dexamethasone, methotrexate, NPCII, abiraterone, prednisone, prednisolone, ruxolitinib, tofacitinib, calcitriol, calcidiol, cholecalciferol, sirolimus, tacrolimus, acetylsalicylic acid, mycophenolate mofetil, cabazitaxel, betamethasone, and NLRP3 inhibitors, including CYO9(4-[[4-oxo-2-thio-3-[[3-(trifluoromethyl)phenyl]methyl]-5- [Thiazolyl]methyl]benzoic acid), INT-MA014 or MCC950 (N-(1,2,3,5,6,7-hexahydro-s-indarin-4-ylcarbamoyl)-4-(2-hydroxy-2-propyl)-2-furansulfonamide) or derivatives thereof, or cannabinoids, including cannabinol, cannabilotriol, cannabidiol, cannabidiol, cannabilotriol, cannabidiol, cannabilotriol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, tetrahydrocannabinol or tetrahydrocannabinol or derivatives thereof.

[0108] The prodrug can be INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-D049, INT-D050, INT-D051, INT-D050, INT-D051, INT-D055, INT-D056, INT-D057, INT-D058, INT-D059, INT-D060, INT-D061, INT-D062, INT-D 063, INT-D064, INT-D065, INT-D066, INT-D067, INT-D053, INT-D068, INT-D069, INT-D070, INT-D071, INT-D07 2. INT-D073, INT-D074, INT-D075, INT-D076, INT-D077, INT-D078, INT-D079, INT-D080, INT-D081 or INT-D082.

[0109] Prodrugs with the structure of Formula I are also provided:

[0110] Formula I:

[0111] M-X1-[L]-X2-R

[0112] in

[0113] M is the drug fraction (D) derived from anticancer drugs or immunomodulators;

[0114] X1 is a linker containing one or more biodegradable groups, which covalently links D to any carbon atom on L;

[0115] L is a scaffold carbon chain derived from a hydroxy fatty acid having 16-20 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0116] X2 is a chemical bond that covalently links S to any carbon atom on L; and

[0117] R is a linear or branched hydrocarbon having 1-25 carbon atoms and optionally one or more cis or trans C=C double bonds, and is derived from an acyl chain.

[0118] R assigns a predetermined LogP value to the prodrug.

[0119] Also provided is a prodrug molecule P for preparing a prodrug, the prodrug scaffold molecule P having the following formula:

[0120] Formula III:

[0121] RG-[L]-X2-R

[0122] RG is a reactive functional group containing at least one reactive atom selected from O, C, N, P, S, Si or B;

[0123] L is a scaffold carbon chain having 5-40 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0124] X2 is a chemical connection that covalently links R to any carbon atom on L; and

[0125] R is a hydrocarbon having 1-40 carbon atoms and optionally one or more cis or trans C=C double bonds.

[0126] A precursor molecule P having the following formula is also provided:

[0127] Formula IIIa:

[0128]

[0129] in

[0130] L1-L2 are scaffold carbon chains L with 5-40 carbon atoms;

[0131] L1 is a carbon chain having 5-30 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0132] L2 is a carbon chain having L minus L1 carbon atoms and optionally one or more cis or trans C=C double bonds; and

[0133] X2 covalently bonds R to L2 at any carbon atom in L2.

[0134] RG can be a hydroxyl, amine, or carboxyl group. In one embodiment, X2 is an ester group. R can be derived from an acyl chain. In one embodiment, the resulting first and second connections are ester connections.

[0135] According to any of the above embodiments, the scaffold lipid is derived from hydroxy lipids.

[0136] A method for preparing a prodrug is also provided, the method comprising: providing a precursor molecule as defined in any of the above embodiments; and conjugating the precursor molecule to a drug D, a linker, or a drug-linker to produce a prodrug.

[0137] It also provides prodrugs produced from the aforementioned precursor molecules.

[0138] It also provides methods for treating cancer, autoimmune diseases, or infections, said methods including administering a prodrug of any of the above-described embodiments.

[0139] Pharmaceutical compositions comprising a lipid conjugate of any of the above embodiments are also provided. In another embodiment, nanoparticles comprising a prodrug of one of the above embodiments are provided. In an optional embodiment, the nanoparticles are liposomes. Brief description of the attached diagram

[0141] Figure 1 depicts various prodrugs that can be prepared according to certain embodiments based on scaffold molecule L;

[0142] Figure 2 depicts various prodrugs that can be prepared according to certain embodiments based on ricinoleyl-based lipid scaffolds;

[0143] Figure 3 depicts the chemical structures of various prodrugs containing castor oil-based lipid scaffolds;

[0144] Figure 4 Electron micrographs showing various molar percentages (10, 20, 30, 40 and 80 mol%) of dexamethasone prodrugs conjugated to castor oil group + hexanoyl group (INT-D034) in lipid nanoparticle (LNP) formulations.

[0145] Figure 5 Electron micrographs showing dexamethasone conjugated to castor oil + hexanoyl group (INT-D034; left panel) and dexamethasone conjugated to castor oil + oleoyl group (INT-D035; right panel) in LNP formulations at a prodrug concentration of 10 mol%.

[0146] Figure 6AThis study shows the dissociation over time of various castor oil-based dexamethasone prodrugs (INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-D049, INT-D050, INT-D051, INT-D085, INT-D086, and INT-D089) formulated in LNP at 10 mol% in human plasma. The residual amount of the prodrug in each LNP formulation was measured at 0 hours (left bar) and 2 hours (right bar) after incubation.

[0147] Figure 6B This study demonstrates the dissociation over time of various castor oil-based dexamethasone (INT-D034, INT-D045) or castor oil-based calcitriol (INT-D053, INT-D083) prodrugs formulated in LNP at 10-99 mol% in human plasma. The residual amount of prodrug in each LNP formulation was measured at 0 hours (left bar) and 2 hours (right bar) after incubation.

[0148] Figure 6C This study illustrates the dissociation over time of various castor oil-based dexamethasone (INT-D034, INT-D045) or castor oil-based calcitriol (INT-D053, INT-D083) prodrugs formulated in LNP at 99 mol% in human plasma. The residual amount of prodrug in each LNP formulation was measured at 0 h (left bar), 2 h (middle bar), and 24 h (right bar) after incubation.

[0149] Figure 7A This is a schematic diagram depicting the decomposition of various castor oil-based dexamethasone prodrugs (INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-D049, D050, D051, D085, and D089) formulated in LNP at 10 mol% after incubation in mouse plasma. The relative amounts of intact prodrugs in each LNP were measured at 0 h (left bar) and 2 h after incubation (right bar), as determined by ultra-high pressure liquid chromatography (UPLC). The data were calibrated against the amounts of the corresponding conjugates in the pre-incubated mixture. Error bars represent three independent experiments.

[0150] Figure 7BThis is a schematic diagram depicting the amount of dexamethasone released from different LNP formulations of castor oil-based dexamethasone prodrugs (INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-D049, INT-D050, and INT-D051) in mouse plasma over time after incubation. The prodrugs were formulated at 10 mol% in LNPs, and free drug was measured after 2 hours of incubation, and the results are reported as area under the curve (AU). Error bars represent three independent experiments.

[0151] Figure 7C This diagram illustrates the degradation of various ricinole-based dexamethasone (INT-D034, INT-D045) or ricinole-based calcitriol (INT-D053, INT-D083) prodrugs formulated in LNPs at 10–99 mol% over time after incubation in mouse plasma. The relative amounts of intact prodrugs in each LNP were measured at 0 h (left bar) and 2 h after incubation (right bar), as determined by ultra-high pressure liquid chromatography (UPLC). Data were normalized to the amount of the corresponding conjugate in the pre-incubated mixture. Error bars represent three independent experiments.

[0152] Figure 8 This diagram illustrates the pro-inflammatory cytokine levels in the macrophage line J774.2 cultured with LNP formulations containing the prodrugs INT-D034 and INT-D035 (D034 and D035), free dexamethasone (Dex-21-P), LNP without the prodrug (control), and untreated cells. The schematic depicts the expression of cytokines IL-1β (upper), TNFα (middle), and IL-6 (lower) after 24 hours of incubation with the components equivalent to 1, 3, or 10 μM dexamethasone, followed by overnight stimulation with 10 ng / mL lipopolysaccharide (LPS). Cytokine levels were measured by qRT-PCR, and the data were calibrated against control LNP treatment without the drug-lipid conjugate.

[0153] Figure 9AThis diagram illustrates the pro-inflammatory cytokine levels in Raw264.7 cells incubated with LNP formulations containing prodrugs INT-D034 and INT-D035 (D034 and D035), free dexamethasone (Dex-21-P), LNP without prodrugs (control), and untreated cells. The schematic depicts the expression of cytokines IL-1β (upper), TNFα (middle), and IL-6 (lower) after 24 hours of incubation with the aforementioned components equivalent to doses of 1, 3, or 10 μM dexamethasone, followed by overnight stimulation with 10 ng / mL LPS. Cytokine levels were measured by qRT-PCR, and the data were calibrated against control LNP treatment without the drug-lipid conjugate.

[0154] Figure 9B This diagram illustrates the pro-inflammatory cytokine levels in Raw264.7 cells incubated with the prodrugs INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, and INT-D049 (D034, D045, D046, D047, D048, and D049), free dexamethasone (Dex-21-P), LNP without the prodrug (control), and untreated cells. The schematic depicts the expression of the cytokine IL-1β in cells 24 hours after incubation with the components equivalent to 1 or 10 μM dexamethasone, followed by overnight stimulation with 10 ng / mL LPS. Cytokine levels were measured by qRT-PCR, and the data were calibrated against control LNP without the drug-lipid conjugate.

[0155] Figure 10 The percentage of CD4+ T cell proliferation is shown as 10–99% of various mol% dexamethasone prodrugs (INT-D034 and INT-D045) and calcitriol (INT-D053 and INT-D083) in a mixed lymphocyte (MLR) reaction assay. Bone marrow-derived dendritic cells (BMDCs) from C57Bl / 6 male mice were first treated with LNPs containing different mol% dexamethasone or calcitriol conjugates for 48 h, and then activated by incubation with LPS for 24 h. They were then harvested and mixed with CD4+ T cells isolated from Balb / cJ male mice (Jackson Laboratories) at a T:BMDC ratio of 5:1 or 10:1.

[0156] Figure 11Electron micrographs of LNPs loaded with two different prodrugs derived from different parent drug moieties, namely dexamethasone and calcitriol. The prodrugs include: INT-D045 and INT-D053 (left panel); INT-D045 and INT-D068 (middle panel); and INT-D045 and INT-083 (right panel). Each prodrug was formulated in the LNP at an equimolar concentration of 10 mol%.

[0157] Figure 12 This chart shows the dissociation over time of 10% mol of castor oil-based dexamethasone (INT-D045) or castor oil-based calcitriol (INT-D053, INT-D068, or INT-D083) conjugates, alone or in combination, formulated in LNP. The residual amount of lipid-drug conjugates in each LNP formulation was measured at 0 h (left bar), 2 h (middle bar), and 24 h (right bar) after incubation in human plasma. The top graph shows the level of dexamethasone conjugates in single or combined formulations. The bottom graph shows the level of calcitriol conjugates in single or combined formulations. Data were calibrated against the amounts of the corresponding conjugates in the pre-incubated mixtures.

[0158] Figure 13 This diagram illustrates the breakdown of castor oil-based dexamethasone (INT-D045) or castor oil-based calcitriol (INT-D053, INT-D068, or INT-D083) conjugates formulated alone or in combination in LNPs at 10% mol. The relative amounts of intact prodrugs in each LNP were measured by UPLC after incubation in mouse plasma at 0 h (left bar), 2 h (middle bar), and 24 h (right bar). The top graph represents the levels of the dexamethasone conjugate in the single or combined formulations. The bottom graph represents the levels of the calcitriol conjugate in the single or combined formulations. Data standards were calibrated against the amounts of the corresponding conjugates in the pre-incubated mixtures. Error bars represent three independent experiments.

[0159] Detailed description

[0160] lipid conjugates of formula I

[0161] The lipid conjugates described herein can be prodrugs, and in some embodiments, a prodrug refers to a compound that becomes active after administration to a subject. However, in addition to the pharmaceutical portion, other molecules of interest M can also be conjugated to the lipid portion, such as the polymers described herein. Regardless of the molecule of interest, the lipid conjugates comprise a scaffold L, which is typically a linear carbon chain; however, branched structures are also included in the compositions described herein. The molecule of interest M is linked to L via a chemical link X1, which in some embodiments may include a direct linker or a linker group. An R hydrocarbon is linked to L via a chemical link X2. Optionally, a second R hydrocarbon is chemically linked to L via X2. Furthermore, a third R hydrocarbon is optionally linked to L via a chemical link as described below.

[0162] In one embodiment, the lipid conjugate has the structure of Formula I as exemplified below.

[0163] Formula I:

[0164] M-X1-[L]-X2-R

[0165] in

[0166] M represents the molecule of interest, including drugs or polymers;

[0167] X1 is any chemical connection that links M to any carbon atom on L, including covalent or ionic bonds or bonds containing hydrogen bonds;

[0168] L is a scaffold carbon chain having 5-40 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0169] X2 is a chemical connection that covalently links R to any carbon atom on L; and

[0170] R is a hydrocarbon having 1-40 carbon atoms and optionally one or more cis or trans C=C double bonds, and

[0171] Optionally, a second R hydrocarbon having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds is chemically linked to L via an X2 chemical link. Further, a third R hydrocarbon having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds is chemically linked to L via an X2 chemical link.

[0172] Optionally, the side chain R′ is chemically linked to any one of the hydrocarbons R via X2. Non-limitingly, a second R′ side chain can be linked to the hydrocarbon R via X2, and a third R′ can be chemically linked to any one of the hydrocarbons R via X2. Various other combinations can be readily conceived by those skilled in the art. The chemical link X2 can include any functional groups and / or linkers described below, as well as other functional groups and / or linkers known to those skilled in the art.

[0173] In another embodiment, R and / or optionally other R or R′ groups are independently hydrocarbon chains having 1-40 carbon atoms, 2-30 carbon atoms, or 5-25 carbon atoms. Similarly, the L scaffold (hereinafter) may have 1-40 carbon atoms, 2-30 carbon atoms, or 5-25 carbon atoms.

[0174] The schematic diagram in Figure 1 illustrates various lipid conjugates of formulas I, Ia, II, and IIa, which can be produced in selected embodiments using the methods of the present invention described herein. As shown, the molecule of interest M or the linker R hydrocarbon and optional second R hydrocarbon or optional additional third R hydrocarbon can occupy different positions on the scaffold backbone L to provide a customized prodrug. As further described (and noted above), one or more hydrocarbons R linked to the scaffold L can have additional carbon-based side chains attached thereto.

[0175] Although the structure depicted in Figure 1 chemically links the molecule of interest to the scaffold molecule L using a linker X1 (also referred to in the art as a "spacer"), optionally, the molecule of interest M can be directly linked to L via the X1 functional group. Furthermore, the chemical linker X1 can comprise any combination of a linker and one or more functional groups, as further described below.

[0176] Specifically, structure A in Figure 1 shows the scaffold molecule L, which in this non-limiting example has 5-30 carbon atoms, wherein the terminal carbon atom is chemically linked to the molecule of interest M via X1 as a linker. The hydrocarbon R is linked to the internal carbons of the scaffold carbon chain L via X2.

[0177] Structure B in Figure 1 depicts a scaffold molecule L with 5–30 carbon atoms, where the terminal carbon atom is connected to the hydrocarbon R via an X2 chemical linker (instead of the molecule of interest M and the linker). The molecule of interest M is connected to the inner carbon of the scaffold via an X1 chemical linker, which acts as a linker.

[0178] Similar to structure A, the structure depicted in structure C of Figure 1 shows a scaffold molecule L, in which the terminal carbon atom is connected to the molecule of interest M via a linker X1, and in which a hydrocarbon R is connected to an internal carbon of the scaffold via X2. However, in this embodiment, a second hydrocarbon R is connected to another internal carbon of the scaffold via X2.

[0179] In structure D, the scaffold molecule L is depicted, where the molecule of interest M is chemically linked to the inner carbon atom of the scaffold via X1, which acts as a linker. Hydrocarbon R is linked to the inner carbon atom of the scaffold via X2. A second hydrocarbon R' is chemically linked to the terminal carbon atom of the scaffold L via X3.

[0180] Structure E in Figure 1 depicts the scaffold molecule L, where the molecule of interest M is chemically linked to the internal carbon atom of the scaffold via X1, acting as a linker. Hydrocarbon R is linked to the internal carbon atom of the scaffold via X2. A second hydrocarbon R is chemically linked to the terminal carbon atom of the scaffold L via X2. Structure E differs from structure D above in that the molecule of interest M is linked to the carbon atom of the scaffold L closer to the terminal carbon than the position where the second hydrocarbon R is linked.

[0181] In another example, structure F in Figure 1 depicts a scaffold molecule L, where the molecule of interest M is chemically linked to the inner carbon of the scaffold via an X1 linker. A hydrocarbon R is linked to the inner carbon of the scaffold via an X2 linker. A second hydrocarbon R is chemically linked to the terminal carbon atom of the scaffold L via an X2 linker. Structure F differs from structure E above in that a third hydrocarbon R is chemically linked to the scaffold L via an X2 linker. It is readily conceivable that other combinations could include a drug-linker at C1 and three hydrocarbon portions linked to the inner carbon of L via their respective X2 links.

[0182] In yet another example shown in structure G of Figure 1, hydrocarbon R has an attached hydrocarbon side chain R′, which is connected via X2. The terminal carbon atom is chemically connected to the molecule of interest M via X1, which acts as a linker. Hydrocarbon R is connected to the inner carbon of the support carbon chain L via X2.

[0183] The above structure AG is an example, as those skilled in the art can readily conceive of other arrangements and implementations that fall within the scope of this disclosure.

[0184] In some embodiments, the point on the scaffold L to which the group R is attached may be at least 3 carbon atoms away from the terminal carbon on L (as measured from the first carbon of L, referred to as C1). To describe such a branching point in the chemical formula of the prodrug (Formula I above), the symbol "L1-L2" may be used to refer to the scaffold molecule L. According to such an embodiment, L1 is at least 3 carbon atoms and S is attached to the carbon atom of L2. In a particularly advantageous embodiment, L1 is at least 4 or 5 carbon atoms.

[0185] In those embodiments in which group R is attached to L at a position at least 3 carbon atoms away from C1, formula I can take the form of formula Ia:

[0186] Formula Ia:

[0187]

[0188] Where M is the molecule of interest; X1 is a chemical link that conjugates or connects M to any carbon atom on L1-L2 through any suitable chemical linking described herein; L1 has at least 3 carbon atoms; L1-L2 have 5-40 carbon atoms; and L2 = L-L1. Chemical link X1 conjugates the molecule of interest M to any carbon atom on L1-L2, and chemical link X2 conjugates R to any carbon atom on L2. R is a hydrocarbon having 1-40 carbon atoms.

[0189] In one embodiment, L1 has 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms. In another embodiment, L1 can be 3-30 carbon atoms, 4-30 carbon atoms, 5-25 carbon atoms, 6-25 carbon atoms, or 7-20 carbon atoms. Optionally, L1 has one or more cis or trans C=C double bonds. In yet another embodiment, L1 is a linear carbon chain.

[0190] Although L2 is typically a linear carbon chain, branched structures are also of interest. As mentioned above, L2 = L - L1. For example, in those embodiments where L is 20 carbon atoms and L1 is 11 carbon atoms, L2 is 9 carbon atoms.

[0191] In an alternative embodiment, the lipid conjugate has a lipid moiety of the structure of Formula II as exemplified below.

[0192] Formula II:

[0193]

[0194] The L lipid scaffold backbone is represented by L1+L2+L3+L4+L5+L6, and L contains 5-40 carbon atoms, 5-30 carbon atoms, 5-25 carbon atoms, and 0-2 cis or trans C=C double bonds.

[0195] L1 is a carbon chain having 1-30 carbon atoms, 3-30 carbon atoms, 4-30 carbon atoms, 5-25 carbon atoms, 6-25 carbon atoms, or 7-20 carbon atoms, and optionally L1 has one or more cis or trans C=C double bonds or 0-2 cis or trans C=C double bonds.

[0196] L2 and L4 are each carbon atoms;

[0197] L3 consists of 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds;

[0198] L5 consists of 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds;

[0199] L6 is -CH3, =CH2, or H;

[0200] Each R is independently a linear or branched hydrocarbon chain having 0-30 carbon atoms and 0-2 cis or trans C=C double bonds, wherein, according to an optional embodiment, each R is independently branched, and each branching point includes an X2 functional group containing a heteroatom.

[0201] Where n is 0-8 and p is 0-8, and n+p is ≥1 or 1-8; or where n is 0-6 and p is 0-6, and n+p is ≥1 or 1-6; or where n is 0-4 and p is 0-4, and n+p is ≥1 or 1-4; and

[0202] X1 and X2 are independently selected from esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas; or wherein X1 contains one or more hydrogen bonds and has a structure of formula V as defined below.

[0203] In one embodiment, at least one of X1 and X2 is biodegradable.

[0204] In one embodiment, X1 and / or X2 are independently selected from esters, ethers, or carbamates. Esters or carbamates can be in any orientation. For example, an ester can be linked to the molecule of interest (M) via its carbonyl group or via its -O- group. Similarly, a carbamate can be linked to the molecule of interest (M) via its nitrogen atom or via its -O- group.

[0205] In one embodiment, the lipid portion of Formula II has a total of less than 300, less than 200, less than 150, less than 100 carbon atoms, less than 75 carbon atoms, or less than 50 carbon atoms (L+R).

[0206] Each R hydrocarbon chain in the lipid moiety is optionally substituted with a heteroatom at one of its internal carbon atoms, provided that no more than eight heteroatoms are substituted in the R hydrocarbon chain of the lipid moiety. In another embodiment, the predicted or experimental logP of the conjugate is greater than 5.

[0207] In yet another embodiment, the lipid conjugate is not an ionizable lipid.

[0208] In an alternative embodiment, the lipid conjugate has a lipid moiety of the structure of formula IIa as exemplified below.

[0209] Formula IIa:

[0210]

[0211] Where L is composed of [CH2] m –L2–L3–L4–[CH2] q –CH3 indicates that the total number of carbon atoms in L is 5-30;

[0212] L2 and L4 are carbon atoms;

[0213] Where m is 0-20; n is 1-4; p is 0-4, and n+p is 1-4;

[0214] L3 consists of 0-10 carbon atoms and has 0-2 cis or trans C=C;

[0215] X1 and X2 are independently selected from ether, ester and carbamate groups;

[0216] Each R is independent of:

[0217] (a) A linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms, wherein each R is fused with one of its respective X2 at any carbon atom in the hydrocarbon chain; or

[0218] (b) A branched structure of formula IIb with a support represented by L':

[0219] Formula IIb:

[0220]

[0221] Where L' is composed of [CH2] r –L2–G3–L4–[CH2] u –CH3 indicates that the total number of carbon atoms in L is 3-30; and

[0222] Where r is 0-20, 2-20, 3-20 or 4-20;

[0223] s is 0-4, t is 0-4; and s+t is either >1 or 1-4;

[0224] u is 1-20;

[0225] G3 consists of 0-10 carbon atoms and has 0-2 cis or trans C=C arrangements;

[0226] Each R' in formula IIb is independently a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms;

[0227] The total number of R' hydrocarbon chains in formula IIb is 1-16;

[0228] Each of the R and R' hydrocarbon chains in the lipid moiety is optionally substituted with a heteroatom, provided that no more than 8, 6, 4, or 2 heteroatoms are substituted in the R and R' hydrocarbon chains, and the predicted or experimental logP of the conjugate is greater than 5; and

[0229] The lipid-conjugates mentioned therein are not ionizable lipids.

[0230] Non-limiting examples of prodrug lipid conjugates having structures of formulas I, Ia, II, and IIa are provided in Table 1 below, and their chemical structures are provided in Figure 3. In such embodiments, the lipid conjugates are derived from dexamethasone and use a succinate linker (X1 chemical linker); however, as discussed further herein, a wide range of pharmaceuticals or other molecules of interest and linkers can be incorporated into said lipid conjugates.

[0231] Table 1: Non-limiting examples of prodrugs

[0232]

[0233]

[0234] Support L of type I, Ia, II or IIa

[0235] In one embodiment, the L of formula I, Ia, II or IIa is derived from a fatty acid having a functional group on its carbon chain that links to R.

[0236] For example, the L in formula I, Ia, II, or IIa can be derived from a hydroxy fatty acid (HFA), which is a fatty acid having an OH group bonded at any position on its carbon chain. Without restriction, the HFA can be an α-hydroxy fatty acid, a β-hydroxy fatty acid, an ω-hydroxy fatty acid, or any (ω-1)-hydroxy fatty acid, or any other known HFA. The HFA can be saturated or unsaturated. Two or more hydroxyl groups can also be present on the carbon chain.

[0237] Non-limiting examples of fatty alcohols from which HFAs can be derived are listed in Table 2 below:

[0238] Table 2: Examples of hydroxy fatty acids (HFAs) and corresponding fatty alcohols

[0239]

[0240] Examples of HFAs having two or more hydroxyl functional groups present in the carbon chain include 9,10-dihydroxyoctadecanoic acid and trihydroxyhexadecanoic acid (also known as 2,15,16-trihydroxypalmitic acid or 2,15,16-trihydroxyhexadecanoic acid).

[0241] Alternatively, the L in formula I, Ia, II, or IIa is derived from a branched fatty acid ester of an HFA, which is referred to in the art as a fatty acid ester of hydroxy fatty acids (FAHFA). These fatty acid esters contain a branched ester linkage between the fatty acid and the HFA. For example, 9-[(9Z)-octadecenoyloxy]octadecanoic acid is a fatty acid ester obtained by condensation of the carboxyl group of oleic acid with the hydroxyl group of 9-hydroxyoctadecanoic acid.

[0242] In an alternative embodiment, L is derived from a fatty amide, which may contain ethanolamine as an amine component.

[0243] The L in formulas I, Ia, II, or IIa can be derived from other fatty acids besides those mentioned above. Furthermore, it is understood that fatty acids can thus be derived from their corresponding triglycerides.

[0244] The L in formula I, Ia, II, or IIa may include an OH group introduced by oxidizing a double bond on the carbon backbone of the lipid. Thus, the precursor of L can be derived from any fatty acid, fatty alcohol, or fatty amide precursor that is unsaturated and oxidized to introduce a reactive OH group.

[0245] The lipid moiety of lipid conjugates, such as prodrugs or lipid-polymer conjugates, can be compatible with lipids incorporated into drug delivery carriers. For example, it can include vesicle-forming lipids, such as phospholipids, that are compatible with components forming lipid nanoparticles, such as liposomes. The lipid moiety can also be compatible with other drug delivery carriers, such as polymer-based nanoparticles, emulsions, micelles, and nanotubes.

[0246] In one embodiment, L may be derived from a precursor fatty acid or other molecule having, for example, 5-30 carbon atoms, 14-20 carbon atoms, or 16-18 carbon atoms.

[0247] lipid-based precursor P

[0248] In an alternative embodiment, the lipid moiety of the lipid conjugate of Formula I above may be derived from a precursor, referred herein as “p” as defined in Formula III:

[0249] Formula III:

[0250] RG-[L]-X2-R

[0251] RG is a reactive functional group containing one or more reactive atoms selected from O, C, N, P, S, Si, or B. In one embodiment, the reactive functional group is selected from hydroxyl, amine, or carboxyl groups. In another embodiment, the reactive functional group is a hydroxyl or carboxyl group. In an optional embodiment, the RG functional group forms a biodegradable chemical link with a linker on the molecule of interest M or directly with such a molecule.

[0252] L is a scaffold carbon chain having 5-40 carbon atoms and optionally one or more cis or trans C=C double bonds;

[0253] X2 is a chemical connection that covalently links R to any carbon atom on L; and

[0254] R is a hydrocarbon having 1-40 carbon atoms and optionally one or more cis or trans C=C double bonds.

[0255] In another embodiment, the lipid portion of formula Ia can be derived from a precursor P having the structure of formula IIIa:

[0256] Formula IIIa:

[0257]

[0258] RG is a reactive functional group containing at least one reactive atom selected from O, C, N, P, S, Si, or B. In one embodiment, the reactive functional group is selected from hydroxyl, amine, or carboxyl. In another embodiment, the reactive functional group is hydroxyl or carboxyl. In an optional embodiment, the RG functional group forms a biodegradable chemical link with a linker on the drug or with the drug. In another optional embodiment, the RG functional group is a hydrogen bond donor or acceptor group. L1 has at least 3 carbon atoms; L1-L2 have 5-40 carbon atoms; and L2 = L-L1. The chemical link X2 causes R to adsorb onto any carbon atom on L2. R is a hydrocarbon having 1-40 carbon atoms.

[0259] In one non-limiting embodiment, RG in Formula III or IIIa is a hydroxyl group. RG may be conjugated to a drug substance or a corresponding reactive group on a linker, such as a carboxyl group. The bond formed in such a reaction (X1 of Formula I or Ia) may be selected from ester or amide bonds, however, other bonds may also be formed.

[0260] The carbon backbone of L in Formula III or L1-L2 in Formula IIIa may further include an additional reactive group RG for attaching the second hydrocarbon R group. Furthermore, a third hydrocarbon R can be attached to the carbon backbone of L via RG. Similarly, the second or third reactive group RG may contain one or more atoms selected from O, C, N, P, S, Si, or B. In a non-limiting example, each RG is independently selected from hydroxyl, amine, or carboxylic acid groups, as well as other suitable groups known to those skilled in the art.

[0261] Furthermore, the two or more hydrocarbon moieties R of formulas III and IIIa may have respective R' side chains connected to them. For example, the R' side chain may be connected to R via an X2 connection, and as previously described with respect to formulas I, Ia, II, and IIa, a second R' side chain may be connected to another R via an X2 connection, and / or a third R' may be connected to any R via an X2 connection. However, those skilled in the art can readily conceive of various other combinations.

[0262] In another embodiment, the lipid moiety of formula II may be derived from the precursor P having the structure of formula IIIb:

[0263] Formula IIIb:

[0264]

[0265] RG is a reactive functional group containing one or more reactive atoms selected from O, C, N, P, Si, or B. In one embodiment, the reactive functional group is selected from hydroxyl, amine, or carboxyl groups. In another embodiment, the reactive functional group is a hydroxyl or carboxyl group. In an optional embodiment, the RG functional group forms a biodegradable chemical link with a linker group on the drug or with the drug. In another embodiment, the RG functional group is a hydrogen bond donor or acceptor group or an atom for forming a hydrogen bond with a respective acceptor or donor group on the molecule of interest M;

[0266] The L lipid scaffold backbone is represented by L1+L2+L3+L4+L5+L6, and L contains 5-40 carbon atoms, 5-30 carbon atoms, 5-25 carbon atoms, and 0-2 cis or trans C=C double bonds.

[0267] L1 is a carbon chain having 1-30 carbon atoms, 3-30 carbon atoms, 4-30 carbon atoms, 5-25 carbon atoms, 6-25 carbon atoms, or 7-20 carbon atoms, and optionally L1 has one or more cis or trans C=C double bonds or 0-2 cis or trans C=C double bonds.

[0268] L2 and L4 are each carbon atoms;

[0269] L3 consists of 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds;

[0270] L5 consists of 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds;

[0271] L6 is -CH3, =CH2, or H;

[0272] Each R is independently a linear or branched hydrocarbon chain having 0-30 carbon atoms and 0-2 cis or trans C=C double bonds, wherein, according to an optional embodiment, each R is independently branched, and each branching point includes an X2 functional group containing a heteroatom.

[0273] Where n is 0-8 and p is 0-8, and n+p is ≥1 or 1-8; or where n is 0-6 and p is 0-6, and n+p is ≥1 or 1-6; or where n is 0-4 and p is 0-4, and n+p is ≥1 or 1-4; and

[0274] X1 and X2 are independently selected from esters, amides, amidines, hydrazones, disulfides, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas; or wherein X1 contains one or more hydrogen bonds and has a structure of formula V as defined below.

[0275] In another embodiment, the lipid moiety of formula IIa may be derived from a precursor P having the structure of formula IIIc:

[0276] Formula IIIc:

[0277]

[0278] RG is a reactive functional group containing one or more reactive atoms selected from O, C, N, P, Si, or B. In one embodiment, the reactive functional group is selected from hydroxyl, amine, or carboxyl groups. In another embodiment, the reactive functional group is a hydroxyl or carboxyl group. In an optional embodiment, the RG functional group forms a biodegradable chemical link with a linker on a molecule of interest, such as a drug.

[0279] Where L is composed of [CH2] m –L2–L3–L4–[CH2] q –CH3 indicates that the total number of carbon atoms in L is 5-30;

[0280] L2 and L4 are carbon atoms;

[0281] Where m is 0-20; n is 1-4; p is 0-4; and n+p is 1-4.

[0282] L3 consists of 0-10 carbon atoms and has 0-2 cis or trans C=C;

[0283] X1 and X2 are independently selected from ether, ester and carbamate groups;

[0284] Each R is independent of:

[0285] (a) A linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms, wherein each R is fused with one of its respective X2 at any carbon atom in the hydrocarbon chain; or

[0286] (b) A branched structure of formula IIb with a support represented by L':

[0287] Formula IIb:

[0288]

[0289] Where L' is composed of [CH2] r –L2–G3–L4–[CH2] u –CH3 indicates that the total number of carbon atoms in L is 3-30; and

[0290] Where r is 0-20, 2-20, 3-20 or 4-20;

[0291] s is 0-4, t is 0-4; and s+t is either >1 or 1-4;

[0292] u is 1-20;

[0293] G3 consists of 0-10 carbon atoms and has 0-2 cis or trans C=C arrangements;

[0294] Each R' in formula IIb is independently a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms;

[0295] The total number of R' hydrocarbon chains in formula IIb is 1-16;

[0296] Each of the R and R' hydrocarbon chains in the lipid moiety is optionally substituted with a heteroatom, provided that no more than eight heteroatoms are substituted in the R and R' hydrocarbon chains, and the predicted or experimental logP of the conjugate is greater than 5; and

[0297] The lipid-conjugates mentioned therein are not ionizable lipids.

[0298] Molecules of interest

[0299] Various molecules of interest can be linked to the lipid moiety. As noted, the lipid conjugate can be a prodrug. The pharmaceutical portion of the prodrug conjugate can be derived from any class of drugs, including any drug used to treat, prevent, improve, alleviate symptoms of a subject's disease or other adverse condition, and / or diagnose those symptoms, for example, after activation. The pharmaceutical portion can be an active agent or an active agent subsequently activated after release from the conjugate. However, other molecules of interest can also be linked to the lipid moiety, including hydrophilic polymers.

[0300] In some embodiments, the characteristic of the molecule of interest M may lie in its nature of connection or binding with a lipid moiety. For example, in some embodiments, the pharmaceutical moiety D may be derived from a drug that loses one or more atoms when conjugated with a reactive group or linker on a scaffold L to form a chemical link X1. In one embodiment, the drug loses a hydroxyl or hydrogen atom when conjugated with P or a linker to form a prodrug of formula I, Ia, IIa, or IIb. However, the pharmaceutical moiety D can be derived from any known drug, as the methods of the invention described herein are applicable to a wide range of conjugations or bindings of pharmaceutical agents with lipid moieties. The drug D can be a small molecule or a macromolecule. The moiety M (the molecule of interest) can be derived from a chemical structure containing one or more reactive functional groups, such as -(C=O)O, -OH, -NH2, -NHR, -PO3H2, etc., known to those skilled in the art, but there is no limitation on the orientation of the atoms.

[0301] For example, when RG is -OH, the prodrug or other lipid conjugates described herein can be formed (directly or via one or more intermediates) through conjugation between the (C=O)OH group on the molecule of interest and the hydroxyl group on the prodrug scaffold P. The general reaction is shown below:

[0302]

[0303] In the above exemplary embodiments, X1 of formula I, Ia, II, or IIa is chemically linked to an ester and has the following structure:

[0304]

[0305] In another exemplary instance, the molecule of interest M may have a hydroxyl group (-OH) that reacts with the carboxyl group ((C=O)OH) on the linker. A second carboxyl group ((C=O)OH) on the linker can react with a hydroxyl group on a carbon atom of the precursor scaffold P via a condensation reaction. The following reaction depicts the application of succinic acid as a linker. The application of this type of linker yields a prodrug having two ester groups according to the following reaction:

[0306]

[0307] In the above non-limiting examples, the X1 chemical link has the following structure:

[0308]

[0309] It should be understood that the above reaction can be carried out in two steps. That is, firstly, the drug is conjugated to the linker, and then the resulting drug-linker conjugate reacts with the prodrug scaffold P to produce the prodrug reaction product.

[0310] The above description is provided for illustrative purposes only, as various linker groups besides succinic acid can also be used to generate prodrugs. In another example, the molecule of interest M or the linker group may have a carboxyl group ((C=O)O) for conjugation with the amino group of L to form an amide or amide-containing linker X1 between the drug moiety and L. As described below, those skilled in the art can envision further reactions of functional groups on the drug or linker with the scaffold L to generate a chemical link of X1.

[0311] Certain molecules of interest may contain more than one reactive functional group for attachment to the precursor scaffold P. In such embodiments, as those skilled in the art will understand, protecting groups may be used during the synthesis of the drug-lipid conjugate to selectively conjugate a designated group on the drug to the scaffold L, leaving an unconjugated group. The drug may also be characterized by its biological effects, including its ability to treat, prevent, and / or improve symptoms in a subject or in vitro cells. The drug portion may be derived from an anticancer drug, such as an antitumor drug. In another embodiment, the drug portion may be derived from an immunomodulatory drug, such as an immunosuppressant, for the treatment of autoimmune diseases, such as Crohn's disease, rheumatoid arthritis, psoriasis, ulcerative colitis, or diabetes. In one embodiment, the immunomodulatory drug is an anti-inflammatory drug.

[0312] As used in this article, drugs that function as anticancer agents can have direct or indirect effects on the growth, proliferation, invasion, and / or survival of tumor cells and / or tumors. Antitumor drugs include alkylating agents, antimetabolites, cytotoxic antibiotics, various plant alkaloids and their derivatives, and immunomodulators.

[0313] Examples of immunosuppressant drugs include glucocorticoids, cell growth inhibitors, antibodies, and drugs that act on immunoglobulins, as known to those skilled in the art. Examples of glucocorticoids include prednisone, prednisolone, and dexamethasone. Methotrexate is an example of a cell growth inhibitor.

[0314] In one embodiment, the drug is partially derived from docetaxel, dexamethasone, methotrexate, NPC1I, abiraterone, prednisone, prednisolone, ruxolitinib, tofacitinib, calcitriol, calcidiol, cholecalciferol, sirolimus, tacrolimus, acetylsalicylic acid, mycophenolate mofetil, cabazitaxel, betamethasone, and NLRP3 inhibitors, including CYO9(4-[[4-oxo-2-thio-3-[[3-(trifluoromethyl)phenyl]methyl]-5-imidazolidinyl]methyl]benzoic acid. ), INT-MA014 or MCC950 (N-(1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propyl)-2-furansulfonamide) and its derivatives, and cannabinoids, including cannabinol, cannabilotriol, cannabidiol, cannabidiol, cannabilotriol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, cannabidiol, tetrahydrocannabinol or tetrahydrocannabinol and its derivatives.

[0315] In another embodiment, the drug has a free hydroxyl group, which is used to conjugate to a linker or group on any carbon of L. However, other functional groups on the drug can also be used for this type of conjugation.

[0316] Other molecules of interest, besides the drug, can be linked to the lipid moiety via X1 to scaffold L using reactive groups similar to those described above. This includes small molecules and those forming macromolecular structures. For example, in some embodiments, the molecule of interest, M, is a polymer forming a lipid-polymer conjugate. The polymer can be a hydrophilic polymer suitable for biological systems. Examples of hydrophilic polymers include polyalkyl ethers, such as polyethylene glycol (PEG), polymethyl ethylene glycol, polypropylene glycol, and polyhydroxypropylene glycol. Other suitable polymers include polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyvinyl methyl ether, polymethyl oxazoline, polyethyl oxazoline, polyhydroxypropyl oxazoline, polyhydroxypropyl methacrylate, polymethyl methacrylate, polydimethyl methacrylate, polyhydroxypropyl methacrylate, polyhydroxyethyl acrylate, hydroxymethyl cellulose, hydroxyethyl cellulose, or polyasparagine. The polymer chains can have a molecular weight of about 300-10,000 Daltons. In some non-limiting embodiments, the polymer can be a block copolymer.

[0317] In yet another implementation, the molecule of interest M is an antibody, peptide, or genetic material such as siRNA.

[0318] In one implementation, the molecule of interest M is genetic material, such as nucleic acid. Nucleic acid includes, but is not limited to, RNA, including small interfering RNA (siRNA), small nuclear RNA (snRNA), microRNA (miRNA), or DNA, such as plasmid DNA or linear DNA. Nucleic acid length is variable and can include nucleic acids from 5 to 50,000 nucleotides in length. Nucleic acid can be in any form, including single-stranded DNA or RNA, double-stranded DNA or RNA, or hybrids thereof. Single-stranded nucleic acids include antisense oligonucleotides.

[0319] In a particularly advantageous implementation, the molecule of interest is siRNA. siRNA is incorporated into endogenous cellular mechanisms, leading to mRNA degradation and thereby inhibiting transcription. Because RNA is readily degraded, incorporating it into a delivery vector as described herein can reduce or prevent such degradation, thereby facilitating delivery to the target site.

[0320] Chemical Linkage X1

[0321] In one embodiment, the molecule of interest M is directly linked to the L scaffold carbon chain via the X1 functional group. In such embodiments, X1 in formula I, Ia, II, or IIa can be one or more functional groups selected from esters, amides, amidines, hydrazones, disulfides, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas.

[0322] In one embodiment, the X1 group is not a disulfide or a thioether. In another embodiment, X1 does not contain a sulfur atom.

[0323] As discussed, the molecule of interest M can be linked to the L scaffold via X1 as a linker. The inclusion of a linker in lipid conjugates is particularly advantageous for molecules that are released from the lipid moiety after administration, such as prodrugs, because they contain components that can facilitate the enzymatic cleavage of the molecule of interest M from the lipid moiety. As described below, one or more of the aforementioned functional groups, including but not limited to those specifically described in Table 3 below, may be included in the linker molecule. Such functional groups are most advantageously cleavable under in vivo conditions.

[0324] Non-limiting examples of lipid conjugates having an X1 chemical linker selected from succinic acid linker, ester, amide, hydrazone, ether, carbamate, carbonate, or phosphodiester group are described in Table 3 below. The chemical links below are shown as components of Formula I or Ia. As discussed, although for simplicity the linker is described as arising from a direct conjugation between the drug and L (except for succinic acid esters), it should be understood that the groups shown in the table may also be incorporated into the linker.

[0325]

[0326]

[0327] As described above, in certain advantageous embodiments, the hydroxyl group (RG=OH in formula III, IIIa, IIIb or IIIc) or the amine group (RG=NH2 in formula III, IIIa, IIIb or IIIc) of the precursor scaffold P reacts with the carboxyl group on the drug via a condensation reaction to form an X1 chemical link, which is an ester or amide group, respectively.

[0328] In such embodiments, the lipid conjugate of formula I, Ia, II, or IIa has the following structure:

[0329]

[0330] Where X = -O or -NH.

[0331] In this type of implementation, X1 is chemically linked to form a component of the prodrug of Formula I:

[0332]

[0333] In an alternative embodiment, L is derived from the reaction of a carboxyl group of a fatty acid with a linker or a hydroxyl or amino group of the molecule of interest. In this embodiment, X1 forms a chemical link between the molecule of interest and P as follows:

[0334]

[0335] Where X = -O or -NH.

[0336] In a particularly advantageous embodiment, X = -O in the above structure. In such embodiments, X1 is an ester bond.

[0337] In one embodiment, the X1 linker is biodegradable, meaning it can cleave after administration to a patient. Without limitation, the ester bond can be hydrolyzed by esterases after administration to a patient, thereby releasing the molecule of interest, including but not limited to drug moiety D, from the lipid conjugate. However, other X1 links can be used to tailor drug release based on their release properties when exposed to the environment of the disease site. For example, the hydrazone bond between drug moiety D and scaffold L can conjugates of formula I, Ia, II, or IIa to pH-sensitive release. At neutral pH, hydrazones show little degradation, while at lower pH, the bond may break. Therefore, X1 chemical links consisting of or containing one or more hydrazone bonds can provide drug release at the low pH conditions often present in tumor tissue.

[0338] In one embodiment, X1 can be cleaved by esterases, alkaline phosphatases, amidases, peptidases, or by exposure to reducing environments and / or high or low pH values.

[0339] As discussed, if the lipid conjugate is a prodrug, then in some embodiments, the X1 chemical linker is most advantageously a linker. A variety of chemical linkers are known to those skilled in the art and can be used in some embodiments described herein. The linker may have 0-12 carbon atoms and at least one cleavable functional group. In one embodiment, the linker has at least two functional groups, a first functional group for conjugating one end of the linker to the molecule of interest M, and a second functional group for conjugating the other end of the linker to a carbon atom on L. The two functional groups can be independently selected from esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acylalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas.

[0340] As those skilled in the art will understand, in some embodiments, if the molecule of interest is drug D, the linker can provide enhanced release of drug D by introducing a biodegradable group. Linkers having one or more ester bonds can be hydrolyzed by esterases upon administration to a patient, thereby releasing drug moiety D from the prodrug conjugate. Similar to the linkage resulting from a direct reaction between D and L, introducing a hydrazone bond between drug moiety D and scaffold L can confer pH-sensitive release of the prodrug of formula I or Ia.

[0341] However, it is understood that the foregoing is merely exemplary. Other examples of linkers are provided in U.S. Patent No. 5,149,794, which is incorporated herein by reference. Non-limiting examples of linkers described in U.S. Patent No. 5,149,794 include aminocaproic acid, polyglycine, polyamide, polyethylene, and short-functionalized polymers having a carbon backbone having a length of 1-12 carbon atoms.

[0342] Further examples applicable to the prodrugs described herein are provided in the following references:

[0343] 1.Rautio et al., "The expanding role of prodrugs in contemporary drug design and development" Nature Reviews Drug Discovery 2018, 17, 559.

[0344] 2.Irby et al., "Lipid-drug conjugate for enhanced drug delivery" Molecular Pharmaceutics 2017, 14, 1325.

[0345] 3.Sun et al., "Chemotherapy agent-unsaturated fatty acid prodrugs and prodrug-nanoplatforms for chemotherapy cancer" Journal of Controlled Release2017, 264, 145.

[0346] 4.Walther et al., "Prodrugs in medicinal chemistry and enzyme prodrugtherapies" Advanced Drug Delivery Reviews 2017, 118, 65.

[0347] 5. Hu et al., "Glyceride-mimetic prodrugs incorporating self-immolative spacers promote lymphatic transport, avoid first-pass metabolism and enhanceoral bioavailability" Angewandte Chemie International Edition 2016, 55, 13700.

[0348] 6. Blencowe et al., "Self-immolative linkers in polymeric delivery systems" Polymer Chemistry 2011, 2, 773.

[0349] Each of the foregoing references is incorporated herein by reference in its entirety. In another embodiment, the X1 chemical linker comprises a functional group and a separate linker. Different combinations of linker and functional groups (e.g., those in Table 3 above) can be used to obtain the desired lipid conjugates of formula I, Ia, II, or IIa.

[0350] In one embodiment, at least one end of the linker is conjugated to an L1-bound second functional group that is an ester or amide. In another embodiment, one functional group on the linker may be hydrolyzed by an enzyme, such as an esterase. In yet another embodiment, both functional groups on the linker are ester-conjugated.

[0351] Although a wide range of known linkage bases can be used in the embodiments described herein, some non-limiting examples of X1 linkage bases are provided below.

[0352] In one embodiment, without limitation, the molecule of interest M-linker X1 (D-X1) of formula I, Ia, II, or IIa is based on the following formula IV:

[0353] Formula IV:

[0354] M-[X4-M1-X5] X1

[0355] Wherein M is the molecule of interest, X4 and X5 are independently selected from any of the functional groups described above, and M1 is an optional spacer group attached to the functional groups X4 and X5 and having 0-12 carbon atoms, or CH2, CH2CH2, N-alkyl, N-acyl, O, or S. X4 and X5 may be the same or different. In one embodiment, any one or both of X4 and X5 are capable of being cleaved in vivo. In another embodiment, X4 and / or X5 are ester groups.

[0356] In the above formula IV, X4, X5, or both functional groups can each be repeating units from 1 to about 20. In addition, the X4-M1-X5 unit can be repeating units from 1 to 20, or if M1 is not present, then X4-X5 can be repeating units.

[0357] In another embodiment, X5 in formula IV is an ester group, wherein M-X1 of formula I, Ia, II or IIb is as follows:

[0358] Formula IVa:

[0359]

[0360] Where M is the molecule of interest, and X4 is a functional group that covalently links M to M1 and is selected from ester, amide, hydrazone, ether, carbonate, carbamate or phosphodiester group; and M1 is a spacer region having a linker having 0-12 carbon atoms, or is CH2, CH2CH2, N-alkyl, N-acyl or O.

[0361] In one embodiment, without limitation, the connecting base X1 of formula I, Ia, II, or IIa has the following structure:

[0362] Formula IVb:

[0363]

[0364] Wherein Z is selected from O or N, Y is CH2, CH2CH2 or C=O, T is 0-6 carbon atoms, and W is O or N. In one embodiment, Z is O, Y is CH2, CH2CH2 or C=O, T is 0-6 carbon atoms, and W is O. In another embodiment, the linker X1 is derived from succinic acid.

[0365] In such embodiments, the linker of formula IVb forms the components of lipid conjugates of formulas I, Ia, and II as follows:

[0366] Formula I:

[0367]

[0368] Formula Ia:

[0369]

[0370] Formula II:

[0371]

[0372] Formula IIa:

[0373]

[0374] Wherein Z is selected from O or N, Y is CH2, CH2CH2 or C=O, T is 0-6 carbon atoms, and W is O or N. In one embodiment, Z is O, Y is CH2, CH2CH2 or C=O, T is 0-6 carbon atoms, and W is O. In another embodiment, the linker X1 is derived from succinic acid.

[0375] In a particularly advantageous embodiment, the X1 linker is a succinate group, and the prodrugs of formulas I, Ia, II, and IIa have the following structures:

[0376] Formula I:

[0377]

[0378] Formula Ia:

[0379]

[0380] Formula II:

[0381]

[0382] Formula IIa:

[0383]

[0384] Besides the succinic acid linker, non-limiting examples of X1-linked structures include the following chemical structures:

[0385]

[0386] Where M is the molecule of interest and L is the lipid scaffold. For simplicity, the remaining lipid portions are not shown in the above structures, but may include any lipid portions of formulas I, Ia, II, and IIb.

[0387] It should be understood that reactions that generate X1 chemical links are not limited to those resulting from the direct reaction between the corresponding functional group (e.g., the drug, polymer, or linker attached thereto) present on the molecule of interest and the corresponding group on the precursor scaffold P. Typically, such conjugates are generated via multi-step synthetic schemes and through various intermediates. Furthermore, the precursor L, such as a fatty alcohol, can be modified to generate its derivative, and this derivative can then react with a reactive functional group on the molecule of interest to generate a lipid conjugate, or vice versa. For example, US2002 / 0177609 (incorporated herein by reference) describes a method involving the derivatization of a fatty alcohol with appropriate linking and leaving groups to form an intermediate and the reaction of that intermediate with a drug to form a conjugate compound. Many different X1 links can be generated in this manner, including drugs conjugated to the scaffold L via one or more carbonate, carbamate, ether, phosphate, ester, guanidine, thiocarbonyl carbamate, phosphonate, oxime, isourea, amide, phosphoramide, or phosphonamide groups. Similarly, in addition to fatty alcohols, other molecules can be modified to introduce reactive groups that cannot be generated by the reaction of existing functional groups present on the drug with fatty acids.

[0388] In another embodiment, the molecule of interest M is connected to the scaffold L of the lipid moiety via an X1 link comprising one or more intermolecular hydrogen bonds. According to such an embodiment, the molecule of interest contains one or more electronegative atoms. The molecule of interest may contain at least one hydrogen bond donor, which is a hydrogen atom covalently linked to a relatively electronegative atom, and L may contain at least one hydrogen bond acceptor, which is a relatively electronegative atom bonded to hydrogen via a hydrogen bond. Conversely, L may contain one or more hydrogen bond donors, and the molecule of interest M may contain one or more hydrogen bond acceptors.

[0389] The hydrogen bonds between L and M of the lipid conjugate can have a structure of formula V:

[0390] Formula V:

[0391]

[0392] E1, E2, E3, E4 and E5 are electronegative atoms selected from O, N and P;

[0393] E1, E2, and E3 are hydrogen bond acceptors, while E4 and E5 are hydrogen bond donors;

[0394] Dashed lines depict hydrogen bonds and solid lines depict covalent bonds;

[0395] Where L is a lipid scaffold of the lipid portion as described in Formula I, Ia, II or IIa;

[0396] n is 0 or 1; o is 0 or 1; and p is 0 or 1; and n + o + p ≥ 2;

[0397] q is 1-10, 2-10, or 4-10;

[0398] L represents the lipid scaffold of the lipid portion;

[0399] M is the molecule of interest; and

[0400] E1 and E3 optionally contain substituents connected thereto, such as alkyl, aryl, alkylene, or H.

[0401] Examples of drug-lipid conjugates containing X1 hydrogen bonds are provided below. In this example, doxorubicin contains a hydrogen-bonded acceptor group, while having a terminal... The lipid portion of the group contains a hydrogen bond donor group. However, it will be understood that those skilled in the art can readily conceive of other atomic configurations for the hydrogen bond donor and acceptor.

[0402] Examples of hydrogen bond X1 linkages in drug-lipid conjugates:

[0403]

[0404] Chemical Linkage X2

[0405] Similarly, X2 is a chemically linked R covalently to any carbon atom on L of formula I, Ia, II, or IIa, and can be formed by reacting a functional group on any carbon of L with a reactive group on R. However, similar to X1, X2 does not require a direct reaction between functional groups on L, but can be formed through a multi-step synthetic scheme.

[0406] Different X2 functional groups can link R to L or L2. For example, X2 can be a functional group selected from: esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonyloxymethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or urea. In one embodiment, the reactive group on L that forms X2 with the reactive group on R is a functional group selected from -OH, -NH2, or -C=O (O). Most advantageously, X2 is -C=O (O), which is formed by the reaction of an acyl group with a hydroxyl group on L. However, such groups are merely exemplary, and other groups known to those skilled in the art may also be used.

[0407] The X2 chemical linker can also be a linker group. If desired, the linker group can have 0-12 carbon atoms and at least one cleavable functional group to release R in formula I, Ib, II, or IIa. In one embodiment, the linker group has at least two functional groups, a first functional group attaching one end of the linker group to the support L, and a second functional group attaching the other end of the linker group to a carbon atom on R. The two functional groups can each be independently selected from esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphonophosphonyl methyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas. In one advantageous embodiment, at least one functional group in the linker is an ester, amide, hydrazone, ether, carbonate, carbamate, or phosphate diester. In another embodiment, at least one functional group of X2 can be cleaved in vivo to release R from the scaffold L. Such a later embodiment may be desirable if R or L is a therapeutic lipid.

[0408] R or R' group

[0409] As noted, in one embodiment, R or R' in formula I, Ia, II, or IIa is a hydrocarbon group having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds. In another embodiment, R is an aliphatic hydrocarbon. In yet another embodiment, R does not contain any heterocyclic structure. In yet another embodiment, R is not biotin.

[0410] In one embodiment, the number of carbon atoms in the R group is selected such that the lipid conjugate of formula I, Ia, II, or IIa has a desired LogP value. As can be seen from Table 1 above, in some embodiments, the logP of the lipid conjugate can generally be related to the number of carbon atoms on the hydrocarbon R. For example, in the examples provided in Table 1, based on L (formula I) or L1-L2 (formula Ia) derived from ricinoleol, if the R hydrocarbon is derived from an acyl group having 2 carbon atoms (i.e., R is 1 carbon atom based on the nomenclature of formula I or Ia above), the LogP is only 8.33. However, the LogP of INT-D048, derived from an acyl chain with 5 carbon atoms, is 10.13 (i.e., based on the S nomenclature of formula I or Ia, where S is 4 carbon atoms). When an oleoyl group with 18 carbon atoms is conjugated to L (i.e., R with 17 carbon atoms based on the R nomenclature of formula I or Ia), the LogP of INT-D035 increases to 15.34. When the acyl chain conjugated to L has 20 carbon atoms, as in INT-D051, the LogP is 15.14 (i.e., S equals 19 carbon atoms). As discussed, by designing prodrugs with desired hydrophobicity, it is easier to control the drug loading and retention properties after administration.

[0411] Therefore, in one embodiment, R in formula I, Ia, II or IIa has 1-40 carbon atoms and is linear or branched, and is selected to provide a lipid conjugate with a desired logP, falling within the range of 5-25, 5-18 or 6-16.

[0412] As discussed, optionally, a second R hydrocarbon having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds can be chemically linked to L via an X2 chemical link. Further, optionally, a third R hydrocarbon having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds can be chemically linked to L via an X2 chemical link.

[0413] Furthermore, one or more R hydrocarbon moieties connected to L may have respective R' side chains connected thereto. For example, the R' side chain may be connected to the first, second, or third R via X2 connection, and the second R' side chain may be connected to any R via X2 connection and / or the third R' may be connected to any R via X2 connection. Various other combinations can be readily conceived by those skilled in the art.

[0414] It should be understood that the R-hydrocarbon need not be derived from an acyl or fatty acid. For example, R can be a cholesterol moiety or other hydrocarbon group. The R-hydrocarbon can also be a therapeutic or preventative moiety, released upon cleavage from a prodrug such as a therapeutically active lipid or sterol.

[0415] As noted, various chemical linkages can be used to link the molecule of interest M to L, one or more R hydrocarbons to L, or one or more R' groups to R. Those skilled in the art will understand that different functional groups or combinations thereof can be used in these linkages. That is, X1 and X2 in the above various embodiments relating to the lipid conjugates of formulas I, Ia, II, and IIa and the precursors P of formulas III, IIIa, IIIb, and IIIc can be independently selected from esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thiocarbonyl carbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, aminophosphates, phosphate esters, phosphonates, phosphate diesters, phosphate phosphonomethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azo compounds, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH2), or ureas. In another alternative embodiment, either of the linkages to X1 and X2 is biodegradable.

[0416] In another embodiment, any X2 is a connection comprising one or more hydrogen bonds. According to this type of embodiment, X2 has the structure of the connection portion of formula VI:

[0417]

[0418] E1, E2, E3, E4 and E5 are electronegative atoms selected from O, N and P;

[0419] E1, E2, and E3 are hydrogen bond acceptors, while E4 and E5 are hydrogen bond donors;

[0420] Dashed lines depict hydrogen bonds and solid lines depict covalent bonds;

[0421] Where L is a lipid scaffold of the lipid portion as described in Formula I, Ia, II or IIa;

[0422] R and R' are hydrocarbon chains as shown in formula IIa;

[0423] n is 0 or 1; o is 0 or 1; and p is 0 or 1; and n + o + p ≥ 2;

[0424] q is 1-10, 2-10, or 4-10;

[0425] L represents the lipid scaffold of the lipid portion;

[0426] M is the molecule of interest; and

[0427] E1 and E3 optionally contain substituents connected thereto, such as alkyl, aryl, alkylene, or H.

[0428] Products, compositions and formulations

[0429] The lipid conjugates described herein can be administered in free form, including as a component of pharmaceutical products or compositions, or as part of a delivery carrier. Such products or compositions typically include known pharmaceutically acceptable salts and / or excipients.

[0430] A variety of delivery systems can be used to prepare pharmaceutical formulations. These include, but are not limited to, nanoparticles (LNPs), including lipid nanoparticles containing vesicles with one or more bilayers, such as liposomes or polymer nanoparticles containing lipids, polymer-based nanoparticles, emulsions, micelles, and carbon nanotubes.

[0431] The lipid conjugates disclosed herein are particularly suitable for incorporation into nanoparticles, such as liposomes or polymer-based systems comprising lipids or other hydrophobic components. In some embodiments, the lipid-like properties of the lipid conjugates can facilitate their loading into these or other delivery carriers. For example, in some embodiments, the loading efficiency of a given nanoparticle is 75%-100%, 80%-100%, or most advantageously 90%-100%.

[0432] In one embodiment, the lipid conjugate is loaded into lipid nanoparticles (e.g., liposomes) by mixing the lipid conjugate with lipid formulation components, including vesicle-forming lipids and optional sterols. Therefore, lipid nanoparticles incorporating these drug-lipid conjugates can be prepared using a variety of well-described formulation methods known to those skilled in the art, including but not limited to extrusion, ethanol injection, and in-line mixing. Such methods are described in the following references: Maclachlan, I. and P. Cullis, “Diffusible-PEG-lipid Stabilized Plasmid LipidParticles”, Adv. Genet., 2005, 53 PA: 157-188; Jeffs, LB et al., “A Scalable, Extrusion-free Method for Efficient Liposomal Encapsulation of Plasmid DNA”, Pharm Res, 2005, 22(3): 362-72; and Leung, AK et al., “Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core”, The Journal of Physical Chemistry.C, Nanomaterials and Interfaces, 2012, 116(34): 18440-18450, each of which is incorporated herein by reference in its entirety.

[0433] Although liposomes contain an internal aqueous solution surrounded by a phospholipid bilayer, lipid nanoparticles can alternatively contain a lipophilic core. This lipophilic core can be used as a reservoir for prodrugs. Solid and liquid lipid nanoparticles can be used to deliver prodrugs as described herein.

[0434] One embodiment provides lipid nanoparticles comprising a phospholipid bilayer, wherein the lipid conjugate forms a hydrophobic oil phase within the bilayer. Such a delivery carrier is described in Examples 3 and 4. The hydrophobic oil phase can be observed by electron microscopy. In one embodiment, the lipid conjugate has a structure of formula I, Ia, II, or IIa. In another embodiment, the lipid nanoparticles are particles having one or more bilayers, such as liposomes.

[0435] The delivery carrier can also be nanoparticles comprising a lipid core stabilized by a surfactant. The lipids forming the vesicles can be used as stabilizers. In another embodiment, the lipid nanoparticles are polymer-lipid hybrid systems comprising a polymer nanoparticle core surrounded by a stabilizing lipid. In such embodiments, the lipid conjugates of this disclosure can be lipid-polymer conjugates.

[0436] Nanoparticles can also be prepared from lipid-free polymers. Such nanoparticles may include concentrated drug cores surrounded by a polymer shell, or may have a solid or liquid dispersed throughout the polymer matrix.

[0437] The lipid conjugates described herein can also be incorporated into emulsions, which are drug delivery carriers comprising oil droplets or oil cores. The emulsions can be lipid-stabilized. For example, an emulsion may comprise a core filled with an emulsifying component, such as a monolayer or bilayer lipid-stabilized oil.

[0438] Micelles are self-assembled particles composed of amphiphilic lipid or polymeric components for delivering drugs contained in a hydrophobic core. As described herein, conjugating drugs to scaffold molecules L having hydrophobic groups R can improve drug loading into micelles.

[0439] Another class of drug delivery carriers known to those skilled in the art for encapsulating the lipid conjugates described herein is carbon nanotubes.

[0440] Various methods for preparing the aforementioned delivery carrier and incorporating the prodrug therein are available and can be readily performed by those skilled in the art.

[0441] Certain lipid conjugates covered in this disclosure can form components of carrier-free systems. In such embodiments, the lipid conjugates can self-assemble into particles. Without limitation, if the drug portion D or the polymer is hydrophilic, the amphiphilic prodrug can assemble into nanoparticles with or without a stabilizer.

[0442] Although the pharmaceutical composition is as described above, the lipid conjugate can be a component of any nutritional product, cosmetic, cleaning product, or food.

[0443] application

[0444] In some embodiments, the lipid conjugate is a prodrug, which is either free or formulated in a drug delivery carrier and administered to treat, prevent, and / or improve a patient's condition. That is, the prodrug, in its free form or formulated in a delivery carrier, can provide preventative, improving, or therapeutic benefits. The pharmaceutical composition containing the prodrug will be administered at any suitable dose. In one embodiment, the prodrug, in its free form or formulated in a drug delivery carrier, is administered parenterally, i.e., intra-arterial, intravenous, subcutaneous, or intramuscularly. In other embodiments, the prodrug, in its free form or formulated in the delivery carrier described herein, can be administered topically. In a further alternative embodiment, the prodrug, in its free form or formulated in the delivery carrier described herein, can be administered orally. In another embodiment, the prodrug, in its free form or formulated in a delivery carrier, is administered to the lungs via an aerosol or powder dispersion.

[0445] In another embodiment, the molecule of interest is a hydrophilic polymer and the conjugate is a lipid-polymer conjugate. The lipid-polymer conjugate can be incorporated into a delivery carrier along with one or more drugs and administered to treat, prevent, and / or improve a patient's condition.

[0446] The term "patient" as used in this article includes both human and non-human subjects.

[0447] The following examples are provided for illustrative purposes only and are by no means intended to limit the scope of the invention. Example

[0448] Example 1: Castor oil-based alcohol as an exemplary scaffold molecule L

[0449] Examples of lipid conjugates are illustrated in Figure 2, demonstrating the diversity of conjugates that can be formed using ricinoleic acid or ricinoleol as precursors to the scaffold L in formulas I, Ia, II, or IIa above. In this schematic, the chemical structures of the X1 and X2 links of formula I are not depicted. Instead, these schematics show hydroxyl groups at C1 and C12 of fatty acids or alcohols (and atoms Z and Y at positions 9 and 10 in the oxidized form of the molecule), which can react with complementary functional groups on the drug-linking group and / or acyl groups, such as carboxylic acids. In this example, the X1 and X2 links can contain ester functional groups based on the condensation reaction between the carboxyl and hydroxyl groups; however, other functional groups can also be formed, depending on the specific functional groups present on the drug, molecular scaffold, side group R, or linking group that reacts to form X1 or X2.

[0450] The following embodiments also use linkers to connect the molecule of interest M to the scaffold molecule L. However, it should be understood that such linkers are optional, as the molecule of interest M may optionally be conjugated to the scaffold molecule L itself.

[0451] Ricinole alcohol is an unsaturated fatty alcohol derived from ricinoleic acid, which is a hydroxy fatty acid (HFA) having 18 carbon atoms and a hydroxyl group substituted at C12. Although ricinoleic acid or ricinole alcohol is described as a precursor scaffold P (X is C=O or CH2) in the structure of Figure 2, other molecules may be used, including, but not limited to, other hydroxy fatty acids, their corresponding fatty alcohols or fatty amines. Furthermore, the scaffold L based on ricinoleic acid or ricinole alcohol does not need to be prepared from a fatty acid or fatty alcohol having hydroxyl groups at both C1 and C12. For example, the precursor of L can be prepared from a corresponding molecule having a hydroxyl group at C1 and an ether substituent at C12 (e.g., the silyl ether I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol (2) intermediate described in Example 2).

[0452] In some embodiments, the double bonds of the backbone of ricinoleic acid or ricinoleol are partially or completely oxidized to provide additional reactive groups that can be used to conjugate the second acyl chain R'. These groups are depicted as Y and Z in the figure.

[0453] In this embodiment, the scaffold molecule L is described as the L1-L2 chain of formula Ia. L1 is the carbon chain from C1 to the carbon before the first branching point, where a side group (e.g., an acyl chain) or a molecule of interest or an M-linker is attached. L2 is the carbon chain including the carbon from the branching point to the scaffold terminal.

[0454] In structure A of Figure 2, X1 is a linker that covalently connects the molecule of interest to ricinoleic acid (X = C = O) or ricinoleol L (X = CH2) at C1. This linker is attached to C1 of ricinoleic acid or ricinoleol via a reactive group that is a hydroxyl group at C1. At C12 of ricinoleic acid or ricinoleol, an acyl-derived side chain R is attached to L2 via a hydroxyl group. In this example, L1 is a linear 11-carbon chain with cis double bonds at C9 and C10 as shown in Figure 2, and L2 is a 7-carbon saturated chain from C12 to C18. X2 is not shown, but it connects C12 of ricinoleic acid or ricinoleol to the acyl-derived R side chain. As described above, the acyl carboxylic acid reacts with the hydroxyl group at C12 of ricinoleic acid or ricinoleol to form an -O (C = O) ester linker. Similarly, in this example, the OH group at C1 of ricinoleic acid or ricinole-ol reacts with the carboxyl group at one end of the linker to form an X1-O (C=O) ester link. Alternatively, the OH group at C1 of L reacts directly with the free carboxylic acid on the molecule of interest M to form an -O (C=O) link (X1).

[0455] In structure B of Figure 2, linker X1 covalently links the molecule of interest M to the hydroxyl group at C12 of ricinoleic acid or ricinoleol. At C1 of the molecule, the R hydrocarbon derived from the acyl side group is linked to L1 via a terminal hydroxyl group at C1. In this example, L1 of the molecular scaffold has 11 carbon atoms, and L2 has 7 carbon atoms. Alternatively, the OH group at C1 of L reacts directly with the carboxylic acid at the molecule of interest M to form a -O (C=O) link.

[0456] In structure C of Figure 2, partially oxidized ricinoleic acid or ricinole alcohol serves as the precursor scaffold P. The double bonds of ricinoleic acid or ricinole-alcohol at C9 and C10 are oxidized to produce a saturated hydrocarbon chain, which is substituted at the C10 position by the reactive group Y and at the C12 position by a hydroxyl group. The side chain R derived from the acyl group is conjugated to C12 of L1-L2 via a hydroxyl group, while the second side chain R' from another acyl chain is conjugated to the C10 position via Y. In this example, Y is the reactive group and contains N, O, S, or P as the first atom in the group. Non-limitingly, if Y is N, the reactive group can be an amine; if Y is O, the reactive group can be a hydroxyl group. Similarly, if Y is P, the reactive group can be a phosphate ester. As discussed, these reactive groups are merely exemplary and other groups can be readily conceived by those skilled in the art.

[0457] The linker group X1 of the molecule of interest M is attached to C1 via a terminal hydroxyl group of ricinoleic acid or ricinoleol. Alternatively, the OH groups at C1 of L1-L2 react with the carboxylic acid on the molecule of interest M itself to form a -O (C=O) linker. In this example, L1 has 9 carbon atoms and L2 has 9 carbon atoms.

[0458] In structure D of Figure 2, partially oxidized ricinoleic acid or ricinole alcohol is again used as a scaffold precursor and contains the molecule of interest M linked via a linker X1 at C1. Alternatively, the OH groups at C1 of L1-L2 react directly with the carboxylic acid on the molecule of interest M to form a -O (C=O) link, instead of utilizing the linker as shown. The first side chain R, derived from the acyl chain, is linked at C12 via a hydroxyl reactive group, and the second side chain R', derived from the acyl chain, is linked at C9 of the ricinole-ol via a Y group, wherein the first atom in this group is N, O, S, or P, as described in structure C. In this example, L1 has 8 carbon atoms and L2 has 10 carbon atoms.

[0459] In structure E of Figure 2, oxidized ricinoleic acid or ricinoleol serves as a precursor for scaffold L, which has a side chain derived from an acyl chain R attached to the C12 position via a hydroxyl group, and a second side chain R' derived from the acyl chain attached to C9 via a Z group. Similar to Y, the Z group is a reactive group, wherein the first atom in this group is N, O, S, or P, as described in structures C and D above. The drug moiety D is attached to C1 via a linker X1 through a chemical link formed with the reactive hydroxyl group at C1. Alternatively, the OH groups at C1 of L1-L2 react directly with the carboxylic acid on drug D to form a -O (C=O) linker, instead of using a linker.

[0460] Example 2: Synthesis of lipid conjugates

[0461] Materials and methods

[0462] Different prodrugs were prepared using the synthetic methods AE listed below.

[0463] All reagents and solvents were purchased from commercial suppliers and, unless otherwise specified, can be used without further purification, except for THF (freshly distilled from Na / benzophenone under nitrogen atmosphere) and Et3N, DMF, and CH2Cl2 (freshly distilled from CaH2 under nitrogen atmosphere). USP grade castor oil was purchased from a local pharmacy (Life). TM (Brand) and use as is. For NMR, chemical shifts are expressed in parts per million (ppm) on the δ scale, and coupling constant J is in Hertz (Hz). Multiplicity is reported as “s” (singleton), “d” (doublet), “dd” (doublet), “dt” (doublet triplet), “ddd” (doublet doublet), “t” (triplet), “td” (triplet doublet), “q” (quartet), “quin” (quintet), “sex” (sextet), “m” (multiplet), and further specified as “app” (obvious) and “br” (broad peak).

[0464] The general steps for the synthesis of lipid conjugates based on hydroxyl and carboxyl derivatives of castor oil (glycerol triricinoleate) are provided in Scheme 1 below. Scheme 1, referred to as General Method AE, describes the steps for preparing the prodrugs of Examples 2A to 2V below.

[0465] Scheme 1: General synthesis of lipid conjugates based on hydroxyl and carboxyl derivatives of castor oil (glycerol triricinoleate).

[0466]

[0467] According to the synthesis reaction described in Scheme 1 above, castor oil, also known as glyceryl triricinoleate (glycerol ester of ricinoleic acid), is the raw material for synthesizing the prodrug shown in Figure 3.

[0468] In step 1) above, sodium methoxide (2.0 mL of 3.0 M MeOH solution, 6.00 mmol, 0.20 equivalent) was added to a stirred 1:1 THF / MeOH (30 mL) solution of castor oil (28.0 g, 30.0 mmol, 1.00 equivalent) at room temperature in a round-bottom flask under an argon atmosphere. After 14 h, the reaction mixture was quenched with a saturated aqueous NH4Cl solution and extracted with Et2O (3 × 150 mL). The combined organic layers were washed with water (1 × 150 mL) and brine (1 × 150 mL), dried over Na2SO4, and concentrated to give a clear, colorless oil (28.0 g, quantitative yield) of (12R)-hydroxyoleic acid methyl ester 1, which was used without further purification. The structure and physical properties of (12R)-hydroxyoleic acid methyl ester are shown below:

[0469] (12R)-hydroxyoleic acid methyl ester (1):

[0470]

[0471] R f =0.50 (SiO2, 70:30 hexane / EtOAc);

[0472] 1 ¹H NMR (300MHz, CDCl₃): δ 5.64–5.50 (m, 1H), 5.49–5.35 (m, 1H), 3.68 (s, 3H), 3.63 (quintet, J = 5.6Hz, 1H), 2.32 (t, J = 7.6Hz, 2H), 2.23 (t, J = 6.6Hz, 2H), 2.13–2.00 (m, 2H), 1.72–1.19 (m, 20H), 0.90 (t, J = 6.4Hz, 3H).

[0473] According to step 2) of the above reaction scheme, in an argon atmosphere, a 15 mL solution of (12R)-hydroxyoleic acid methyl ester (9.37 g, 30.0 mmol) in THF at room temperature was added to a stirred, ice-cold THF suspension (90 mL) of LiAlH4 (1.25 g, 33.0 mmol, 1.10 equivalents) in a round-bottom flask at room temperature over 20-30 min. After the addition was complete, the cold bath was removed. After 14 h, the reaction mixture was cooled in an ice bath, diluted with 150 mL of Et2O, quenched with a quenching solution (1.25 mL H2O, 1.25 mL 1 M NaOH aqueous solution, 3.75 mL H2O), stirred at room temperature for 1 h, and filtered through a C salt filter. Simultaneously, the mixture was thoroughly washed with Et2O. The filtrate was concentrated using a rotary evaporator to obtain crude diol, a pale yellow oil (quantitative yield), which was used without further purification.

[0474] According to step 3) of the above reaction scheme, a 20 mL solution of tert-butyldimethylsilyl chloride (3.96 g, 26.2 mmol, 1.00 equivalent) in DMF at room temperature was added to a 25 mL solution of the above diol (8.21 g, 28.9 mmol, 1.10 equivalent) and i-Pr2Net (5.73 mL, 32.8 mmol, 1.25 equivalent) in DMF at 10–15 °C in a round-bottom flask under an argon atmosphere for 30 min. The reaction mixture was then heated for 14 h, quenched with a saturated aqueous NH4Cl solution, and extracted with 1:1 Et2O / hexane (3 × 100 mL). The combined organic layers were washed with H2O (3 × 100 mL) and brine (1 × 100 mL), dried with Na2SO4, and concentrated using a rotary evaporator to give crude primary silyl ether as a pale yellow oil. The crude product was purified by filtration through a silica gel pad (220 mL SiO2, 99:1 → 95:5 hexane / EtOAc) to obtain a clear, colorless oil (8.38 g, 80% yield) consisting of silyl ether 2. The structure and physical properties of silyl ether 2 are shown below:

[0475] I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol (2):

[0476]

[0477] R f =0.16 (SiO2, 95:5 hexane / EtOAc);

[0478] 1 ¹H NMR (300MHz, CDCl₃): δ 5.64–5.50 (m, 1H), 5.49–5.35 (m, 1H), 3.68 (s, 3H), 3.63 (quintet, J = 5.6Hz, 1H), 2.32 (t, J = 7.6Hz, 2H), 2.23 (t, J = 6.6Hz, 2H), 2.13–2.00 (m, 2H), 1.72–1.19 (m, 20H), 0.90 (t, J = 6.4Hz, 3H).

[0479] According to step 4) of the above reaction scheme, N,N'-dicyclohexylcarbodiimide (DCC) (495 mg, 2.40 mmol, 1.20 equivalence) was added to a chilled CH2Cl2 (6 mL) solution of RCO2H (279 mg, 2.40 mmol, 1.20 equivalence) in a round-bottom flask under an argon atmosphere. The chill was then removed, and the resulting mixture was stirred for 15 min. In this example, RCO2H is hexanoic acid; however, other acyl groups can be used to generate the desired hydrocarbon side chain S. The reaction mixture was then cooled in an ice bath, and a CH2Cl2 (2 mL) solution of silyl ether, 1-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol 2 (797 mg, 2.00 mmol), was added, followed by DMAP (366 mg, 3.00 mmol, 1.50 equivalence). The reaction mixture was then warmed to room temperature for 14 h. The reaction mixture was diluted with Et2O and stirred for 10 min, then filtered through a C salt. The filtrate was concentrated using a rotary evaporator to give a crude ester as a white semi-solid. The crude product was purified by filtration through a silica gel pad (20 mL SiO2, 95:5 hexane / EtOAc) to give a clear, colorless oily intermediate ester (quantitative yield) with R f =0.53 (SiO2, 90:10 hexane / EtOAc).

[0480] According to step 5) of the above reaction scheme, under an argon atmosphere, a pure HF·pyridine solution (0.74 mL of 70% HF pyridine solution, 6.00 mmol, 3.00 equivalent) was added to a stirred, ice-cold solution of THF (6 mL), pyridine (0.48 mL, 6.00 mmol, 3.00 equivalent), and the above-mentioned silyl ether (2.00 mmol) in a round-bottom flask. After 2 h, the reaction mixture was quenched with saturated NaHCO3 and aqueous solution. The mixture was extracted with Et2O (2 × 10 mL), and the combined organic extracts were washed with H2O (1 × 10 mL) and brine, dried with Na2SO4, and concentrated by a rotary evaporator to give crude primary alcohol. The crude product was purified by filtration through a silica gel pad (20 mL, 90:10 hexane / EtOAc) to give primary alcohol 3 (quantitative yield) with the following structure and physical properties as a clear, colorless oil:

[0481] (12R)-Hexadecyloxyoleyl alcohol (3):

[0482]

[0483] According to step 6) of the above reaction scheme, solid succinic anhydride (400 mg, 4.00 mmol, 2.00 equivalent) and DMAP (611 mg, 5.00 mmol, 2.50 equivalent) were added to a stirred solution of (12R)-hexanoyloxyoleyl alcohol (3) (765 mg, 2.00 mmol, 1.00 equivalent) in CH2Cl2 (6 mL) at room temperature under an argon atmosphere in a round-bottom flask. After 14 hours, the reaction system was quenched with 1 M HCl aqueous solution and extracted with CH2Cl2 (2 × 15 mL). The combined organic extracts were then washed with 1 M HCl aqueous solution (1 × 15 mL) and H2O (2 × 15 mL), dried over Na2SO4, and concentrated using a rotary evaporator to give the intermediate hemisuccinate (quantitative yield) as a pale yellow oil, which was used without further purification. This intermediate has R f =0.32 (SiO2, 50:50 hexane / EtOAc).

[0484] According to step 7) of the reaction protocol, solid DCC (99 mg, 0.48 mmol, 1.20 equivalence) was added to a stirred, ice-cold CH₂Cl₂ (2 mL) solution of the above hemisuccinate (232 mg, 0.48 mmol, 1.20 equivalence) in a round-bottom flask under an argon atmosphere. The ice bath was then removed, and the resulting mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and solid dexamethasone (157 mg, 0.40 mmol) and DMAP (73 mg, 0.60 mmol, 1.50 equivalence) were added. The reaction mixture was heated for 14 h, diluted with Et₂O, stirred for 10 min, and then filtered through a C₂ salt. The filtrate was concentrated to give a crude product, a pale yellow oil. The crude product was purified by rapid column chromatography (50 mL SiO2, 80:20 → 50:50 hexane / EtOAc) to obtain a clear, colorless oil, which was the desired prodrug 4 (328 mg, 95% yield), and had the following structure and properties:

[0485] 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]57ctadic57rene-17-yl)-2-oxoethyl((R,Z)-12-(hexanoyloxy)57ctadic-9-en-1-yl) ester (4):

[0486]

[0487] R f=0.38 (SiO2, 50:50 hexane / EtOAc);

[0488] 1 H NMR (300MHz, CDCl3): δ7.22 (dd, J=10.2, 3.9, 1H), 6.32 (dd, J=10.2, 1.7, 1H), 6 .1(s, 1H), 5.44-5.17(m, 9H), 5.00-4.81(m, 2H), 4.43-4.22(m, 4H), 4.21-4.06( m, 2H), 3.16-3.01 (m, 1H), 2.84-2.51 (m, 11H), 2.50-2.23 (m, 9H), 2.21-1.48 (m , 25H), 1.45-1.15(m, 34H), 1.14-1.00(m, 1H), 1.03(s, 3H), 0.95-0.81(m, 10H).

[0489] The prodrug is based on a castor oil-based scaffold L with a hexanoyl (C6:0) side chain (INT-D034) conjugated to dexamethasone via a succinate linker.

[0490] In the above example, the RCO2H added in step 4) of the above reaction is hexanoic acid to generate a hexanoyl side chain (C6:0). However, other fatty acids can be used to generate the desired hydrocarbon side chain R on the castor oil-based scaffold.

[0491] Acylation of A-(R)-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol 3 (4a-h) using the general method:

[0492] In a round-bottom flask under an argon atmosphere, DCC (1.20 equivalents) was added to a stirred, chilled CH₂Cl₂ solution of the desired carboxylic acid (1.20 equivalents). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and a CH₂Cl₂ solution of the aforementioned alcohol 3 (1.00 equivalents, 0.25 M CH₂Cl₂ solution) was added, followed by DMAP (1.50 equivalents). The reaction mixture was then warmed to room temperature for 14 h. The reaction mixture was diluted with Et₂O, stirred for 10 min, and then passed through… Filtration. The filtrate was concentrated using a rotary evaporator to obtain crude ester, a white semi-solid. The crude product was purified by filtration through a silica gel pad (95:5 hexane / EtOAc) to obtain pure ester.

[0493] General method for desilylation-succinylation (5a-h) of B-(12R)-acyloxy oleyl alcohol 4a-h:

[0494] In a round-bottom flask under an argon atmosphere, a solution of HF·pyridine (3.00 equivalents of 70% HF in pyridine) was added to a stirred, ice-cold solution of pyridine (3.00 equivalents) and 12-acylricinoleol silyl ether (1.00 equivalents) in THF (0.30 M relative to the starting silyl ether). When TLC showed depletion of the feed (2–8 h), the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The mixture was extracted with Et2O (2 × 10 mL), followed by washing of the combined organic extracts with H2O (1 × 10 mL) and brine. The extracts were dried over Na2SO4 and concentrated using a rotary evaporator to give a crude primary alcohol. The crude product was purified by filtration through a silica gel pad (90:10 hexane / EtOAc), concentrated using a rotary evaporator, and dried under high vacuum to give a primary alcohol as a clear, colorless oil, which was used for subsequent succinylation without further purification.

[0495] In a round-bottom flask under an argon atmosphere, solid succinic anhydride (2.00 equivalents) and DMAP (2.50 equivalents) were added to a stirred, room-temperature solution of 12-acylricinoleol (1.00 equivalents) in CH₂Cl₂ (0.30 M relative to the initial primary alcohol). After 14 hours, the reaction was quenched with 1 M HCl aqueous solution and extracted with CH₂Cl₂ (2 × 15 mL). The combined organic extracts were then washed with 1 M HCl aqueous solution (1 × 15 mL) and H₂O (2 × 15 mL), dried over Na₂SO₄, and concentrated using a rotary evaporator. The residue was redissolved in hexane, treated with activated carbon, and then... Filter and concentrate the filtrate to obtain the intermediate hemisuccinate, which is a colorless to pale yellow oil and is used without further purification.

[0496] General method for acylation of C-(12R)-methyl ricinoleate 2 (6a-c):

[0497] In a round-bottom flask under an argon atmosphere, DCC (1.20 equivalents) was added to a stirred, ice-cold CH2Cl2 solution of the desired carboxylic acid (1.20 equivalents). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and a CH2Cl2 solution of (12R)-methyl ricinoleate (1.00 equivalents, 0.30 M CH2Cl2 solution) was added, followed by DMAP (1.50 equivalents). The reaction mixture was then warmed to room temperature for 14 h. The reaction mixture was diluted with hexane, stirred for 10 min, and then passed through… Filtration. The filtrate was concentrated using a rotary evaporator to obtain crude diester, a white semi-solid, which was purified by filtration through a silica gel pad (95:5 hexane / EtOAc) to obtain pure ester.

[0498] General method for conjugating D-dexamethasone with hemisuccinate 5a-h:

[0499] In an argon atmosphere, DCC (1.20 equivalents) was added to a stirred, ice-cold solution of 1.20 equivalents of hemisuccinate 12-acylricinoleate in CH₂Cl₂ (0.2 M dexamethasone solution) in a round-bottom flask. The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and solid dexamethasone (1.00 equivalents) and DMAP (1.50 equivalents) were added. The reaction mixture was warmed for 14 h, diluted with Et₂O, stirred for 10 min, and then passed through… Filtration. The filtrate was concentrated to obtain a crude product, which was a pale yellow oil. It was then purified by rapid column chromatography (SiO2, 80:20→50:50 hexane / EtOAc) to obtain a clear, colorless oil, which was the desired dexamethasone conjugate.

[0500] General methods for conjugating E-dexamethasone with ricinoleic acid 12a-b, 13:

[0501] In a round-bottom flask under an argon atmosphere, DCC (1.10 equivalents) was added to a stirred, ice-cold solution of acyloxystearic acid (1.10 equivalents) in CH₂Cl₂ (0.1 M dexamethasone solution). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and solid dexamethasone (1.00 equivalents) and DMAP (1.50 equivalents) were added. The reaction mixture was warmed for 14 h, diluted with Et₂O, stirred for 10 min, and then passed through… Filtration. The filtrate was concentrated to obtain a crude product, which was a pale yellow oil. This crude product was then purified by rapid column chromatography to obtain a clear, colorless oil, which was the desired conjugate.

[0502] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4a):

[0503] In a round-bottom flask under an argon atmosphere, acetyl chloride (0.43 mL, 6.00 mmol, 1.20 equivalence) was added dropwise to a stirred, ice-cold solution of silyl ether 3 (2.00 g, 5.00 mmol, 1.00 equivalence), acetyl chloride (0.43 mL, 6.00 mmol, 1.20 equivalence), triethylamine (0.83 mL, 6.00 mmol, 1.2 equivalence), and DMAP (733 mg, 6.00 mmol, 1.20 equivalence) in CH₂Cl₂ (10 mL). The mixture was then warmed to room temperature. After 14 h, the reaction mixture was diluted with CH₂Cl₂, washed with saturated NH₄Cl aqueous solution (1 × 15 mL), water (2 × 15 mL), dried over Na₂SO₄, and concentrated using a rotary evaporator. The residue was redissolved in the eluent and passed through a silica gel pad (30 mL SiO2, 97:3 hexane / EtOAc) to give ester 4a (1.83 g, 83%), which was a pale yellow oil.

[0504] R f =0.45 (SiO2, 95:5 hexane / EtOAc);

[0505] 1 H NMR (300MHz, CDCl3): δ5.65-5.53(m, 1H), 5.49-5.36(m, 1H), 3.70-3.56(m, 3H), 2.23(t , J=6.8Hz, 2H), 2.13-2.00(m, 2H), 1.58-1.23(m, 22H), 0.97-0.86(m, 12H), 0.07(s, 6H).

[0506]

[0507] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4b):

[0508] According to general method A, 2.37 g of ester 4b (2.39 g, quantitative yield) was obtained in CH2Cl2 (15 mL) by reacting silyl ether 3 (2.00 g, 5.00 mmol), hexanoic acid (697 mg, 6.00 mmol), DCC (1.24 g, 6.00 mmol) and DMAP (916 mg, 7.50 mmol) with silyl ether 3 (2.00 g, 5.00 mmol), hexanoic acid (697 mg, 6.00 mmol), DCC (1.24 g, 6.00 mmol) and DMAP (916 mg, 7.50 mmol) as a clear, colorless oil.

[0509] R f =0.43 (SiO2, 95:5 hexane / EtOAc);

[0510] 1¹H NMR (300MHz, CDCl₃): δ 5.56–5.42 (m, 1H), 5.41–5.27 (m, 1H), 4.90 (quintet, J = 6.3 Hz, 1H), 3.61 (t, J = 6.6 Hz, 2H), 2.37–2.22 (m, 4H), 2.10–1.96 (m, 2H), 1.71–1.45 (m, 6H), 1.43–1.19 (m, 22H), 0.91 (br s, 15H), 0.07 (s, 6H).

[0511]

[0512] Lauric acid (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4c):

[0513] According to general method A, ester 4c (1.38 g, quantitative yield) was obtained by reacting silyl ether 3 (997 mg, 2.50 mmol), lauric acid (601 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol), and DMAP (458 mg, 3.75 mmol) in CH2Cl2 (8 mL) to a clear, colorless oil.

[0514] R f =0.56 (SiO2, 95:5 hexane / EtOAc);

[0515] 1 ¹H NMR (300MHz, CDCl₃): δ 5.56–5.41 (m, 1H), 5.41–5.26 (m, 1H), 4.90 (quintet, J = 6.2 Hz, 1H), 3.61 (t, J = 6.6 Hz, 2H), 2.37–2.21 (m, 4H), 2.11–1.95 (m, 2H), 1.72–1.43 (m, 12H), 1.43–1.13 (m, 38H), 0.91 (br s, 15H), 0.07 (s, 6H).

[0516]

[0517] Stearic acid (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4d):

[0518] According to general method A, silyl ether 3 (997 mg, 2.50 mmol), stearic acid (853 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol), and DMAP (458 mg, 3.75 mmol) in 2:1 THF / CH2Cl2 (6 mL) yielded ester 4d (1.56 g, 94%), which was a clear, colorless oil.

[0519] R f =0.48 (SiO2, 90:10 hexane / EtOAc);

[0520] 1 ¹H NMR (300MHz, CDCl₃): δ 5.57–5.41 (m, 1H), 5.41–5.25 (m, 1H), 4.90 (quintet, J = 6.3 Hz, 1H), 3.61 (t, J = 6.5 Hz, 2H), 2.39–2.20 (m, 4H), 2.11–1.96 (m, 2H), 1.72–1.43 (m, 8H), 1.43–1.13 (m, 44H), 0.91 (br s, 15H), 0.07 (s, 6H).

[0521]

[0522] Oleic acid (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4e):

[0523] According to general method A, ester 4e (1.64 g, quantitative) was obtained from silyl ether 3 (997 mg, 2.50 mmol), oleic acid (847 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in CH2Cl2 (10 mL) to a clear, colorless oily substance.

[0524] R f =0.41 (SiO2, 95:5 hexane / EtOAc);

[0525] 1 ¹H NMR (300MHz, CDCl₃): δ 5.56–5.25 (m, 4H), 4.90 (quintet, J = 6.2Hz, 1H), 3.61 (t, J = 6.5Hz, 2H), 2.42–2.19 (m, 8H), 2.11–1.93 (m, 6H), 1.70–1.44 (m, 8H), 1.44–1.17 (m, 40H), 0.91 (br s, 15H), 0.06 (s, 6H).

[0526]

[0527] Linoleic acid (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4f):

[0528] According to general method A, ester 4f (1.06 g, 76%) was obtained from silyl ether 3 (847 mg, 2.12 mmol), linoleic acid (715 mg, 2.55 mmol), DCC (526 mg, 2.55 mmol) and DMAP (389 mg, 3.19 mmol) in CH2Cl2 (7 mL) as a clear, colorless oil.

[0529] R f =0.46 (SiO2, 95:5 hexane / EtOAc);

[0530] 1 ¹H NMR (300MHz, CDCl₃): δ 5.67–5.24 (m, 6H), 4.90 (quintet, J = 6.2Hz, 1H), 3.61 (t, J = 6.6Hz, 2H), 2.79 (t, J = 5.9Hz, 2H), 2.40–2.17 (m, 4H), 2.15–1.94 (m, 4H), 1.71–1.44 (m, 8H), 1.43–1.17 (m, 26H), 0.91 (br s, 15H), 0.07 (s, 6H).

[0531]

[0532] Linolenic acid (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4g):

[0533] According to general method A, 4 g (1.52 g, 92%) of ester was obtained from silyl ether 3 (997 mg, 2.50 mmol), linolenic acid (835 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in CH2Cl2 (8 mL) as a clear, colorless oil.

[0534] R f =0.34 (SiO2, 95:5 hexane / EtOAc);

[0535] 1¹H NMR (300MHz, CDCl₃): δ 5.58–5.26 (m, 8H), 4.90 (quintet, J = 6.2 Hz, 1H), 3.61 (t, J = 6.5 Hz, 2H), 2.83 (t, J = 5.8 Hz, 4H), 2.35–2.22 (m, 4H), 2.17–1.97 (m, 6H), 1.69–1.44 (m, 6H), 1.43–1.18 (m, 26H), 1.00 (t, J = 7.5 Hz, 3H), 0.91 (br s, 12H), 0.07 (s, 6H).

[0536]

[0537] Arachidonic acid (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl ester (4h):

[0538] According to general method A, silyl ether 3 (797 mg, 2.00 mmol), arachidonic acid (670 mg, 2.20 mmol), DCC (227 mg, 2.20 mmol) and DMAP (366 mg, 3.00 mmol) in CH2Cl2 (7 mL) were subjected to rapid column chromatography (99:1 → 95:5 hexane / EtOAc) to obtain an ester (730 mg, 53%) as a clear, colorless oil.

[0539] R f =0.57 (SiO2, 95:5 hexane / EtOAc);

[0540] 1 ¹H NMR (300MHz, CDCl₃): δ 5.57–5.26 (m, 10H), 4.91 (quintet, J = 6.3Hz, 1H), 3.61 (t, J = 6.5Hz, 2H), 2.94–2.84 (m, 6H), 2.38–2.22 (m, 4H), 2.20–1.96 (m, 6H), 1.71 (quintet, J = 7.4Hz, 2H), 1.63–1.46 (m, 4H), 1.45–1.16 (m, 26H), 0.91 (br s, 15H), 0.07 (s, 6H).

[0541]

[0542] (R,Z)-4-((12-acetoxyoctadec-9-en-1-yl)oxy)-4-oxobutyric acid (5a):

[0543] According to general method B, silyl ether 4a (1.79 g, 4.07 mmol) was desilylated with HF·pyridine solution (1.52 mL, 12.2 mmol), pyridine (0.98 mL, 12.2 mmol), and THF (10 mL) to give the intermediate primary alcohol (1.34 g), which was then acylated with succinic anhydride (814 mg, 8.14 mmol), DMAP (1.24 g, 10.2 mmol), and CH2Cl2 (10 mL) to give carboxylic acid 5a (1.72 g, quantitative yield).

[0544] R f =0.23 (SiO2, 50:50 hexane / EtOAc);

[0545] 1 H NMR (300MHz, CDCl3): δ5.57-5.42 (m, 1H), 5.42-5.27 (m, 1H), 4.89 (quin., J=6.2Hz, 1H), 4.11 (t, J=6.7Hz, 2 H), 2.76-2.57(m, 4H), 2.11-1.97(m, 2H), 2.05(s, 3H), 1.72-1.46(m, 4H), 1.46-1.16(m, 18H), 0.90(m, 3H).

[0546]

[0547] (R,Z)-4-((12-(hexanoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutyric acid (5b):

[0548] According to general method B, pyridine (1.21 mL, 15.0 mmol) and THF (13 mL) silyl ether 4b (2.35 g, 5.00 mmol) were desilylated to give intermediate primary alcohol (2.01 g), which was then acylated with succinic anhydride (1.00 g, 10.0 mmol), DMAP (1.53 g, 12.5 mmol) and CH2Cl2 (13 mL) to give carboxylic acid 5b (2.20 g, 92% yield).

[0549] R f =0.32 (SiO2, 50:50 hexane / EtOAc);

[0550] 1¹H NMR (300MHz, CDCl₃): δ 5.56–5.42 (m, 1H), 5.41–5.27 (m, 1H), 4.90 (quintet, J = 6.4 Hz, 1H), 4.11 (t, J = 6.5 Hz, 2H), 2.76–2.58 (m, 4H), 2.38–2.22 (m, 4H), 2.11–1.96 (m, 2H), 1.73–1.47 (m, 6H), 1.46–1.15 (m, 22H), 0.97–0.82 (m, 6H).

[0551]

[0552] (R,Z)-4-((12-(lauroyloxy)octadec-9-en-1-yl)oxy)-4-oxobutyric acid (5c):

[0553] According to general method B, silyl ether 4c (1.38 g, 2.50 mmol) was desilylated with HF·pyridine solution (0.93 mL, 7.50 mmol), pyridine (0.60 mL, 7.50 mmol), and THF (8 mL) to give the intermediate primary alcohol (1.21 g), which was then acylated with succinic anhydride (500 mg, 5.00 mmol), DMAP (764 mg, 6.25 mmol), and CH2Cl2 (8 mL) to give carboxylic acid 5c (1.33 g, 94%).

[0554] R f =0.44 (SiO2, 50:50 hexane / EtOAc);

[0555] 1 ¹H NMR (300MHz, CDCl₃): δ 5.57–5.42 (m, 1H), 5.41–5.26 (m, 1H), 4.90 (quintet, J = 6.2 Hz, 1H), 4.12 (t, J = 6.6 Hz, 2H), 2.78–2.59 (m, 4H), 2.37–2.22 (m, 4H), 2.11–1.96 (m, 2H), 1.73–1.45 (m, 6H), 1.45–1.12 (m, 28H), 0.98–0.80 (m, 6H).

[0556]

[0557] (R,Z)-4-oxo-4-((12-(stearoyloxy)octadec-9-en-1-yl)oxy)butyric acid (5d):

[0558] According to general method B, silyl ether 4d (1.66 g, 2.50 mmol) was desilylated with HF·pyridine solution (0.93 mL, 7.50 mmol), pyridine (0.60 mL, 7.50 mmol), and THF (8 mL) to give the intermediate primary alcohol (1.30 g), which was then acylated with succinic anhydride (500 mg, 5.00 mmol), DMAP (764 mg, 6.25 mmol), and CH2Cl2 (8 mL) to give carboxylic acid 5d (1.29 g, 79% yield).

[0559] R f =0.35 (SiO2, 50:50 hexane / EtOAc);

[0560] 1 ¹H NMR (300MHz, CDCl₃): δ 5.56–5.42 (m, 1H), 5.41–5.27 (m, 1H), 4.90 (quintet, J = 6.3 Hz, 1H), 4.11 (t, J = 6.5 Hz, 2H), 2.77–2.58 (m, 4H), 2.39–2.19 (m, 4H), 2.12–1.95 (m, 2H), 1.73–1.45 (m, 6H), 1.44–1.11 (m, 46H), 0.98–0.80 (m, 6H).

[0561]

[0562] (R,Z)-4-oxo-4-((12-(oleoyloxy)octadec-9-en-1-yl)oxy)butyric acid (5e):

[0563] According to general method B, silyl ether 4e (663 mg, 1.00 mmol) was desilylated with HF·pyridine solution (0.37 mL, 3.00 mmol), pyridine (0.24 mL, 3.00 mmol), and THF (5 mL) to give the intermediate primary alcohol (546 mg), which was then acylated with succinic anhydride (200 mg, 2.00 mmol), DMAP (305 mg, 2.50 mmol), and CH2Cl2 (5 mL) to give carboxylic acid 5e (630 mg, 97% yield).

[0564] R f =0.42 (SiO2, 50:50 hexane / EtOAc);

[0565] 1¹H NMR (300MHz, CDCl₃): δ 5.57–5.25 (m, 4H), 4.90 (quintet, J = 6.2Hz, 1H), 4.11 (t, J = 6.5Hz, 2H), 2.77–2.59 (m, 4H), 2.39–2.20 (m, 4H), 2.13–1.93 (m, 6H), 1.72–1.46 (m, 6H), 1.46–1.02 (m, 34H), 0.97–0.80 (m, 6H).

[0566]

[0567] 4-(((R,Z)-12-(linoleyloxy)octadec-9-en-1-yl)oxy)-4-oxobutyric acid (5f):

[0568] According to general method B, silyl ether 4f (1.06 g, 1.60 mmol) was desilylated with HF·pyridine solution (0.60 mL, 4.80 mmol), pyridine (0.39 mL, 4.80 mmol), and THF (8 mL) to give the intermediate primary alcohol (890 mg), which was then acylated with succinic anhydride (320 mg, 3.20 mmol), DMAP (489 mg, 4.00 mmol), and CH2Cl2 (8 mL) to give carboxylic acid 5f (1.04 g, quantitative yield).

[0569] R f =0.35 (SiO2, 50:50 hexane / EtOAc);

[0570] 1 ¹H NMR (300MHz, CDCl₃): δ 5.57–5.26 (m, 6H), 4.90 (quintet, J = 6.3Hz, 1H), 4.11 (t, J = 6.7Hz, 2H), 2.79 (t, J = 6.0Hz, 2H), 2.75–2.58 (m, 6H), 2.38–2.20 (m, 4H), 2.14–1.94 (m, 6H), 1.72–1.46 (m, 8H), 1.46–1.14 (m, 30H), 0.98–0.81 (m, 6H).

[0571]

[0572] 4-(((R,Z)-12-(linolenic acid)octadec-9-en-1-yl)oxy)-4-oxobutyric acid (5g):

[0573] According to general method B, 4 g (1.54 g, 2.34 mmol) of silyl ether was desilylated with HF·pyridine solution (0.87 mL, 7.01 mmol), pyridine (0.57 mL, 7.01 mmol), and THF (6 mL) to give an intermediate primary alcohol (1.31 g), which was then acylated with succinic anhydride (468 mg, 4.68 mmol), DMAP (714 mg, 5.84 mmol), and CH2Cl2 (6 mL) to give a carboxylic acid (1.47 g, quantitative yield).

[0574] R f =0.35 (SiO2, 50:50 hexane / EtOAc);

[0575] 1 ¹H NMR (300MHz, CDCl₃): δ 5.56–5.25 (m, 8H), 4.90 (quintet, J = 6.2Hz, 1H), 4.11 (t, J = 6.5Hz, 2H), 2.82 (t, J = 5.7Hz, 4H), 2.37–2.22 (m, 4H), 2.16–1.95 (m, 6H), 1.74–1.46 (m, 6H), 1.46–1.15 (m, 30H), 0.99 (t, J = 7.6Hz, 3H), 0.94–0.83 (m, 6H).

[0576]

[0577] 4-(((R,Z)-12-(arachidonicyloxy)octadec-9-en-1-yl)oxy)-4-oxobutyric acid (5h):

[0578] According to general method B, silyl ether was desilylated for 4 h (711 mg, 1.04 mmol) with HF·pyridine solution (0.39 mL, 3.11 mmol), pyridine (0.25 mL, 3.11 mmol), and THF (5 mL) to give the intermediate primary alcohol (593 mg), which was then acylated with succinic anhydride (201 mg, 2.01 mmol), DMAP (306 mg, 2.51 mmol), and CH2Cl2 (5 mL) to give the carboxylic acid for 5 h (582 mg, 87% yield).

[0579] R f =0.31 (SiO2, 50:50 hexane / EtOAc);

[0580] 1¹H NMR (300MHz, CDCl₃): δ 5.58–5.24 (m, 10H), 4.90 (quintet, J = 6.2Hz, 1H), 4.11 (t, J = 6.7Hz, 2H), 2.93–2.75 (m, 6H), 2.76–2.58 (m, 4H), 2.39–2.22 (m, 4H), 2.20–1.96 (m, 6H), 1.71 (quintet, J = 7.4Hz, 2H), 1.69–1.47 (m, 4H), 1.46–1.13 (m, 26H), 0.99–0.80 (m, 6H).

[0581]

[0582] (12R)-hexanoyloxymethyl oleate (6a):

[0583] According to general method C, methyl ricinoleate (2.00 g, 6.40 mmol), hexanoic acid (898 mg, 7.68 mmol), DCC (1.58 g, 7.68 mmol) and DMAP (1.17 g, 9.60 mmol) in CH2Cl2 (10 mL) were filtered through silica gel (95:5 hexane / EtOAc) to give ricinoleate 6a (2.52 g, 96% yield) as a clear, colorless oil.

[0584] R f : 0.62 (SiO2, 70:30 hexane: EtOAc);

[0585] 1 H (300MHz, CDCl3): δ 5.54-5.42 (m, 1H), 5.40-5.28 (m, 1H), 4.90 (quintet, J = 6.2Hz, 1H), 3.69 (s, 3H), 2.37-2.23 (m, 6H), 2.11-1.97 (m, 2H), 1.72-1.48 (m, 6), 1.43-1.20 (m, 20), 0.96-0.84 (m, 6H).

[0586]

[0587] (12R)-Linoleoyloxymethyl oleate (6b):

[0588] According to general method C, methyl ricinoleate (500 mg, 1.60 mmol), linoleic acid (538 mg, 1.92 mmol), DCC (396 mg, 1.92 mmol) and DMAP (293 mg, 2.40 mmol) in CH2Cl2 (5 mL) were filtered through silica gel (95:5 hexane / EtOAc) to obtain ricinoleate 6c (875 g, 93% yield) as a pale yellow oil.

[0589] R f : 0.67 (SiO2, 80:20 hexane: EtOAc);

[0590] 1 H (300MHz, CDCl3): δ 5.54-5.42 (m, 1H), 5.40-5.28 (m, 1H), 4.90 (quintet, J = 6.2Hz, 1H), 3.69 (s, 3H), 2.37-2.23 (m, 6H), 2.11-1.97 (m, 2H), 1.72-1.48 (m, 6), 1.43-1.20 (m, 20), 0.96-0.84 (m, 6H).

[0591]

[0592] (12R)-Hexanoyloxyoleic acid (7a):

[0593] Methyl ester 6a (1.97 g, 4.79 mmol, 1.00 equivalent) and t-BuOH (12 mL) were added to an argon-purged round-bottom flask, followed by the addition of 2.0 M NaOH aqueous solution (1.80 mL, 3.60 mmol, 0.75 equivalent). After 17 h, the pH of the reaction solution was adjusted to 2 using 1 M HCl aqueous solution, and the mixture was extracted with Et₂O (3 × 30 mL). The combined organic layers were washed with water (1 × 30 mL) and brine (1 × 30 mL), dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The product (98:2:0 → 50:45:5 hexane:EtOAc:MeOH) was filtered through a silica gel sieve to give carboxylic acid 7a (1.30 g, 92% yield) as a pale yellow oil.

[0594] R f =0.24 (SiO2, 75:20:5 hexane / EtOAc / MeOH);

[0595] 1¹H NMR (300MHz, CDCl₃): δ 5.55–5.28 (m, 6H), 4.90 (quintet, J = 6.2Hz, 1H), 3.69 (s, 3H), 2.79 (t, J = 5.8Hz, 2H), 2.40–2.21 (m, 6H), 2.16–1.93 (m, 6H), 1.72–1.46 (m, 8H), 1.46–1.18 (m, 32H), 1.00–0.80 (m, 6H).

[0596]

[0597] (12R)-Linoleoyloxyoleic acid (7b):

[0598] Methyl ester 6b (5.97 g, 10.4 mmol, 1.00 equivalent) and t-BuOH (26 mL) were added to an argon-purged round-bottom flask, followed by the addition of 2.0 M NaOH aqueous solution (4.70 mL, 9.30 mmol, 0.90 equivalent). After 17 h, the pH of the reaction solution was adjusted to 2 using 1 M HCl aqueous solution, and the mixture was extracted with Et₂O (3 × 30 mL). The combined organic layers were washed with water (1 × 30 mL) and brine (1 × 30 mL), dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The residue was purified by rapid column chromatography (SiO₂, 95:5:0 → 80:15:5 hexane:EtOAc:MeOH) to give carboxylic acid 7b (4.48 g, 85% yield) as a pale yellow oil.

[0599] R f : 0.35 (SiO2, 75:20:5 hexane / EtOAc / MeOH);

[0600] 1 H(CDCl3, 300MHz): δ5.55-5.28 (m, 6H), 4.90 (quintet, J = 6.2Hz, 1H), 2.79 (t, J = 6.0Hz, 2H), 2.43-2.21 (m, 6H), 2.14-1.96 (m, 6H), 1.73-1.47 (m, 6H), 1.46-1.18 (m, 30H), 0.99-0.81 (m, 6H).

[0601]

[0602] 9,10-Dihydroxystearic acid methyl ester (8):

[0603] In a 500 mL Erlenmeyer flask, KOH (7.01 g, 125 mmol, 5.00 equivalents) was added to a rapidly stirred mixture of oleic acid (7.06 g, 25.0 mmol) and water (175 mL) at room temperature, and then cooled to ~10 °C. A solution of KMnO4 (7.11 g, 45.0 mmol, 1.80 equivalents) in water (75 mL) was added dropwise over 10 min. After stirring for another 10–15 min, the reaction was quenched by adding a saturated aqueous solution of NaHSO3, and then the pH was adjusted to ≤2 by adding concentrated HCl using a cooling bath. The white flocculent mixture was stirred at room temperature for 1 h, and the solid was collected by vacuum filtration and air-dried overnight. The resulting white solid was thermogravimetrically filtered and recrystallized from EtOH to give (±)-syn-9,10-dihydroxystearic acid as white crystals (5.86 g, 74% yield).

[0604] Concentrated H₂SO₄ (0.06 mL, 1.00 mmol, 0.05 equivalent) was added to a suspension of the above-mentioned dihydroxy acid (6.33 g, 20.0 mmol) in MeOH (50 mL), and the mixture was heated under reflux. After 14 h, the mixture was cooled to room temperature and concentrated under reduced pressure using a rotary evaporator, so that the resulting residue was partitioned between EtOAc and a saturated aqueous solution of NaHCO₃. The organic layer was washed with water (1 × 75 mL) and brine, dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator to give methyl ester 8 (6.44 g, 97% yield) as a white solid.

[0605] R f =0.45 (SiO2, 50:50 hexane / EtOAc);

[0606] 1 H NMR (300MHz, CDCl3): δ3.68 (s, 3H), 3.61 (app br s, 2H), 2.32 (t, J=7.4Hz, 2H), 2.06-1.85 (app br s, 2H), 1.73-1.16 (m, 26H), 0.96-0.81 (m, 3H).

[0607]

[0608] 9,10,12R-methyl trihydroxystearate (9):

[0609] In a 1 L Erlenmeyer flask, KOH (5.61 g, 100 mmol, 2.00 equivalents) was added to a rapidly stirred mixture of ricinoleic acid (14.9 g, 50.0 mmol) and water (500 mL) at room temperature, and then cooled to ~10 °C. A solution of KMnO4 (13.4 g, 85.0 mmol, 1.70 equivalents) in water (250 mL) was added dropwise over 15 min. After stirring for another 10–15 min, the reaction was quenched by adding a saturated aqueous solution of Na2SO3, and then the pH was adjusted to ≤2 by adding concentrated HCl using a cooling bath. The white flocculent mixture was stirred at room temperature for 4 h, and the solid was collected by vacuum filtration and air-dried overnight. The resulting white solid was thermogravimetrically filtered with EtOH to obtain crude 9,10,12-trihydroxystearic acid, which was used without further purification.

[0610] Concentrated H₂SO₄ (0.13 mL, 2.50 mmol, 0.05 equivalent) was added to a suspension of the above-mentioned dihydroxy acid (6.33 g, 20.0 mmol) in MeOH (120 mL), and the mixture was heated under reflux. After 14 h, the mixture was cooled to room temperature and concentrated under reduced pressure using a rotary evaporator, so that the resulting residue was partitioned between EtOAc and a saturated aqueous solution of NaHCO₃. The organic layer was washed with water (1 × 75 mL) and brine, dried with Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The resulting pale yellow solid was ground four times with warm Et₂O to give methyl ester 9 (9.52 g, 55% yield), a white solid.

[0611] R f =0.33 (SiO2, 50:50 hexane / EtOAc);

[0612] 1 H NMR (300MHz, CDCl3): δ4.07-3.58 (m, 3H), 3.68 (s, 3H), 2.31 (t, J=7.5Hz, 2H), 1.86-1.14 (m, 24H), 0.90 (br t, 3H).

[0613]

[0614] 9,10-Dihexanoyloxymethyl stearate (10a):

[0615] In an argon atmosphere, DCC (2.27 g, 11.0 mmol, 2.20 equivalence) was added to a stirred, ice-cold CH₂Cl₂ (13 mL) solution of hexanoic acid (1.28 g, 11.0 mmol, 2.20 equivalence). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and glycol 8 (1.65 g, 5.00 mmol) was added, followed by DMAP (1.53 g, 12.5 mmol, 2.50 equivalence). The reaction mixture was then warmed to room temperature for 14 h. The reaction mixture was diluted with Et₂O, stirred for 10 min, and then... Filtration. The filtrate was washed with 1M HCl (2×30mL) aqueous solution, 1M NaOH aqueous solution (2×30mL), H2O (1×30mL), and brine. The filtrate was dried with Na2SO4 and concentrated under reduced pressure using a rotary evaporator to obtain triester 10a (2.61g, quantitative yield), which was a clear, colorless oil.

[0616] R f =0.66 (SiO2, 70:30 hexane / EtOAc);

[0617] 1 H NMR (300MHz, CDCl3): δ 5.08-4.92 (m, 2H), 3.68 (s, 3H), 2.40-2.20 (m, 6H), 1.74-1.44 (m, 12H), 1.44-1.13 (m, 28H), 1.01-0.80 (m, 9H).

[0618]

[0619] 9,10-Dilinoleoyloxymethyl stearate (10b):

[0620] In an argon atmosphere, DCC (4.33 g, 21.0 mmol, 2.10 equivalences) was added to a stirred, ice-cold CH₂Cl₂ (25 mL) solution of linoleic acid (5.89 g, 21.0 mmol, 2.20 equivalences). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and glycol 8 (3.30 g, 10.0 mmol) was added, followed by DMAP (3.05 g, 25.0 mmol, 2.50 equivalences). The reaction mixture was then warmed to room temperature for 14 h. The reaction mixture was diluted with hexane, stirred for 10 min, and then... Filtration. The filtrate was concentrated using a rotary evaporator to obtain a crude product, which was a white semi-solid. This product was purified by filtration through a silica gel pad (95:5 hexane / EtOAc) to give triester 10b (7.24 g, 85% yield), which was a clear, colorless oil.

[0621] R f =0.57 (SiO2, 70:30 hexane / EtOAc);

[0622] 1 H NMR (300MHz, CDCl3): δ5.49-5.27 (m, 8H), 5.05-4.94 (m, 2H), 3.68 (s, 3H), 2.79 (t, J=5.9Hz, 4H), 2.39-2.23(m, 6H), 2.15-1.97(m, 8H), 1.72-1.45(m, 10H), 1.45-1.15(m, 50H), 0.98-0.82(m, 9H).

[0623]

[0624] 9,10,12R-Trihexanoyloxymethyl stearate (11):

[0625] In an argon atmosphere, DCC (2.64 g, 12.8 mmol, 3.20 equivalence) was added to a stirred, ice-cold CH₂Cl₂ (13 mL) solution of hexanoic acid (1.49 g, 12.8 mmol, 3.20 equivalence). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and triol 9 (1.39 g, 4.00 mmol) was added, followed by DMAP (1.71 g, 14.0 mmol, 3.50 equivalence). The reaction mixture was then warmed to room temperature for 14 h. The reaction mixture was diluted with hexane, stirred for 10 min, and then... Filtration. The filtrate was washed with 1M HCl (2×30mL) aqueous solution, 1M NaOH aqueous solution (2×30mL), H2O (1×30mL), and brine. It was dried with Na2SO4 and concentrated under reduced pressure using a rotary evaporator to give triester 11 (1.99g, 78% yield), which was a clear, colorless oil.

[0626] R f =0.77 (SiO2, 70:30 hexane / EtOAc);

[0627] 1 H NMR (300MHz, CDCl3): δ5.13-4.84(m, 3H), 3.68(s, 3H), 2.38-2.19(m, 8H), 1. 92-1.69 (m, 2H), 1.69-1.42 (m, 12H), 1.42-1.16 (m, 28H), 1.00-0.82 (m, 12H).

[0628]

[0629] 9,10-Dihexanoyloxystearic acid (12a):

[0630] In a round-bottom flask under an argon atmosphere, 0.91 mL of 2.0 M KOH aqueous solution (1.82 mmol, 1.00 equivalent) was added to a 7 mL solution of t-BuOH containing 1.05 g of triester 10a (2.00 mmol, 1.10 equivalent) at room temperature. After stirring for 20 h, the reaction mixture was acidified to pH ≤ 2 by adding 3 M HCl aqueous solution and extracted with Et2O (3 × 20 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The crude residue (90:5:5 → 85:10:5 hexane / EtOAc / MeOH) was purified by rapid column chromatography to give carboxylic acid 12a (802 mg, 86% yield) as a clear, colorless oil.

[0631] R f =0.22 (SiO2, 85:10:5 hexane / EtOAc / MeOH);

[0632] 1 H NMR (300MHz, CDCl3): δ5.08-4.93 (m, 2H), 2.36 (t, J=7.8Hz, 2H), 2.30 (t, J=7.6Hz, 4H), 1.72-1.44 (m, 10H), 1.44-1.16 (m, 30H), 0.97-0.83 (m, 9H).

[0633]

[0634] 9,10-Dilinoleoyloxystearic acid (12b):

[0635] In a round-bottom flask under an argon atmosphere, 3.00 mL of 2.0 M KOH aqueous solution (6.00 mmol, 1.00 equivalent) was added to a 7 mL solution of t-BuOH containing 5.64 g (6.60 mmol, 1.10 equivalent) of triester 10b at room temperature. After stirring for 20 h, the reaction mixture was acidified to pH ≤ 2 by adding 3 M HCl aqueous solution and extracted with hexane (3 × 75 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The crude residue was purified by rapid column chromatography (90:10:0 → 85:10:5 hexane / EtOAc / MeOH) to give carboxylic acid 12b (2.39 g, 68% yield) as a clear, colorless oil.

[0636] R f=0.33 (SiO2, 85:10:5 hexane / EtOAc / MeOH);

[0637] 1 H NMR (300MHz, CDCl3): δ5.49-5.25 (m, 8H), 5.07-4.93 (m, 2H), 2.79 (t, J=5.9Hz, 4H), 2.36 (t, J=7.7Hz, 2H ), 2.30 (t, J=7.5Hz, 4H), 2.13-2.00 (m, 8H), 1.72-1.45 (m, 10H), 1.45-1.15 (m, 50H), 0.98-0.81 (m, 9H).

[0638]

[0639] 9,10,12R-trihexanoyloxystearic acid (13):

[0640] In a round-bottom flask under an argon atmosphere, 1.47 mL of 2.0 M KOH aqueous solution (2.94 mmol, 1.00 equivalent) was added to a 10 mL solution of t-BuOH in 1.98 g (3.10 mmol, 1.10 equivalent) of triester 11 at room temperature. After stirring for 20 h, the reaction mixture was acidified to pH ≤ 2 by adding 3 M HCl aqueous solution and extracted with hexane (3 × 30 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The crude residue was purified by rapid column chromatography (90:10:0 → 85:10:5 → 75:20:5 hexane / EtOAc / MeOH) to give carboxylic acid 13 (1.40 g, 78% yield) as a clear, colorless oil.

[0641] R f =0.32 (SiO2, 80:15:5 hexane / EtOAc / MeOH);

[0642] 1 H NMR (300MHz, CDCl3): δ 5.13-4.82 (m, 3H), 2.42-2.18 (m, 8H), 1.92-1.69 (m, 2H), 1.69-1.43 (m, 12H), 1.43-1.14 (m, 28H), 0.99-0.81 (m, 12H).

[0643] Example 2A: Synthesis of INT-D047

[0644]

[0645] (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl)succinic acid (R,Z)-12-acetoxyoctadec-9-en-1-yl ester (INT-D047):

[0646] According to general method D, dexamethasone (294 mg, 0.75 mmol), hemisuccinate 5a (384 mg, 0.90 mmol), DCC (186 mg, 0.90 mmol), DMAP (137 mg, 1.12 mmol), and CH2Cl2 (4 mL) were subjected to rapid column chromatography (SiO2, 80:20→50:50 hexane / EtOAc) to obtain INT-D047 (541 mg, 90% yield), as a clear, colorless oil.

[0647] R f =0.36 (SiO2, 50:50 hexane / EtOAc);

[0648] 1 ¹H NMR (300 MHz, CDCl₃): δ 7.21 (d, J = 10.1 Hz), 6.36 (dd, J = 10.2, 1.7 Hz), 6.13 (s, 1H), 5.58–5.43 (m, 1H), 5.43–5.28 (m, 1H), 4.92 (s, 2H), 4.89 (quintet, J = 6.4 Hz), 4.45–4.34 (m, 1H), 4.11 (t, J = 6.7 Hz). Hz, 2H), 3.20-3.04 (m, 1H), 2.86-2.55 (m, 5H), 2.52-2.26 (m, 4H), 2.24-2.12 (m, 1H), 2.11-1.9 9 (m, 1H), 2.05 (s, 3H), 1.90-1.46 (m, 12H), 1.44-1.17 (m, 15H), 1.06 (s, 3H), 0.99-0.84 (m, 6H).

[0649] Example 2B: Synthesis of INT-D046

[0650]

[0651] (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl)succinic acid (R,Z)-12-(dodecanoyloxy)octadecano-9-en-1-yl ester (INT-D046):

[0652] According to general method D, dexamethasone (157 mg, 0.40 mmol), hemisuccinate 5c (272 mg, 0.48 mmol), DCC (99 mg, 0.48 mmol), DMAP (73 mg, 0.60 mmol), and CH2Cl2 (2 mL) were subjected to rapid column chromatography (SiO2, 80:20 → 50:50 hexane / EtOAc) to obtain INT-D046 (363 mg, 96% yield), as a clear, colorless oil.

[0653] R f =0.48 (SiO2, 50:50 hexane / EtOAc);

[0654] 1 ¹H NMR (300MHz, CDCl₃): δ 7.21 (d, J = 10.1 Hz), 6.36 (dd, J = 10.2, 1.7 Hz), 6.13 (s, 1H), 5.57–5.42 (m, 1H), 5.40–5.28 (m, 1H), 4.92 (s, 2H), 4.90 (quintet, J = 6.4 Hz), 4.44–4. 33(m, 1H), 4.11(t, J=6.9Hz, 2H), 3.20-3.03(m, 1H), 2.85-2.54(m, 5H), 2.53-1.94 (m, 10H), 1.93-1.48 (m, 15H), 1.45-1.14 (m, 28H), 1.06 (s, 3H), 0.98-0.82 (m, 9H).

[0655] Example 2C: Synthesis of INT-D050

[0656]

[0657] ((R,Z)-12-(stearoyloxy)octadec-9-en-1-yl)succinic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D050): JZ-25-009

[0658] According to general method D, dexamethasone (392 mg, 1.00 mmol), hemisuccinate 5d (781 mg, 1.28 mmol), DCC (248 mg, 1.28 mmol), DMAP (183 mg, 1.50 mmol) and CH2Cl2 (5 mL) were subjected to rapid column chromatography (SiO2, 80:20→50:50 hexane / EtOAc) to obtain INT-D050 (933 mg, 91% yield), as a clear, colorless oil.

[0659] R f =0.42 (SiO2, 50:50 hexane / EtOAc);

[0660] 1 ¹H NMR (300MHz, CDCl₃): δ 7.22 (d, J = 10.1 Hz), 6.36 (dd, J = 10.1, 1.5 Hz), 6.13 (s, 1H), 5.55–5.41 (m, 1H), 5.40–5.27 (m, 1H), 4.93 (s, 2H), 4.89 (quintet, J = 6.2 Hz), 4.44–4. 33 (m, 1H), 4.10 (t, J=6.9Hz, 2H), 3.20-3.04 (m, 1H), 2.85-2.55 (m, 5H), 2.53-1.95 (m, 11H), 1.91-1.45 (m, 15H), 1.43-1.15 (m, 44H), 1.06 (s, 3H), 0.98-0.81 (m, 9H).

[0661] Example 2D: Synthesis of INT-D035

[0662]

[0663] ((R,Z)-12-(oleoyloxy)octadec-9-en-1-yl)succinic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D035):

[0664] According to general method D, dexamethasone (133 mg, 0.34 mmol), hemisuccinate 5e (264 mg, 0.41 mmol), DCC (84 mg, 0.41 mmol), DMAP (62 mg, 0.51 mmol), and CH2Cl2 (2 mL) were subjected to rapid column chromatography (SiO2, 80:20 → 50:50 hexane / EtOAc) to obtain INT-D035 (330 mg, 95% yield), as a clear, colorless oil.

[0665] R f =0.49 (SiO2, 50:50 hexane / EtOAc);

[0666] 1 ¹H NMR (300 MHz, CDCl₃): δ 7.22 (d, J = 10.1 Hz), 6.36 (dd, J = 10.2, 1.8 Hz), 6.13 (s, 1H), 5.55–5.26 (m, 4H), 4.92 (s, 2H), 4.89 (quintet, J = 6.3 Hz), 4.44–4.34 (m, 1H) , 4.11(t, J=6.8Hz, 2H), 3.20-3.04(m, 1H), 2.85-2.54(m, 5H), 2.54-1.94(m, 1 3H), 1.92-1.46(m, 16H), 1.44-1.16(m, 36H), 1.06(s, 3H), 0.99-0.81(m, 9H).

[0667] Example 2E: Synthesis of INT-D045

[0668]

[0669] ((R,Z)-12-(linoleyloxy)octadec-9-en-1-yl)succinic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D045):

[0670] According to general method D, dexamethasone (157 mg, 0.40 mmol), hemisuccinate 5f (310 mg, 0.48 mmol), DCC (99 mg, 0.48 mmol), DMAP (73 mg, 0.60 mmol), and CH2Cl2 (2 mL) were subjected to rapid column chromatography (SiO2, 80:20 → 50:50 hexane / EtOAc) to obtain INT-D045 (278 mg, 68% yield), as a clear, colorless oil.

[0671] R f =0.50 (SiO2, 50:50 hexane / EtOAc);

[0672] 1 ¹H NMR (300 MHz, CDCl₃): δ 7.22 (d, J = 10.1 Hz), 6.36 (dd, J = 10.2, 1.8 Hz), 6.13 (s, 1H), 5.56–5.25 (m, 6H), 4.93 (s, 2H), 4.89 (quintet, J = 6.3 Hz), 4.46–4.31 (m, 1H) , 4.10 (t, J=6.8Hz, 2H), 3.20-3.04 (m, 1H), 2.88-2.54 (m, 7H), 2.53-1.91 (m, 1 5H), 1.90-1.46(m, 14H), 1.47-1.12(m, 34H), 1.06(s, 3H), 0.99-0.81(m, 9H).

[0673] Example 2F: Synthesis of INT-D049

[0674]

[0675] ((R,Z)-12-(linolenic acid)octadec-9-en-1-yl)succinic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D049):

[0676] According to general method D, dexamethasone (294 mg, 0.75 mmol), hemisuccinate 5 g (264 mg, 0.90 mmol), DCC (84 mg, 0.90 mmol), DMAP (137 mg, 1.12 mmol), and CH2Cl2 (4 mL) were subjected to rapid column chromatography (SiO2, 80:20→50:50 hexane / EtOAc) to obtain INT-D049 (740 mg, 96% yield), which was a clear, colorless oil.

[0677] R f =0.42 (SiO2, 50:50 hexane / EtOAc);

[0678] 1 ¹H NMR (300 MHz, CDCl₃): δ 7.21 (d, J = 10.1 Hz), 6.36 (dd, J = 10.2, 1.8 Hz), 6.13 (s, 1H), 5.56–5.25 (m, 8H), 4.93 (s, 2H), 4.89 (quintet, J = 6.3 Hz), 4.46–4.32 (m, 1H), 4.10 (t, J = 10.1 Hz). =6.9Hz, 2H), 3.22-3.03(m, 1H), 2.90-2.53(m, 9H), 2.53-1.91(m, 17H), 1.90-1.44 (m, 14H), 1.46-1.12 (m, 28H), 1.06 (s, 3H), 0.99 (t, J=7.6Hz, 3H), 0.96-0.81 (m, 6H).

[0679] Example 2G: Synthesis of INT-D051

[0680]

[0681] ((R,Z)-12-(arachidonicyloxy)octadec-9-en-1-yl)succinic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D051):

[0682] According to general method D, dexamethasone (303 mg, 0.77 mmol), hemisuccinate 5f (570 mg, 0.85 mmol), DCC (175 mg, 0.85 mmol), DMAP (142 mg, 1.16 mmol), and CH2Cl2 (5 mL) were subjected to rapid column chromatography (SiO2, 80:20 → 50:50 hexane / EtOAc) to give INT-D051 (758 mg, 94% yield), as a clear, colorless oil.

[0683] R f =0.29 (SiO2, 50:50 hexane / EtOAc);

[0684] 1 ¹H NMR (300 MHz, CDCl₃): δ 7.22 (d, J = 10.2 Hz), 6.36 (dd, J = 10.2, 1.8 Hz), 6.13 (s, 1H), 5.56–5.26 (m, 10H), 4.93 (s, 2H), 4.88 (quintet, J = 6.3 Hz), 4.43–4.33 (m, 1H) , 4.10(t, J=6.9Hz, 2H), 3.20-3.03(m, 1H), 2.94-2.55(m, 11H), 2.54-1.95(m, 17H), 1.91-1.42(m, 14H), 1.47-1.15(m, 28H), 1.05(s, 3H), 1.00-0.81(m, 9H).

[0685] Example 2H: Synthesis of INT-D055

[0686]

[0687] (R,Z)-12-hexanoyloxyoleic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D055):

[0688] Carboxylic acid 7a (114 mg, 0.30 mmol, 1.20 equivalence) and CH2Cl2 (1.2 mL) were added to an argon-purged round-bottom flask and cooled in an ice bath. DCC (63 mg, 0.30 mmol, 1.2 equivalence) was added to the flask and stirred at ambient room temperature for 15 min. The flask was then cooled in an ice bath again, and a mixture of dexamethasone (99 mg, 0.25 mmol, 1.00 equivalence) and DMAP (47 mg, 0.38 mmol, 1.50 equivalence) in CH2Cl2 (1.3 mL), prepared in a separate argon-purged round-bottom flask, was added via syringe. After 17 h, the reaction mixture was diluted with Et2O and... The product was filtered and then concentrated under reduced pressure using a rotary evaporator. The crude product was purified by rapid column chromatography (80:20→50:50 hexane / EtOAc) to give INT-D055 as a pale yellow, viscous oil (185 mg, 96% yield).

[0689] R f : 0.15 (SiO2, 70:30 hexane: EtOAc);

[0690] 1 H (300MHz, CDCl3): δ7.22 (d, J=10.2Hz, 1H), 6.36 (dd, J=10.2, 1.6Hz, 1H), 6.14 (s, 1H), 5.55-5.42 (m, 1H), 5.40-5.28 (m, 1H), 4.96-4.82 (m, 3H), 4. 44-4.34(m, 1H), 3.21-3.03(m, 1H), 2.73-2.55(m, 1H), 2.53-1.97(m, 13H) , 1.91-1.48(m, 12H), 1.44-1.18(m, 21H), 1.07(s, 3H), 0.98-0.83(m, 9H).

[0691] Example 2I: Synthesis of INT-D089

[0692]

[0693] (R,Z)-12-linoleyloxyoleic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D089):

[0694] Carboxylic acid 7b (600 mg, 1.07 mmol, 1.20 equivalence) and CH2Cl2 (4.4 mL) were added to an argon-purged round-bottom flask and cooled in an ice bath. DCC (221 mg, 1.07 mmol, 1.20 equivalence) was added to the flask, and stirring was maintained at ambient room temperature for 15 min. The flask was then cooled again in an ice bath, and a mixture of dexamethasone (350 mg, 0.89 mmol, 1.00 equivalence) and DMAP (163 mg, 1.34 mmol, 1.50 equivalence) in CH2Cl2 (4.5 mL), prepared in a separate argon-purged round-bottom flask, was added via syringe. After 17 h, the reaction mixture was diluted with hexane and... The product was filtered and then concentrated under reduced pressure using a rotary evaporator. The crude product was purified by rapid column chromatography (80:20→50:50 hexane / EtOAc) to give INT-D089 as a pale yellow, viscous oil (750 mg, 90% yield).

[0695] R f : 0.46 (silica, 50:50 hexane: EtOAc);

[0696] 1 H (300MHz, CDCl3): δ7.22 (d, J=10.3Hz, 1H), 6.36 (dd, J=10.2, 1.5Hz, 1H), 6.13(s, 1H), 5.55-5.28(m, 6H), 4.97-4.82(m, 3H), 4.46-4.33(m, 1H), 3.21 -3.04(m, 1H), 2.79(t, J=5.9Hz, 2H), 2.71-2.55(m, 1H), 2.53-1.97(m, 16H ), 1.91-1.46 (m, 14H), 1.45-1.19 (m, 30H), 1.07 (s, 3H), 0.98-0.84 (m, 9H).

[0697] Example 2J: Synthesis of INT-D085

[0698]

[0699] Dihexanoic acid 1-(2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethoxy)-1-oxooctadecane-9,10-diyl ester (INT-D085):

[0700] According to general method E, dexamethasone (235 mg, 0.60 mmol), carboxylic acid 12a (338 mg, 0.66 mmol), DCC (136 mg, 0.66 mmol), DMAP (110 mg, 0.90 mmol) and CH2Cl2 (6 mL) were obtained by rapid column chromatography (SiO2, 80:20→50:50 hexane / EtOAc), INT-D085 (336 mg, 63% yield), as a clear, colorless oil.

[0701] R f =0.52 (SiO2, 50:50 hexane / EtOAc);

[0702] 1 H NMR (300MHz, CDCl3): δ7.22 (d, J=10.1Hz), 6.35 (dd, J=10.2, 1.7Hz, 1H), 6.12 (s, 1H), 5.07-4.94 (m, 2H), 4.90 (s, 2H), 4.44-4.3 2 (m, 1H), 3.21-3.02 (m, 1H), 2.63 (dt, J=13.4, 5.4Hz, 1H), 2.53-2.26 (m, 6H), 2.30 (t, J=7.3Hz, 4H), 2.24-2.09 (m, 1H), 2.03 (br s, 1H), 1.92-1.44 (m, 18H), 1.43-1.15 (m, 30H), 1.06 (s, 3H), 0.99-0.79 (m, 12H).

[0703] Example 2K: Synthesis of INT-D086

[0704]

[0705] (9Z,9'Z,12Z,12'Z)-bis(octadecano-9,12-dienoic acid)1-(2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethoxy)-1-oxooctadecane-9,10-diyl ester (INT-D086):

[0706] According to general method E, dexamethasone (235 mg, 0.60 mmol), carboxylic acid 12b (555 mg, 0.66 mmol), DCC (136 mg, 0.66 mmol), DMAP (110 mg, 0.90 mmol), and CH2Cl2 (6 mL) were subjected to rapid column chromatography (SiO2, 80:20 → 50:50 hexane / EtOAc) to obtain INT-D086 (584 mg, 80% yield), as a clear, colorless oil.

[0707] R f =0.28 (SiO2, 70:30 hexane / EtOAc);

[0708] 1 H NMR (300MHz, CDCl3): δ7.22 (d, J=10.1Hz, 1H), 6.35 (dd, J=10.1, 1.6Hz, 1H), 6.12 (s, 1H), 5 .48-5.24(m, 8H), 5.06-4.93(m, 2H), 4.90(s, 2H), 4.44-4.31(m, 1H), 3.21-3.02(m, 1H), 2.7 8(t, J=5.9Hz, 6H), 2.63 (dt, J=13.7, 5.9Hz, 1H), 2.52-2.33 (m, 6H), 2.30 (t, J=7.4Hz, 4H), 2 .24-1.97 (m, 9H), 1.94-1.45 (m, 18H), 1.44-1.16 (m, 52H), 1.07 (s, 3H), 0.98-0.82 (m, 12H).

[0709] Example 2L: Synthesis of INT-D056

[0710]

[0711] 9,10,12R-Trihexanoyloxystearic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D056):

[0712] According to general method E, dexamethasone (175 mg, 0.44 mmol), carboxylic acid 13 (307 mg, 0.49 mmol), DCC (101 mg, 0.49 mmol), and DMAP (82 mg, 0.67 mmol) in CH2Cl2 (5 mL) were subjected to rapid column chromatography (SiO2, 80:20 → 50:50 hexane / EtOAc) to obtain INT-D056 as a clear, colorless oil (318 mg, 90% yield).

[0713] R f =0.50 (SiO2, 50:50 hexane / EtOAc);

[0714] 1 H NMR (300MHz, CDCl3): δ7.23 (d, J=10.2Hz, 1H), 6.33 (dd, J=10.1, 1.7Hz, 1H), 6.10 (s, 1H), 5.11-4.89 (m, 3H), 4.90 (s, 2H), 4.42-4.29 (m, 1H), 3 .20-3.00 (m, 1H), 2.61 (dt, J=13.5, 5.4Hz, 1H), 2.52-2.05 (m, 12H), 1. 93-1.42 (m, 20H), 1.42-1.14 (m, 28H), 1.04 (s, 3H), 0.98-0.79 (m, 15H).

[0715] Example 2M: Synthesis of INT-D059

[0716]

[0717] ((12S,Z)-12-(((12S)-9,10,12-tris(hexanoyloxy)octadecanoyl)oxy)octadec-9-en-1-yl)succinic acid 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentadien[a]phenanthrene-17-yl)-2-oxoethyl ester (INT-D059):

[0718] According to general method E, dexamethasone (137 mg, 0.35 mmol), hemisuccinate derived from ricinoleol 2 and carboxylic acid 13 (382 mg, 0.38 mmol), DCC (79 mg, 0.38 mmol), DMAP (64 mg, 0.52 mmol), and CH2Cl2 (3.5 mL) were subjected to rapid column chromatography (SiO2, 70:30 → 50:50 hexane / EtOAc) to give INT-D059 (336 mg, 66% yield) as a clear, colorless oil.

[0719] R f =0.50 (SiO2, 50:50 hexane / EtOAc);

[0720] 1 H NMR (300MHz, CDCl3): δ7.22 (d, J=10.1Hz, 1H), 6.36 (dd, J=10.2, 1.7Hz, 1H), 6.13 (s, 1H ), 5.55-5.41(m, 1H), 5.40-5.27(m, 1H), 5.12-4.83(m, 5H), 4.93(s, 2H), 4.44-4.33(m, 1 H), 4.10 (t, J=6.8Hz, 2H), 3.20-3.04 (m, 1H), 2.84-2.54 (6H), 2.52-2.10 (m, 18H), 2.10 -1.97(m, 2H), 1.93-1.43(m, 32H), 1.43-1.16(m, 62H), 1.05(s, 3H), 0.99-0.81(m, 21H).

[0721] Example 2N: Synthesis of INT-D060

[0722]

[0723] 2-Acetoxybenzoic acid (R,Z)-12-hexanoyloxyoctadec-9-en-1-yl ester (INT-D060):

[0724] In a round-bottom flask under an argon atmosphere, a solution of HF·pyridine (0.53 mL of 70% HF in pyridine, 4.20 mmol, 3.00 equivalence) was added to a stirred, ice-cold solution of pyridine (0.34 mL, 4.20 mmol, 3.00 equivalence) and silyl ether 4b (700 mg, 1.41 mmol) in THF (7 mL). When TLC showed depletion of the starting material (2–8 h), the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The mixture was extracted with Et2O (2 × 10 mL), followed by washing of the combined organic extracts with H2O (1 × 10 mL) and brine. The extracts were dried over Na2SO4 and concentrated using a rotary evaporator to give a crude primary alcohol. The crude product was purified by filtration through a silica gel pad (90:10 hexane / EtOAc) and concentrated using a rotary evaporator to give an intermediate primary alcohol (518 mg) as a clear, colorless oil, which was used without further purification.

[0725] The above-mentioned alcohol, pyridine (0.22 mL, 2.70 mmol, 2.00 equivalent), and CH2Cl2 (6 mL) were added to a flame-dried and argon-purged round-bottom flask, which was then cooled in an ice bath. A funnel was fitted to the flask, and a solution of acetyl salicyl chloride (541 mg, 2.73 mmol, 2.00 equivalent) in CH2Cl2 (7.5 mL), prepared in another argon-purged round-bottom flask, was added dropwise over 15 minutes. After 16.5 h, the reaction solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by two consecutive rapid column chromatography operations (first 90:10 hexane / EtOAc, then 95:5 hexane / EtOAc) to give INT-D060 as a pale yellow oil (557 mg, 76% yield).

[0726] R f : 0.60 (SiO2, 70:30 hexane: EtOAc);

[0727] 1H (300MHz, CDCl3): δ 8.02 (dd, J = 7.9, 1.4Hz, 1H), 7.55 (td, J = 7.6, 1.3Hz, 1H), 7.31 (t, J = 7.6Hz, 1H), 7.10 (d, J = 7.9Hz, 1H), 5.54–5.40 (m, 1H), 5.40–5.26 (m, 1H), 4.89 (quintet, J = 6.2Hz, 1H) ), 4.27 (t, J = 6.7 Hz, 2H), 2.35 (s, 3H), 2.33-2.21 (m, 4H), 2.09-1.96 (m, 2H), 1.74 (quintet, J = 7.1 Hz, 2H), 1.62 (quintet, J = 7.3 Hz, 2H), 1.58-1.48 (m, 2H), 1.48-1.16 (m, 22H), 0.97-0.80 (m, 6H).

[0728] Example 2O: Synthesis of INT-D061

[0729]

[0730] 2-Acetoxybenzoic acid (R,Z)-12-(linoleyloxyoctadec-9-en-1-yl ester (INT-D061):

[0731] In a round-bottom flask under an argon atmosphere, a solution of HF·pyridine (0.39 mL of 70% HF in pyridine, 3.20 mmol, 3.00 equivalent) was added to a stirred, ice-cold solution of pyridine (0.26 mL, 3.20 mmol, 3.00 equivalent) and silyl ether 4f (700 mg, 1.06 mmol) in THF (5 mL). When TLC showed depletion of the starting material (2–8 h), the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The mixture was extracted with Et2O (2 × 10 mL), followed by washing with the combined organic extracts in H2O (1 × 10 mL), brine, drying with Na2SO4, and concentrating by a rotary evaporator to give crude primary alcohol. The crude product was purified by filtration through a silica gel pad (90:10 hexane / EtOAc), and concentrated by a rotary evaporator to give intermediate primary alcohol (553 mg) as a clear, colorless oil, which was used without further purification.

[0732] The above-mentioned alcohol (553 mg, 1.01 mmol, 1.00 equivalent), pyridine (0.13 mL, 1.7 mmol, 1.60 equivalent), and CH2Cl2 (4 mL) were added to an argon-purged round-bottom flask, and the mixture was then cooled in an ice bath. A solution of acetyl salicyl chloride (327 mg, 1.65 mmol, 1.63 equivalent) prepared in another argon-purged round-bottom flask in CH2Cl2 (6 mL) was added dropwise over 15 minutes. After 18 h, the reaction solvent was removed under reduced pressure using a rotary evaporator. The crude product was purified by two consecutive rapid column chromatography operations (95:5 hexane / EtOAc) to give INT-D061 as a pale yellow oil (516 mg, 72% yield).

[0733] R f : 0.56 (SiO2, 70:30 hexane: EtOAc);

[0734] 1 H (300MHz, CDCl3): δ 8.03 (dd, J = 7.9, 1.5Hz, 1H), 7.57 (td, J = 7.6, 1.6Hz, 1H), 7.32 (td, J = 7.6, 1.0Hz, 1H), 7.11 (d, J = 7.9Hz, 1H), 5.54-5.27 (m, 6H), 4.90 (quintet, J = 6.2Hz, 1H), 4.2 8 (t, J = 6.7 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 2.36 (s, 3H), 2.34–2.21 (m, 4H), 2.12–1.96 (m, 6H), 1.75 (quintet, J = 7.1 Hz, 2H), 1.69–1.49 (m, 4H), 1.49–1.18 (m, 32H), 0.98–0.81 (m, 6H).

[0735]

[0736] (E)-6-(4-tert-butyldimethylsilyloxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid (14):

[0737] DMF (0.98 mL), imidazole (159 mg, 2.34 mmol, 7.50 equivalence), and mycophenolic acid (100 mg, 0.312 mmol, 1.00 equivalence) were added to a flame-dried and argon-purged round-bottom flask. TBSCl (282 mg, 1.87 mmol, 6.00 equivalence) was then added to the mixture. After 1 h, the reaction mixture was extracted from 10 mL of water with Et₂O (2 × 10 mL). The combined organic layers were washed with water (3 × 10 mL) and brine (1 × 10 mL), dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The crude residue was dissolved in THF (0.60 mL) and stirred for 1 h with water (0.60 mL) and acetic acid (0.60 mL). The mixture was then extracted from 1 × 10 mL of water with Et₂O (2 × 10 mL). The combined organic layers were washed with water (5 × 10 mL) and brine (1 × 10 mL), dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator. The crude residue was purified by rapid column chromatography (80:20 → 20:80 hexane / EtOAc) to give mycophenolic silyl ether (14) as a white solid (121 mg, 89% yield).

[0738] R f : 0.36 (SiO2, 50:50 hexane: EtOAc);

[0739] 1 H (300MHz, CDCl3): δ5.23 (t, J=6.3Hz, 1H), 5.09 (s, 2H), 3.76 (s, 3H), 3.41 (d, J=6.3Hz, 2H) , 2.50-2.39(m, 2H), 2.38-2.27(m, 2H), 2.17(s, 3H), 1.78(s, 3H), 1.05(s, 9H), 0.26(s, 6H).

[0740] Example 2P: Synthesis of INT-D062

[0741]

[0742] (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhexanoic acid (12R)-hexanoyloxy oleyl ester (INT-D062):

[0743] In a round-bottom flask under an argon atmosphere, a solution of HF·pyridine (0.11 mL of 70% HF in pyridine, 0.91 mmol, 3.00 equivalent) was added to a stirred, ice-cold solution of pyridine (0.07 mL, 0.91 mmol, 3.00 equivalent) and silyl ether 4b (150 mg, 0.30 mmol) in THF (1.5 mL). When TLC showed depletion of the starting material (2–8 h), the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The mixture was extracted with Et2O (2 × 5 mL), followed by washing with H2O (1 × 5 mL) and brine. The combined organic extracts were dried over Na2SO4 and concentrated using a rotary evaporator to give a crude primary alcohol. The crude product was purified by filtration through a silica gel pad (90:10 hexane / EtOAc) and concentrated using a rotary evaporator to give an intermediate primary alcohol (102 mg) as a clear, colorless oil, which was used without further purification.

[0744] CH₂Cl₂ (1.2 mL) and mycophenolic silyl ether (14) (105 mg, 0.24 mmol, 1.00 equivalent) were added to a round-bottom flask cooled in an ice bath purged with argon. DCC (50 mg, 0.24 mmol, 1.00 equivalent) was added to the flask, and the ice bath was removed. After 15 minutes, the ice bath was replaced under the flask, and a solution of the above-mentioned alcohol (102 mg, 0.267 mmol, 1.10 equivalent) and DMAP (44 mg, 0.36 mmol, 1.50 equivalent) in CH₂Cl₂ (1.2 mL) was added. After 15.5 h, the reaction mixture was concentrated under reduced pressure. The crude product was diluted with hexane (4 volumes) and passed through… Filter, then concentrate under reduced pressure using a rotary evaporator. Perform rapid column chromatography (85:15 hexane / EtOAc) on the residue, combine fractions containing the product, and concentrate.

[0745] The residue was transferred to a round-bottom flask and purged with argon. CH₂Cl₂ (1.5 mL) and pyridine (0.06 mL, 0.7 mmol, 2.88 equivalents) were added to the flask, and the mixture was cooled in a water bath. Benzoyl chloride (0.05 mL, 0.50 mmol, 2.06 equivalents) was then added to the flask. After 18 h, the reaction mixture was concentrated under reduced pressure using a rotary evaporator. The crude residue was extracted with Et₂O (3 × 10 mL) and water (1 × 10 mL). The combined organic layers were washed with 1 M HCl aqueous solution (1 × 10 mL), 1 M NaOH aqueous solution (1 × 10 mL), water (1 × 10 mL), and brine (1 × 10 mL), dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator.

[0746] The crude residue was dissolved in THF (1.4 mL) in a round-bottom flask purged with argon and cooled in an ice-water bath. Pyridine (0.07 mL, 0.80 mmol, 3.29 equivalents) and HF·pyridine (0.10 mL, 70% HF, 0.83 mmol, 3.42 equivalents) were added to the flask, and the ice bath was removed. After 2 h, a saturated aqueous solution of NaHCO3 was slowly added to the reaction mixture until bubbling ceased. The reaction mixture was extracted with Et2O (1 × 10 mL), and the combined organic layers were washed with 1 M HCl aqueous solution (1 × 10 mL), water (1 × 10 mL), and brine (1 × 10 mL), dried over Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The residue obtained by rapid column chromatography (90:10→80:20 hexane / EtOAc) was purified to give INT-D062, which was a clear, colorless oil (100 mg, 60% yield, 3 steps).

[0747] R f : 0.22 (silica gel, 80:20 hexane:EtOAc);

[0748] 1 H (300MHz, CDCl3): δ 7.69 (s, 1H), 5.55-5.41 (m, 1H), 5.41-5.17 (m, 4H), 4.90 (quintet, J = 6.2Hz, 1H), 4.02 (t, J = 6.8Hz, 2H), 3.78 (s, 3H), 3.40 (d, J = 6.9Hz, 2H), 2.47-2.36 (m, 2H), 2.36-2.23 (m, 6H), 2.17 (s, 3H), 2.10-1.97 (m, 2H), 1.82 (s, 3H), 1.71-1.47 (m, 6H), 1.43-1.18 (m, 22H), 0.97-0.83 (m, 6H).

[0749] Example 2Q: Synthesis of INT-D063

[0750]

[0751] (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid (12R)-linoleyloxy oleyl ester (INT-D063):

[0752] In a round-bottom flask under an argon atmosphere, a solution of HF·pyridine (0.07 mL of 70% HF in pyridine, 0.60 mmol, 3.00 equivalent) was added to a stirred, ice-cold solution of pyridine (0.05 mL, 0.60 mmol, 3.00 equivalent) and silyl ether 4f (125 mg, 0.19 mmol) in THF (1.5 mL). When TLC showed depletion of the starting material (2–8 h), the reaction mixture was quenched with a saturated aqueous solution of NaHCO3. The mixture was extracted with Et2O (2 × 10 mL), followed by washing with H2O (1 × 10 mL) and brine. The combined organic extracts were dried over Na2SO4 and concentrated using a rotary evaporator to give crude primary alcohol. The crude product was purified by filtration through a silica gel pad (90:10 hexane / EtOAc) and concentrated using a rotary evaporator to give intermediate primary alcohol (95 mg) as a clear, colorless oil, which was used without further purification.

[0753] CH₂Cl₂ (0.5 mL) and mycophenolic silyl ether (14) (68 mg, 0.16 mmol, 1.00 equivalent) were added to a round-bottom flask cooled in an ice bath purged with argon. DCC (32 mg, 0.16 mmol, 1.00 equivalent) was added to the flask, and the ice bath was removed. After 15 minutes, the ice bath was replaced under the flask, and a solution of the above-mentioned alcohol DMAP (29 mg, 0.24 mmol, 1.50 equivalent) in CH₂Cl₂ (1 mL) was added. After 19 hours, the reaction mixture was concentrated under reduced pressure. The crude product was diluted with hexane (4 volumes) and passed through… Filter, then concentrate under reduced pressure using a rotary evaporator. Perform rapid column chromatography (85:15 hexane / EtOAc) on the residue, combine fractions containing the product, and concentrate.

[0754] The residue was transferred to a round-bottom flask and purged with argon. CH₂Cl₂ (1 mL) and pyridine (0.03 mL, 0.40 mmol, 2.50 equivalents) were added to the flask, and the mixture was cooled in a water bath. Benzoyl chloride (0.03 mL, 0.20 mmol, 1.25 equivalents) was then added to the flask. After 18 h, the reaction mixture was concentrated under reduced pressure using a rotary evaporator. The crude residue was extracted with Et₂O (3 × 10 mL) and water (1 × 10 mL). The combined organic layers were washed with 1 M HCl aqueous solution (1 × 10 mL), 1 M NaOH aqueous solution (1 × 10 mL), water (1 × 10 mL), and brine (1 × 10 mL), dried over Na₂SO₄, and concentrated under reduced pressure using a rotary evaporator.

[0755] The crude residue was dissolved in THF (1 mL) in a round-bottom flask purged with argon and cooled in an ice-water bath. Pyridine (0.04 mL, 0.50 mmol, 3.13 equivalents) and HF·pyridine (0.06 mL, 70% HF, 0.50 mmol, 3.13 equivalents) were added to the flask, and the ice bath was removed. After 2 h, a saturated aqueous solution of NaHCO3 was slowly added to the reaction mixture until bubbling ceased. The reaction mixture was extracted with Et2O (1 × 10 mL), and the combined organic layers were washed with 1 M HCl aqueous solution (1 × 10 mL), water (1 × 10 mL), and brine (1 × 10 mL), dried over Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The residue (90:10 hexane / EtOAc) was purified by rapid column chromatography to give INT-D063 as a clear, colorless oil (66 mg, 50% yield, 3 steps).

[0756] R f : 0.17 (SiO2, 85:15 hexane: EtOAc);

[0757] 1 H (300MHz, CDCl3): δ 7.69 (s, 1H), 5.55-5.17 (m, 9H), 4.90 (quintet, J = 6.3Hz, 1H), 4.02 (t, J = 6.7Hz, 2H), 3.78 (s, 3H), 3.40 (d, J = 6.7Hz, 2H), 2.79 (t, J = 6.0Hz, 2H), 2.46-2.36 (m, 2H), 2.36-2.23 (m, 6H), 2.17 (s, 3H), 2.13-1.96 (m, 6H), 1.82 (s, 3H), 1.70-1.47 (m, 6H), 1.45-1.19 (m, 32H), 0.96-0.81 (m, 6H).

[0758] Example 2R: Synthesis of INT-D065

[0759]

[0760] Benzoic acid (4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)amino)-2-(((R,Z)-12-(((9Z,12Z)-octadecano-9,12-dienoyl)oxy)octadecano-9-dienoyl)oxy)-3-phenylpropionyl)oxy)-4,6,11-trihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecano-1H-7,11-methylenecyclodecadieno[3,4]benzo[1,2-b]oxet-12-yl ester (INT-D065):

[0761] In a round-bottom flask under an argon atmosphere, Et3N (0.10 mL, 0.75 mmol, 2.50 equivalences) and Mukaiyama reagent (100 mg, 0.39 mmol, 1.30 equivalences) were added to a 3 mL solution of docetaxel (242 mg, 0.30 mmol) and 12R-linoleyloxyoleic acid 7b (202 mg, 0.36 mmol, 1.20 equivalences) at room temperature. After stirring for 14 h, the reaction mixture was diluted with EtOAc. The residue was filtered and concentrated under reduced pressure using a rotary evaporator. The crude residue (SiO2, 80:20→50:50 hexane / EtOAc) was purified by rapid column chromatography to give INT-D065 as a clear, colorless oil (243 mg, 60% yield).

[0762] R f =0.45 (SiO2, 50:50 hexane / EtOAc);

[0763] 1¹H NMR (300MHz, CDCl₃): δ 8.12 (d, J = 7.3Hz, 2H), 7.62 (t, J = 7.4Hz, 1H), 7.56–7.46 (m, 2H), 7.44–7.35 (m, 2H), 7.35–7.26 (m, 3H), 6.27 (br t, J = 8.0Hz, 1H), 5.70 (d, J = 7.1Hz, 1H), 5.55–5.27 (m, 9H), 5.23 (s, 1H), 4.98 (m, 1H), 4.90 (quintet, J = 6.2Hz, 1H), 4.39–4.16 (m, 4H), 3.95 (d, J = 6.9Hz, 1H), 2.79 (t ... t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t , J=5.8Hz, 2H), 2.67-2.52(m, 1H), 2.46(s, 3H), 2.44-2.23(m, 8H), 2.22-1.71( m, 18H), 1.70-1.45 (m, 7H), 1.44-1.12 (m, 45H), 1.13 (s, 3H), 0.97-0.82 (m, 6H).

[0764] Example 2S: Synthesis of INT-D053

[0765]

[0766] (R,Z)-12-acetoxyoctadec-9-enoic acid (1R,3S,Z)-3-hydroxy-5-(2-(((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylhept-2-yl)-7a-methyloctahydro-4H-indene-4-ethylene)ethylene)-4-methylenecyclohexyl ester and (R,Z)-12-acetoxyoctadec-9-enoic acid (1S,5R,Z)-5-hydroxy-3-(2-(((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylhept-2-yl)-7a-methyloctahydro-4H-indene-4-ethylene)ethylene)-2-methylenecyclohexyl ester (INT-D053): JZ-25-057, 029

[0767] In an argon atmosphere, DCC (50 mg, 0.24 mmol, 1.20 equivalence) was added to a stirred, ice-cold 1:1 CH2Cl2 / THF (4 mL) solution of (12R)-acetoxyoleic acid (82 mg, 0.24 mmol, 1.20 equivalence). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and solid calcitriol (83 mg, 0.20 mmol) and DMAP (29 mg, 0.24 mmol, 1.20 equivalence) were added. The reaction mixture was warmed for 14 h, diluted with EtOAc, stirred for 10 min, and then passed through... Filtration. The filtrate was concentrated to obtain a crude product, which was a pale yellow oil. It was then purified by rapid column chromatography (SiO2, 80:20 → 65:35 hexane / EtOAc) to give a ~1:1 mixture of 1- and 3-acylated conjugates (61 mg, 41% yield), which was a clear, colorless oil.

[0768] R f =0.33 (SiO2, 60:40 hexane / EtOAc);

[0769] 1 ¹H NMR (300MHz, CDCl₃): δ 6.44–6.25 (m, 2H), 6.02 (d, J = 11.2 Hz, 1H), 5.92 (d, J = 11.2 Hz, 1H), 5.56–5.40 (m, 3H), 5.40–5.27 (m, 4H), 5.26–5.16 (m, 1H), 5.07–4.97 (m, 2H), 4.87 (quintet, J = 6.2 Hz, 2H), 4.45– 4.34(m, 1H), 4.23-4.10(m, 1H), 2.89-2.74(m, 2H), 2.68-2.51(m, 2H), 2.48-2.18(m, 11H), 2.17-1 .77(m, 25H), 1.76-1.13(m, 90H), 1.12-0.99(m, 2H), 0.99-0.80(m, 13H), 0.55(s, 3H), 0.52(s, 3H).

[0770] Example 2T: Synthesis of INT-D068

[0771]

[0772] Linoleic acid (R,Z)-18-(((1R,3S,Z)-3-hydroxy-5-(2-(((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylhept-2-yl)-7a-methyloctahydro-4H-inden-4-ethylene)ethylidene)-4-methylenecyclohexyl)oxy)-18-oxooctadec-9-en-7-yl ester and linoleic acid (R ,Z)-18-(((1S,5R,Z)-5-hydroxy-3-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylhept-2-yl)-7a-methyloctahydro-4H-inden-4-ethylene)ethylidene)-2-methylenecyclohexyl)oxy)-18-oxooctadec-9-en-7-yl ester (INT-D068):

[0773] In a round-bottom flask under an argon atmosphere, DCC (50 mg, 0.24 mmol, 1.20 equivalence) was added to a stirred, ice-cold solution of (12R)-linoleyloxyoleic acid (135 mg, 0.24 mmol, 1.20 equivalence) in CH2Cl2 / THF (4 mL). The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and solid calcitriol (83 mg, 0.20 mmol) and DMAP (29 mg, 0.24 mmol, 1.20 equivalence) were added. The reaction mixture was warmed for 14 h, diluted with EtOAc, stirred for 10 min, and then passed through... Filtration. The filtrate was concentrated to obtain a crude product, which was a pale yellow oil. It was then purified by rapid column chromatography (SiO2, 95:5→90:10→70:30 hexane / EtOAc) to give a ~1:1 mixture of 1- and 3-acylated conjugates (75 mg, 39% yield), which was a clear, colorless oil.

[0774] R f =0.26 (SiO2, 70:30 hexane / EtOAc);

[0775] 1 ¹H NMR (300MHz, CDCl₃): δ 6.43–6.26 (m, 2H), 6.02 (d, J = 11.2Hz, 1H), 5.92 (d, J = 11.2Hz, 1H), 5.57–5.26 (m, 15H), 5.26–5.16 (m, 1H), 5.07–4.97 (m, 2H), 4.88 (quintet, J = 6.2Hz, 2H), 4.47–4.34 (m, 1H). H), 4.23-4.10(m, 1H), 2.89-2.70(m, 6H), 2.68-2.52(m, 2H), 2.47-2.20(m, 15H), 2.16-1.77(m , 25H), 1.77-1.12(m, 118H), 1.12-1.01(m, 2H), 1.00-0.79(m, 19H), 0.55(s, 3H), 0.52(s, 3H).

[0776] Example 2U: Synthesis of INT-D070

[0777]

[0778] 3-Fluorobenzyl isothiocyanate (15):

[0779] In a round-bottom flask under an argon atmosphere, Et3N (2.75 mL, 3.30 mmol, 3.30 equivalents) was added to a chilled THF (10 mL) solution of 3-fluorobenzylamine (750 mg, 6.00 mmol). Then, a THF (10 mL) solution of carbon disulfide (0.45 mL, 7.20 mmol, 1.20 equivalents) was added via a syringe pump over 30 minutes. The reaction mixture was warmed to room temperature, and after 3 hours, it was cooled to ice. Toluenesulfonyl chloride (1.26 g, 7.20 mmol, 1.20 equivalents) was added. After another 3 hours, 10 mL of 1 M HCl aqueous solution was added, and the reaction mixture was extracted with ethyl acetate (3 × 10 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The crude product was purified by rapid column chromatography (98:2→96:4 hexane / EtOAc) to obtain isothiocyanate 15 (846 mg, 84% yield), which was a clear, colorless oil.

[0780] R f =0.45 (SiO2, 80:20 hexane / EtOAc);

[0781] 1 H NMR (300MHz, CDCl3): δ7.42-7.35 (m, 1H), 7.13-7.04 (m, 3H), 7.74 (s, 2H).

[0782]

[0783] 3-(3-fluorobenzyl)-2-thiothiazolidin-4-one (16):

[0784] In a round-bottom flask under an argon atmosphere, mercaptoacetic acid (0.17 mL, 2.40 mmol, 0.75 equivalence) was added to an ice-cold mixture of Et3N (0.90 mmol, 6.40 mmol, 2.00 equivalence) and water (10 mL), followed by the addition of a THF solution of isothiocyanate 15 (539 mg, 3.20 mmol) (5 mL) over 5 minutes. The reaction mixture was warmed to room temperature until it turned pale orange. The mixture was adjusted to pH ≤ 2 by adding 6 M HCl aqueous solution. The reaction mixture was refluxed for 14 h, then cooled to room temperature and extracted with EtOAc (3 × 10 mL). The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure using a rotary evaporator. The crude product was purified by silica gel filtration (50:50 hexane / EtOAc) to give Rhodamine 16 (466 mg, 80% yield) as a yellow solid.

[0785] R f=0.52 (SiO2, 70:30 hexane / EtOAc);

[0786] 1 H NMR (300MHz, CDCl3): δ7.72 (s, 1H), 7.65 (d, J=7.7Hz, 1H), (d, J=7.7Hz, 1H), 7.46 (t, J=7.8Hz, 1H), 5.25 (s, 2H), 4.04 (s, 2H).

[0787]

[0788] (Z)-4-((3-(3-fluorobenzyl)-4-oxo-2-thiothiazolidin-5-ylidene)methyl)benzoic acid (INT-MA014):

[0789] In a round-bottom flask, 4-carboxybenzaldehyde (151 mg, 1.01 mmol, 1.10 equivalents) was added to a solution of Rhodamine 16 (221 mg, 0.92 mmol) and piperidine (0.01 mL, 0.14 mmol, 0.15 equivalents) in EtOH (4 mL), and the mixture was heated under reflux. After 1.5 h, the reaction mixture was concentrated under reduced pressure using a rotary evaporator. The crude product was then filtered through silica gel (90:8:2CH2Cl2 / MeOH / HOAc) and concentrated under reduced pressure. Rhodamine carboxylic acid INT-MA014 (179 mg, 52% yield) was precipitated from the hot EtOH as a yellow solid.

[0790] R f =0.26 (SiO2, 50:40:10 hexane / EtOAc / MeOH);

[0791] 1 H NMR (300MHz, CDCl3): δ8.08 (d, J=8.2Hz, 2H), 7.91 (s, 1H), 7.77 (d, J=8.3Hz, 2H), 7.43-7.36 (m, 1h), 7.20-7.11 (m, 3H), 5.27 (s, 2H).

[0792]

[0793] 4-((Z)-(3-(3-fluorobenzyl)-4-oxo-2-thiothiazolidin-5-ylidene)methyl)benzoic acid 12R-linoleyloxyoleyl ester (INT-D070)–JZ-25-169

[0794] In a round-bottom flask under an argon atmosphere, i-Pr2NEt (0.37 mL, 2.10 mmol, 1.50 equivalence) and BOP reagent (682 mg, 1.54 mmol, 1.10 equivalence) were added to a DMF (3.5 mL) solution of carboxylic acid INT-MA014 (523 mg, 1.40 mmol) and 12R-linoleyloxyoleyl alcohol (843 mg, 1.54 mmol, 1.10 equivalence) at room temperature. After stirring for 14 h, the reaction mixture was diluted with water and extracted with t-BuOMe (3 × 15 mL). The combined organic layers were washed with water (4 × 10 mL) and brine (1 × 10 mL), dried over Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The crude residue was purified by rapid column chromatography (SiO2, 99:1→95:5 hexane / EtOAc) to obtain INT-D070, which was a yellow oil (1.18 g, 93% yield).

[0795] R f =0.47 (SiO2, 90:10 hexane / EtOAc);

[0796] 1 H NMR (300MHz, CDCl3): δ8.15 (d, J=8.3Hz, 2H), 7.78 (s, 1H), 7.58 (d, J=8.2Hz, 2H ), 7.37-7.26(m, 2H), 7.23-7.15(m, 1H), 7.06-6.96(m, 1H), 5.55-5.23(m, 8H), 4 0.90 (quintet, J = 6.2 Hz, 1H), 4.36 (t, J = 6.7 Hz, 2H), 2.79 (t, J = 5.9 Hz, 2H), 2.36–2.22 (m, 4H), 2.15–1.96 (m, 6H), 1.79 (m, 2H), 1.70–1.14 (m, 36H), 0.98–0.80 (m, 6H).

[0797] Example 2V: Synthesis of INT-H001

[0798]

[0799] 1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-hydroxypropyl)thiourea (15): JZ-25-145

[0800] In a round-bottom flask under an argon atmosphere, 3-aminopropanol (0.33 mL, 4.40 mmol, 1.10 equivalence) was added to a solution of 3,5-bis(trifluoromethyl)phenyl isothiocyanate (1.08 g, 4.00 mmol) and Et3N (0.61 mL, 4.40 equivalence, 1.10 equivalence) in MeCN (8 mL). After 14 h, the reaction mixture was diluted with H2O and extracted with EtOAc (3 × 15 mL). The combined organic layers were washed with H2O (1 × 15 mL) and brine, dried over Na2SO4, and concentrated under reduced pressure using a rotary evaporator. The crude semi-solid was filtered through a silica gel pad (75:25 EtOAc / hexane), the filtrate was concentrated under reduced pressure, and then recrystallized from t-BuOMe / hexane to give thiourea 15 (1.18 g, 85% yield) as a white solid.

[0801] 1 ¹H NMR (300MHz, DMSO-d6): δ 10.1 (br s, 1H), 8.26 (br s, 3H), 7.72 (br s, 1H), 4.59 (br s, 1H), 3.66–3.39 (m, 4H), 1.72 (quintet, J = 6.4 Hz, 2H);

[0802] 13 C NMR (75.5MHz, DMSO-d6): δ180.4, 142.0, 130.1 (q, J=34Hz), 123.3 (q, J=273Hz), 121.7 (br), 115.9 (br), 58.7, 41.6, 31.3.

[0803]

[0804] 1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-hydroxypropyl)thiourea (INT-H001):

[0805] In an argon atmosphere, DCC (68 mg, 0.33 mmol, 1.10 equivalence) was added to a stirred, ice-cold CH2Cl2 solution of carboxylic acid 12b (252 mg, 0.30 mmol, 1.10 equivalence) in a round-bottom flask. The ice bath was then removed, and the mixture was stirred for 15 min. The reaction mixture was then cooled in an ice bath, and thiourea 15 (114 mg, 0.33 mmol) and DMAP (44 mg, 0.36 mmol, 1.20 equivalence) were added. The reaction mixture was warmed for 14 h, diluted with t-BuOMe, stirred for 10 min, and then passed through... Filtration. The filtrate was concentrated to obtain a crude product, which was a pale yellow oil. It was then purified by rapid column chromatography (SiO2, 80:18:2 hexane / EtOAc / MeOH) to obtain INT-H001 (222 mg, 63% yield), which was a clear, colorless oil.

[0806] R f =0.35 (SiO2, 80:18:2 hexane / EtOAc / MeOH);

[0807] 1 H NMR (300MHz, CDCl3): δ8.06 (br s, 1H), 7.88 (s, 2H), 7.72 (s, 1H), 6.90 (br t, 1H), 5.49-5.24 (m, 8H), 5.10-4.90 (m, 2H), 4.20 (t, J=5.6Hz, 2H), 3.79-3.64 (m, 2H), 2.79 (t, J=5.9 Hz, 4H), 2.29 (m, 6H), 2.14-1.93 (m, 10H), 1.70-1.45 (m, 10H), 1.45-1.16 (m, 48H), 0.96-0.83 (m, 9H).

[0808] Example 2W: Synthesis of Bisubstituted Calcitriol INT-D087

[0809] Examples of synthetic schemes for preparing calcitriol lipid conjugates with two lipid moieties co-substituted are provided below:

[0810]

[0811] Example 3: Formulation of prodrugs in lipid nanoparticles (LNPs)

[0812] The lipid-like properties of prodrugs allow them to be conveniently loaded into LNP systems by simply mixing with lipid formulation components. That is, in some embodiments, loading can be achieved without any further modification to known formulation processes. Therefore, LNPs containing these drug-lipid conjugates can be prepared using a variety of well-described formulation methods, including but not limited to extrusion, ethanol injection, and online mixing.

[0813] LNPs were prepared by dissolving 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC) or 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), cholesterol, and 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-poly(ethylene glycol) (PEG-DSPE) in ethanol. DSPC, DMPC, and PEG-DSPE were purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol was obtained from Sigma (St. Louis, MO).

[0814] The drug-lipid conjugates INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-D049, INT-D050, INT-D051, INT-D053, INT-D060, INT-D061, INT-D062, INT-D063, INT-D083, INT-D085, INT-D086, INT-D088, and INT-D089 (see Figure 3 and Example 7 for structures) were dissolved in isopropanol or THF. LNPs were prepared by rapidly mixing DSPC or DMPC, cholesterol, the drug-lipid conjugates, and PEG-DSPE (molar ratio 49 / 40 / 10 / 1) with phosphate-buffered saline (PBS) using a cross-mixer. The formulation was dialyzed against PBS to remove residual ethanol. In formulations containing more than 10 mol% of drug-lipid conjugates, the amount of phospholipids or cholesterol is thus reduced.

[0815] The physicochemical properties of the LNPs prepared as described above were then characterized. Particle size was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK), after the buffer was replaced with phosphate-buffered saline. Digitally weighted size and distribution data were used. Lipid concentration was determined by measuring total cholesterol using a cholesterol E enzyme assay kit from Wako Chemicals USA (Richmond, VA). The morphology of the LNP formulation containing LD-DEX was analyzed by cryo-transmission electron microscopy (cryoTEM).

[0816] Table 4 below shows that the prodrugs described herein can be formulated in LNPs with high encapsulation efficiency and low polydispersity, both of which are desirable physicochemical properties for drug delivery carriers.

[0817] Table 4. Physicochemical parameters of LNPs containing 10 mol% castor oil-dexamethasone conjugate

[0818]

[0819]

[0820] Example 4: Formation of monodisperse LNPs with novel macromolecular structures from prodrugs

[0821] Using the rapid mixing technique described in Example 3, the hexanoyl prodrug INT-D034 (Figure 3A) was mixed with neutral phospholipids and cholesterol at a prodrug concentration of 0-99 mol% to produce a monodisperse LNP formulation. Figure 4 All INT-D034 formulations exhibited high encapsulation efficiency, with particle sizes ranging from ~29-87 nm and polydispersity index (PDI) of 0.1 or less (Table 5 below). Electron micrographs of the LNP formulations showed that with increasing INT-D034 content, the spherical electron-dense region adjacent to the film expanded, suggesting that the prodrug INT-D034 exists as a hydrophobic oil phase in the LNP lipid bilayer. Figure 4 ).

[0822] Table 5: Particle size and polydispersity index of LNPs containing different amounts of prodrugs

[0823]

[0824]

[0825]

[0826]

[0827] To determine whether this novel ultrastructure was consistent with another castor oil-based conjugate, INT-D035, INT-D035 (according to Figure 3B, having an R-hydrocarbon derived from an oleoyl group rather than the hexanoyl group in INT-D034) was incorporated into the LNP as described in Example 3 at an equal amount of prodrug (10 mol%). Similar to the INT-D034 formulation, the INT-D035 formulation also exhibited a spherical, electron-dense region immediately adjacent to the membrane. Figure 5 These results indicate that the castor oil-based conjugates possess suitable properties as a hydrophobic oil phase within the LNP lipid bilayer.

[0828] Other prodrugs containing different R groups, including INT-D045, INT-D049, INT-D050, INT-D051, INT-D053, INT-D060, INT-D061, INT-D062, INT-D063, INT-D083, INT-D085, and INT-D086, can be effectively incorporated to achieve 99 mol% in LNP (Table 5).

[0829] Example 5: Dissociation of prodrug in LNP as a function of S-group hydrophobicity (LogP)

[0830] The release of ricinole-dexamethasone conjugates from LNPs was examined using a assay involving human plasma containing lipoproteins as lipid aggregates in which lipid exchange occurs. Plasma lacks active esterases that may digest ricinole-dexamethasone conjugates, thus hindering the detection and monitoring of intact conjugates.

[0831] LNP formulations containing 10–99 mol% INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048 or INT-D049, INT-D050, INT-D051, INT-D053, INT-D083, INT-D085, INT-D086 or INT-D089 (see Figure 3 and Example 7 for structures) were incubated in human plasma at 37°C with 1.2 mM total lipids for 0, 2, or 24 hours. Size exclusion chromatography was performed to separate LNPs from lipoproteins (1.5 x 27 cm Sepharose CL-4B size exclusion column). Thirty 2 mL fractions were collected, and three volumes of isopropanol / methanol (1:1 v / v) were added to each fraction.

[0832] Using a photodiode array detector (PDA) Acquity TM The UPLC system was used to quantify drug-lipid conjugates via ultra-high pressure liquid chromatography (UPLC); Empower was employed. TM Data acquisition software version 2.0 (Waters, USA). Use Acquity TM Separation was performed using a BEH C18 column (1.7 μm, 2.1 × 100 mm) at a flow rate of 0.5 mL / min, with a linear gradient from 80 / 20 (% A / B) to 0 / 100 (% A / B). Mobile phase A consisted of water, while mobile phase B consisted of methanol / acetonitrile (1:1, v / v). The method was run at a column temperature of 55 °C for 6 min, and analytes were measured by monitoring a PDA detector at a wavelength of 239 nm.

[0833] Figure 6 shows the amount of each intact castor oil-dexamethasone conjugate that remained associated with the LNP in each fraction, as quantified by UPLC. Castor oil-dexamethasone conjugates with different LogP values ​​exhibited different levels of dissociation (Figure 6). Conjugates with higher predicted LogP values ​​(i.e., more hydrophobic) dissociated less from the LNP compared to those with lower predicted LogP values. For example, compared to ~40% INT-D047 (LogP 8.33) (below) Figure 6A Compared to Table 6), over 90% of INT-D086 (LogP = 21.2) remained in the LNP. These results demonstrate that the prodrug based on the stent design described herein provides a reliable method for controlling drug release from the LNP. In cases where prolonged circulation of the LNP within the body system is required to reach disease sites (e.g., distal tumors), it is desirable for the drug to remain bound to the LNP and not exhibit premature drug leakage, as this can be directly associated with low therapeutic activity.

[0834] Table 6: Biophysical parameters of LNP formulations containing prodrugs

[0835]

[0836]

[0837] Example 6: The prodrug is biodegradable and active in vitro.

[0838] To provide therapeutic activity, the active drug must ultimately be released from the conjugate. An exemplary castor oil-based conjugate contains a biodegradable, esterase-sensitive linker between the active drug and the castor oil-based scaffold. The biodegradability of the castor oil-based conjugate was examined using mouse plasma, as it contains an active esterase that cleaves the linker.

[0839] The LNP formulation containing INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-D049, INT-D050, INT-D051, INT-D053, INT-D083, INT-D085, INT-D086, or INT-D089 was incubated with mouse plasma for 0 or 2 hours, and then the intact conjugate and released dexamethasone or calcitriol were quantified by UPLC as described above. Figure 7A , Figure 7B and Figure 7C Different levels of intact castor oil-drug conjugates were detected, indicating varying levels of plasma esterase degradation. Figure 7A ).

[0840] Notably, different amounts of free dexamethasone detected in mouse plasma were equivalent to Figure 7B The decomposition level exhibited by the prodrug.

[0841] Dexamethasone is known to suppress unwanted immune responses. The activity of a castor oil-based conjugate in a lipopolysaccharide (LPS)-mediated immunostimulated cell model was then demonstrated.

[0842] The cultured macrophage cell line J774.2 ( Figure 8 ) and Raw264.7 Figure 9A Cells were incubated with the immunostimulants LPS and LNP in or without the castor oil-based dexamethasone conjugate INT-D034 / INT-D035. After 24 hours, cells were harvested and the expression of pro-inflammatory cytokines IL1β, TNFα, and IL-6 was analyzed. RNA was isolated from the cells, and the levels of pro-inflammatory cytokines IL1β, TNFα, and IL-6 were determined by qRT-PCR.

[0843] Cells incubated with the control formulation (i.e., without the castor oil-dexamethasone conjugate) showed elevated levels of all three cytokines, suggesting an inflammatory response. Conversely, cells treated with the LNP formulation containing either INT-D034 or INT-D035 showed dose-dependently reduced levels of pro-inflammatory cytokines. Similar reductions in IL-1β levels of INT-D045, INT-D046, INT-D047, INT-D048, and INT-D049 were observed in Raw 264.7. Figure 9B These results suggest that castor oil-based dexamethasone conjugates can be processed intracellularly to release the active drug (dexamethasone), thereby suppressing unwanted immune responses.

[0844] Dexamethasone and calcitriol can tolerate antigen-presenting cells (APCs). The activity of castor oil-based dexamethasone and calcitriol conjugates was then demonstrated in a mixed lymphocyte response (MLR) assay for assessing immune tolerance. First, bone marrow-derived dendritic cells (BMDCs) from C57Bl / 6 male mice (Charles River) were treated with LNPs containing different mol% dexamethasone or calcitriol conjugates for 48 hours, followed by activation by incubation with LPS for 24 hours. These were then harvested and mixed with CD4+ T cells isolated from Balb / cJ male mice (Jackson Laboratories) at a T:BMDC ratio of 5:1 or 10:1. The level of T cell proliferation after 3 days was quantified using flow cytometry. Figure 10As shown, LNPs containing 10-99 mol% dexamethasone conjugates (INT-D034 or INT-D045) or calcitriol conjugates (INT-D053 or INT-D083) were able to inhibit allogeneic T cell proliferation, indicating that these castor oil-based conjugates can be processed intracellularly to release dexamethasone or calcitriol to tolerate BMDC.

[0845] Therefore, the prodrugs described in this article can not only be effectively loaded into LNPs in large quantities to achieve controlled drug release, but also have activity, as shown in in vitro models of immune stimulation and in vitro models of immune tolerance.

[0846] Example 7: Another prodrug example

[0847] Various types of drugs can be used as prodrugs as described herein. Examples of such compounds are shown below and include acetylsalicylic acid, MCC950, INT-MA014, calcitriol, ruxolitinib, tofacitinib, sirolimus, docetaxel, mycophenolic acid, cannabidiol, and tetrahydrocannabinol. Exemplary prodrugs of such compounds are also described below:

[0848]

[0849]

[0850]

[0851]

[0852] These prodrugs can be synthesized using ester or carbonate X1 linkers, as shown in the reaction scheme below. The biodegradation mechanism of the ruxolitinib prodrug with an ester X1 linker is also described below. First, an esterase cleaves the ester group on the prodrug. The resulting hemiacetalamine then spontaneously decomposes to release the free drug.

[0853] Exemplary synthesis of ruxolitinib prodrug using esters and carbonates:

[0854]

[0855] Example 8 - More than one prodrug can be formulated in the same LNP.

[0856] As mentioned above, the lipid-like properties of prodrugs can be conveniently loaded into LNP systems by simply mixing them with lipid formulation components. It has been determined that one or more prodrugs from different corresponding parent drugs can be loaded into the same LNP system because these prodrugs possess lipid-like properties. Table 7 shows LNP formulations produced by mixing two different prodrugs in an equimolar ratio (i.e., 10 mol% each). In particular, it has been demonstrated that prodrugs of dexamethasone and calcitriol can be encapsulated together at very high levels (close to 100%) to produce monodisperse nanoparticle formulations with a diameter of 44-50 nm and a PDI < 0.1. Figure 11 Electron micrographs show that these combined formulations exhibit spherical electron-dense regions immediately adjacent to the membrane, similar to those found in... Figure 4 and 5 The results show the observations in formulations containing a single prodrug. Without limitation, these morphological data suggest that prodrugs of different parent drugs can coexist as hydrophobic oil phases within the lipid bilayer of LNPs. Furthermore, it has been determined that dexamethasone and calcitriol conjugates in varying proportions (1-10 mol%) can be formulated together with high encapsulation efficiency to form monodisperse nanoparticles with diameters of ~50-60 nm (Table 8).

[0857] LNP formulations containing 10 mol% dexamethasone conjugate (INT-D045) were incubated with or without 10 mol% calcitriol conjugates (INT-D053, INT-D068, or INT-D083) in human or mouse plasma at 37°C for 0, 2, or 24 hours to determine the dissociation and biodegradation of the lipid-conjugates, as in Example 5. Figure 12 and Figure 13 As described in [reference needed]. Compared to formulations containing only one lipid-drug conjugate, combination formulations (i.e., formulations containing more than one lipid-drug conjugate) exhibited similar levels of lipid dissociation or biodegradation. These data suggest that a lipid-drug conjugate can retain its function when encapsulated in the same lipid nanoparticle as another lipid-drug conjugate.

[0858] Table 7 - Particle size and polydispersity index of LNPs containing prodrug combinations

[0859]

[0860] Table 8 - Particle size and polydispersity index of LNPs with different prodrug combination molar ratios

[0861]

[0862]

[0863] The foregoing embodiments are merely exemplary. That is, various changes can be made without departing from the scope of certain aspects of the invention as described herein.

Claims

1. A lipid-conjugate for incorporation into nanoparticles, having one of the following structures: 。 2. A pharmaceutical product comprising the conjugate of claim 1.

3. A nanoparticle comprising the conjugate of claim 1.

4. The nanoparticles of claim 3, wherein the nanoparticles... (i) are lipid nanoparticles; or (ii) Contains one or more double layers.

5. The nanoparticles of any one of claims 3-4, wherein the nanoparticles are lipid nanoparticles exhibiting spherical electron-dense regions at the membrane, wherein the lipid nanoparticles comprise a bilayer, and the lipid conjugate is present in a hydrophobic oil phase within the nanoparticles.

6. Use of the conjugate of claim 1 for manufacturing nanoparticles incorporating the conjugate.

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