Lipid conjugate prepared from scaffold moiety

Abstract

To provide a drug-lipid conjugate that allows a broader range of drugs to be easily incorporated into a drug delivery medium.SOLUTION: For example, a drug-lipid conjugate represented by the following formula is illustrated.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention provides lipid conjugates, formulations of lipid conjugates, and precursor molecules for preparing such conjugates. [Background technology]

[0002] Many drugs have the potential to cure cancer, autoimmune diseases and other diseases, but they may not be effective because they do not reach the disease site.For example, when a drug is administered intravenously, only a small amount (e.g., less than 0.1%) of the drug often actually reaches its target, while the rest of the drug is distributed throughout other parts of the body, leading to reduced therapeutic efficacy and undesirable side effects.

[0003] Drug delivery systems, including lipid nanoparticles (LNPs) and polymer-based vehicles, have the potential to overcome this problem by encapsulating drugs and targeting them to parts of the body in need of treatment, such as tumor sites or sites of inflammation. This effect can be achieved by taking advantage of the leaky vasculature and impaired lymphatic drainage that are often present at these disease sites. Regardless of the mechanism, localizing drugs to specific sites may result in higher efficacy and lower toxicity.

[0004] However, in many known drug delivery vehicles, only a small subset of known drugs can be incorporated, and in the case of LNPs with bilayers, loading techniques are mostly limited to multi-step, live-loading techniques, and usually require that the drug has an amine group. In one live-loading method, a transmembrane pH gradient is established such that the interior of the LNP is acidic and the external pH value is adjusted to physiological conditions. Uncharged amine-containing drugs incubated with these LNPs diffuse into the vesicles and become charged inside the LNP due to protonation of the amines. The charged drug can no longer cross the bilayer and is trapped inside the LNP. However, many widely prescribed and important drugs do not have amine groups and cannot be simply encapsulated and retained in LNPs using this method. Thus, the therapeutic benefits of many potentially effective drugs remain largely unrealized.

[0005] One way to make a wider range of drugs accessible for incorporation into drug delivery vehicles is to conjugate them with lipid moieties. Many drug delivery vehicles contain hydrophobic components, and lipid moieties on the conjugates can enhance the incorporation of drugs into such components. A known strategy is to conjugate the terminal C1 carboxyl terminus of a fatty acid with a hydroxyl or amine 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 are linked to certain drugs to generate drug-lipid conjugates (Irby et al., 2017, “Lipid-Drug Conjugate for Enhancing Drug Delivery”, Mol. Pharm.14(5):1325-1338). The drug can also be attached to the lipid moiety via a linker group that acts as a spacer between the drug and the lipid. Linker groups for such purposes are known in the art and are described in U.S. Patent No. 5,149,794, which is incorporated herein by reference.

[0006] The ability to control drug release from a delivery vehicle is an important factor for achieving optimal therapeutic efficacy, and it is generally known that hydrophobic compounds are more likely to remain with the membrane or other hydrophobic components of the delivery vehicle than less hydrophobic ones. Thus, the overall hydrophobicity of a drug-lipid conjugate may affect its releasability from the drug delivery vehicle after administration. In clinical applications, drug-lipid conjugates need to be transported through the bloodstream to reach disease sites such as distant tumors. It is necessary for the drug to exhibit a long circulation lifetime in the blood and it is important that the drug remain stably associated with the delivery vehicle for as long as possible. Other clinical applications, such as those requiring localized delivery, may require more rapid release. However, from a practical standpoint, it is often difficult to precisely tune the hydrophobicity of the molecule.

[0007] The present inventors have identified a simple and broadly applicable method for imparting desirable physical properties to drug-conjugates to enable the clinical use of many potentially effective drugs. In addition to drugs, such methods can be applied to a variety of other molecules of interest. Examples include hydrophilic polymers, genetic material, proteins such as polypeptides and antibodies, and other molecules of interest.

[0008] The compositions and methods of the present disclosure seek to address this problem and / or provide a beneficial alternative to those previously disclosed. Summary of the Invention

[0009] In the embodiments described herein, a scaffold molecule, designated "L", is provided, which forms the carbon backbone of the lipid portion of the lipid conjugate to which one or more groups can be attached. The carbon backbone of L can have 5-40 or 5-30 carbon atoms and can have one or more cis or trans C=C double bonds. L is modular in the sense that it can function as a molecular backbone to which various combinations of hydrocarbon groups (R and / or R') and molecules of interest (M) (including, without limitation, drug moieties (D) or polymers (possibly via linkers)) can be attached via respective functional groups along its carbon backbone.

[0010] In one embodiment, the inventive approach described herein allows for more precise tuning of the hydrophobicity of a molecule of interest, such as a prodrug. By selecting an appropriate hydrocarbon R for attachment to the scaffold L, the molecule of interest can be designed to have a desired octanol / water LogP value, including but not limited to:

[0011] It would be advantageous to be able to more precisely tune the hydrophobicity of a molecule of interest, such as a drug or other target molecule. The inventors have shown in embodiments, without limitation, that lipid conjugates can be designed with hydrophobicity that allows loading into a particular delivery vehicle to approach 100% encapsulation. Furthermore, retention of lipid conjugates in the delivery vehicle after administration to a patient can also be more precisely controlled. For example, it has been found that the predicted LogP values ​​of certain lipid conjugates described herein generally correlate with their retention ability in drug delivery vehicles. Thus, more precise control of drug release can be achieved by tuning the LogP value of the prodrug, such as by selecting the appropriate R group as described herein.

[0012] Generally, the lipid portion of the molecule of interest determines the overall hydrophobicity of the conjugate, and therefore a wide range of compounds can be selected for incorporation into the prodrug, including drugs, macromolecules and other molecules.

[0013] Also described herein is a novel pharmaceutical and drug delivery composition comprising lipid conjugate.The conjugate can be incorporated into a pharmaceutical composition comprising pharmaceutically acceptable salts and / or excipients, or into a drug delivery vehicle that forms a component of a pharmaceutical composition.Alternatively, the conjugate can be incorporated into consumer products, including, but not limited to, food, nutritional supplements, cosmetics, or cleaning products.

[0014] As described herein, the present disclosure is also based on the discovery that LNP formulations incorporating lipid conjugates exhibit globular electron-dense regions in the membrane. The lipid nanoparticles comprise two layers: a lipid conjugate and a hydrophobic oil phase composed of the lipid conjugate. In one embodiment, the lipid nanoparticle is a liposome, and in a further embodiment, the lipid conjugate has the structure of Formula I, Ia, II or IIa as shown below.

[0015] In certain embodiments, lipid conjugates are disclosed that include a branched lipid moiety having a scaffold L, which is a scaffold that links one or more R hydrocarbon chains, the lipid moiety having the formula IId:

[0016] [ka]

[0017] The structure is where the L lipid scaffold is represented by L1+L2+L3+L4+L5+L6, where L contains 5-40 carbon atoms and 0-2 cis or trans C=C double bonds; L1 is a carbon chain having 3 to 30 carbon atoms, and L1 may have one or more cis or trans C=C double bonds, or 0 to 2 cis or trans C=C double bonds; L2 and L4 are each carbon atoms; L3 is 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L5 has 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L6 is -CH 3 , =CH 2 or H; 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, and when one or more of R is branched, each branch point contains an X2 functional group; n is 0 to 8, p is 0 to 8, and n+p is ≧1 or 1 to 8; Each X2 is independently a carbon-based functional group including an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphate, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene, or alkyne, methylene (CH 2 ) or urea; or X2 is a linkage containing at least one hydrogen bond; The conjugate is not an ionizable lipid.

[0018] In certain embodiments, X2 is independently a group that is biodegradable after administration to a patient. X2 can independently be a carbamate, ether, or ester linkage.

[0019] In a further embodiment, L is linked to the molecule of interest M of the conjugate at L1 by X1 to form M-X1-L, where X1 is an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphate, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide. , imine, azo, alkane, alkene or alkyne, carbon-based functional groups, methylene (CH 2 ) or urea; Or, L is linked to the molecule of interest by a hydrogen bond between L and M of the lipid conjugate. In some embodiments, X1 is an ester, ether or carbamate.

[0020] In another embodiment, the second L is linked to the molecule of interest by X1. The second L may have the structure of formula IId:

[0021] In one embodiment, L1 has from 3 to 30 carbon atoms, alternatively from 4 to 30 carbon atoms.

[0022] In yet another embodiment, L is linked to the molecule of interest M by a hydrogen bond between L and M of the lipid conjugate, and L-X1-M is represented by the formula V:

[0023] [ka]

[0024] The structure is wherein E1, E2, E3, E4 and E5 are independently electronegative atoms selected from O, N and P; E1, E2 and E3 are hydrogen bond acceptors, and E4 and E5 are hydrogen bond donors; Dotted lines indicate hydrogen bonds, solid lines indicate covalent bonds; L is the lipid scaffold of the lipid moiety; n is 0 or 1, o is 0 or 1, p is 0 or 1, and n+o+p≧2; q is 1 to 10 or 2 to 10 or 4 to 10; L is the lipid scaffold of the lipid moiety; M is the molecule of interest; E1 and E3 are each selected from alkyl, aryl, alkylene, or H, and may have a substituent linked thereto.

[0025] In one embodiment, at least one R is branched and each branch point of R is independently selected from an ester, an ether, or a carbamate.

[0026] In another alternative embodiment, the lipid portion is non-cylindrical and is flared or frusto-conical in the L1 to L6 orientation.

[0027] According to one embodiment, X2 is not a disulfide or thioether group.

[0028] According to a further alternative embodiment, the lipid moiety is derived from a lipid having one or more reactive groups selected from hydroxyl, amino, and / or amide attached to its internal carbon atoms to function as a scaffold carbon chain in the lipid moiety, and at least one other carbon atom in the hydrocarbon structure. The hydrocarbyl chain is derived from an acyl lipid, and the X1 linkage is formed by reaction of a reactive group on the scaffold carbon chain with a carboxylic acid of the acyl chain.

[0029] Further provided herein is a lipid conjugate comprising a branched lipid moiety having a backbone L that is a scaffold for attachment of one or more R hydrocarbon chains, the lipid moiety comprising: Formula IIe:

[0030] [ka]

[0031] The structure is In the formula, L is [CH 2 ] m -L2-L3-L4-[CH 2 ] q -CH 3 It is shown that the carbon in L The total number of elementary atoms is 5 to 30; L2 and L4 are carbon atoms; m is 0-20, n is 1-4, p is 0-4, n+p is 1-4; L3 is 0-10 carbon atoms and has 0-2 cis or trans C=C; X2 is independently selected from an ether, an ester, and a carbamate group; Each R, independently; (a) a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms; each R is linked to one of the respective X2 at any carbon atom in the hydrocarbon chain; or (b) A branched structure of formula IIb having a scaffold designated L':

[0032] [ka]

[0033] In the formula, L' is [CH 2 ] r -L2-G 3 -L4-[CH 2 ] u -CH 3 It is indicated by L The total number of carbon atoms is 3 to 30; r is 0-20, 2-20, 3-20, or 4-20; s is 0-4, t is 0-4, and s+t is >1 or 1-4; u is between 1 and 20; G 3 has 0-10 carbon atoms and 0-2 cis or trans C=C; Each R' of formula IIb is independently a linear or branched terminal hydrocarbon chain having 0 to 5 cis or trans C=C and 1 to 30 carbon atoms; The total number of R′ hydrocarbon chains in formula IIb is 1 to 16; Each of the R and R' hydrocarbon chains in the lipid moiety may be substituted with a heteroatom, provided that no more than eight heteroatoms are substituted in the R and R' hydrocarbon chains and the conjugate predicted or experimental logP > 5; The lipid conjugate is not an ionizable lipid.

[0034] According to any of the above-mentioned embodiments, the scaffold lipid L is derived from a hydroxy lipid.

[0035] In yet another embodiment, the lipid conjugate has the structure of any one of the lipid conjugates shown in FIG.

[0036] According to a further aspect, there is provided a pharmaceutical composition comprising the above-mentioned conjugate.For example, the conjugate can be formulated in a nanoparticle, such as a lipid particle.According to another embodiment, the nanoparticle comprises one or more bilayers.

[0037] Further provided is a method for treating cancer or an infectious disease, comprising administering the conjugate described above.

[0038] According to a further embodiment, the compound of formula I: M-X1-[L]-X2-R Prodrugs are provided having the structure During the ceremony, M is a drug moiety, D; X1 is a chemical linkage covalently linking D to any carbon atom of L; L is a scaffold carbon chain having 5 to 40 carbon atoms and optionally having one or more cis or trans C═C double bonds; X2 is a chemical linkage covalently attaching R to any carbon atom of L; R is a linear or branched hydrocarbon having 1 to 40 carbon atoms and optionally having one or more cis or trans C=C double bonds; X1 and X2 are independently selected from a functional group or a linker.

[0039] According to a further embodiment, the compound of formula Ia:

[0040] [ka]

[0041] The above prodrugs are provided having the structure: During the ceremony, L1-L2 is a scaffold carbon chain L having 5 to 40 carbon atoms; L1 is a carbon chain having 5 to 40 carbon atoms and optionally having one or more cis or trans C=C double bonds; L2 is a carbon chain equal to L minus the L1 carbon atom and may have one or more cis or trans C=C double bonds, and X2 covalently bonds R to L2 at any carbon atom on L2.

[0042] In some embodiments, the prodrug has a logP of at least 5.

[0043] In another embodiment, the prodrug further comprises a second R hydrocarbon side chain having 1-40 carbon atoms covalently attached to L via chemical linkage X2.

[0044] The prodrug further comprises a 1-40 carbon atom covalently attached to L via a chemical linkage X2. It may include a third side chain R having atoms.

[0045] The prodrug may contain an R' side chain linked to a first R via an X2 linkage. The prodrug may contain an additional R' side chain linked to another R via an X2 linkage.

[0046] The X1 and X2 linkages can independently be any of a carbon-based functional group including an ester, amide, amidine, hydrazone, disulfide, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphate, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene or alkyne, methylene (CH 2 ) or urea.

[0047] The X1 and X2 linkages of the prodrug may contain at least one group that is biodegradable following administration to a patient.

[0048] In one embodiment, the prodrug X1 is a linker and may be biodegradable.

[0049] The (M-X1) moiety of formula I or Ia has the following formula IV:

[0050] [ka]

[0051] may have: wherein X4 and X5 are independently a carbon-based functional group including an ester, an amide, an amidine, a hydrazone, a disulfide, an ether, a carbonate, a carbamate, a thionocarbamate, a guanidine, a guanine, an oxime, an isourea, an acylsulfonamide, a phosphoramide, a phosphoramidate, a phosphate, a phosphonate, a phosphodiester, a phosphate phosphonooxymethyl ether, an N-Mannich adduct, an N-acyloxyalkylamine, a sulfonamide, an imine, an azo, an alkane, an alkene, or an alkyne, a methylene (CH 2 ) or urea, M1 is any spacer group having 0 to 12 carbon atoms linked to the X4 and X5 functional groups, or M1 is CH 2 , C.H. 2 CH 2 , N-alkyl, N-acyl, O or S.

[0052] In some embodiments, R is -CMe 3 , -Me, or a linear carbon chain having 2 to 40 carbon atoms and optionally having 1 to 6 cis or trans double bonds.

[0053] The drug moiety, D, may be derived from an anti-cancer drug or an immunomodulatory drug.

[0054] Drug portion D was selected from the group consisting of docetaxel, dexamethasone, methotrexone, NPC1I, abiraterone, prednisone, prednisolone, ruxolitinib, tofacitinib, calcitriol, calcifediol, cholecalciferol, sirolimus, tacrolimus, acetylsalicylic acid, mycophenolic acid, cabazitaxel, betamethasone, and CY09 (4-[4-oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]methyl]-5-thiazolidinylidene]methyl)benzoic acid], INT-MA014, or MCC950 (N-(1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide). The NLRP3 inhibitors or derivatives thereof may be derived from cannabinoids such as cannabigerol, cannabichromene, cannabidiol, cannabidivarin, cannabicyclol, cannabicitran, cannabielsoin, cannabinol, tetrahydrocannabinol, or tetrahydrocannabivarin.

[0055] The prodrugs are 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-D063, IN It can be T-D064, INT-D065, INT-D066, INT-D067, INT-D053, INT-D068, INT-D069, INT-D070, INT-D071, INT-D072, INT-D073, INT-D074, INT-D075, INT-D076, INT-D077, INT-D078, INT-D079, INT-D080, INT-D081 or INT-D082.

[0056] Further, Formula I: M-X1-[L]-X2-R Further provided are prodrugs having the structure: During the ceremony, M is a drug moiety, D, derived from an anticancer drug or an immunomodulatory drug; X1 is a linker containing one or more biodegradable groups that covalently attaches D to any carbon atom on L; L is a scaffold carbon chain derived from a hydroxy fatty acid having 16 to 20 carbon atoms and optionally having one or more cis or trans C=C double bonds; X2 is a chemical linkage covalently linking S to any carbon atom of L; R is a linear or branched hydrocarbon having 1 to 25 carbon atoms and optionally having one or more cis or trans C=C double bonds, derived from an acyl chain; R gives a predefined LogP value to the prodrug.

[0057] Additionally, compounds of formula III: RG-[L]-X2-R A precursor scaffold molecule P having the formula: RG is a reactive functional group containing at least one reactive atom selected from O, C, N, P, S, Si, or B; L is a scaffolding carbon chain having 5 to 40 carbon atoms and optionally having one or more cis or trans C=C double bonds; X2 is a chemical linkage covalently attaching R to any carbon atom of L; R is a hydrocarbon having 1 to 40 carbon atoms and may have one or more cis or trans C=C double bonds.

[0058] Further, the compound of formula IIIa:

[0059] [ka]

[0060] The precursor molecule P of During the ceremony, L1-L2 is a scaffold carbon chain L having 5 to 40 carbon atoms; L1 is a carbon chain having 5 to 30 carbon atoms and optionally having one or more cis or trans C=C double bonds; L2 is a carbon chain consisting of L minus the carbon atom of L1, which may contain one or more cis or trans C=C double bonds; X2 covalently bonds R to L2 at any carbon atom on L2.

[0061] RG may be a hydroxyl group, an amine or a carboxyl group, and in one embodiment, X2 is an ester group. R may be derived from an acyl chain. In one embodiment, the first and second linkages formed thereby are ester linkages.

[0062] According to any of the above embodiments, the scaffold lipid is derived from a hydroxy lipid.

[0063] Further provided is a method for preparing a prodrug, comprising providing a precursor molecule defined in any of the above embodiments; and attaching the precursor molecule to a drug D, a linker, or a drug-linker to generate the prodrug.

[0064] Further provided are prodrugs generated from the precursor molecules described above.

[0065] Further provided is a method for treating cancer, an autoimmune disease, or an infection comprising administering a prodrug of any one of the above embodiments.

[0066] Further provided is a pharmaceutical composition comprising any one of the lipid conjugates of the above embodiments. In additional embodiments, provided is a nanoparticle comprising a prodrug of one of the above embodiments. In another embodiment, the nanoparticle is a liposome. [Brief description of the drawings]

[0067] [Figure 1A]1 shows various prodrugs that can be prepared according to certain embodiments based on the scaffold molecule L. [Figure 1B] 1 shows various prodrugs that can be prepared according to certain embodiments based on the scaffold molecule L. [Figure 1C] 1 shows various prodrugs that can be prepared according to certain embodiments based on the scaffold molecule L. [Figure 2A] 1 shows various prodrugs that can be prepared according to certain embodiments based on a ricinoleyl lipid scaffold. [Figure 2B] 1 shows various prodrugs that can be prepared according to certain embodiments based on a ricinoleyl lipid scaffold. [Figure 2C] 1 shows various prodrugs that can be prepared according to certain embodiments based on a ricinoleyl lipid scaffold. [Figure 2D] 1 shows various prodrugs that can be prepared according to certain embodiments based on a ricinoleyl lipid scaffold. [Figure 3A] 1 shows the chemical structures of various prodrugs that contain a ricinoleyl lipid scaffold. [Figure 3B] 1 shows the chemical structures of various prodrugs that contain a ricinoleyl lipid scaffold. [Figure 4] Electron micrographs of prodrugs including dexamethasone linked to ricinoleyl+hexanoyl (INT-D034) at various molar percentages (10, 20, 30, 40 and 80 mol%) in lipid nanoparticle (LNP) formulations are shown. [Diagram 5] Electron micrographs of prodrugs containing dexamethasone linked to ricinoleyl + hexanoyl (INT-D034; left panel) and dexamethasone linked to ricinoleyl + oleoyl (INT-D035; right panel) in LNP formulations at 10 mol% prodrug concentration are shown. [Figure 6A]Figure 1 shows the dissociation over time of various ricinoleyl-dexamethasone prodrugs (INT-D034, INT-D035, INT-D045, INT-D046, INT-DQ47, INT-D048, INT-D049, IIMT-D050, INT-0051, INT-D085, INT-D086, and INT-D089) formulated at 10 mol% in LNPs after incubation in human plasma. The amount of prodrug remaining in each LNP formulation was measured at 0 hours (left bar) and 2 hours (right bar) after incubation. [Figure 6B] Dissociation over time of various ricinoleyl-dexamethasone (INT-D034, IIMT-D045) or ricinoleyl-calcitriol (INT-D053, INT-D083) prodrugs formulated at 10-99 mol% in LNPs after incubation in human plasma. The amount of prodrug remaining in each LNP formulation was measured at 0 hours (left bar) and 2 hours (right bar) after incubation. [Figure 6C] Figure 1 shows the dissociation over time of various ricinoleyl-dexamethasone (INT-D034, INT-D045) or ricinoleyl-calcitriol (INT-D053, INT-D083) prodrugs formulated at 99 mol% in LNPs after incubation in human plasma. The amount of prodrug remaining in each LNP formulation was measured at 0 hours (left bar), 2 hours (middle bar), and 24 hours (right bar) after incubation. [Figure 7A]FIG. 1 shows the degradation over time of various ricinoleyl-dexamethasone prodrugs (INT-D034, INT-D035, 1NT-D045, INT-D046, INT-D047, INT-D048, INT-0049, D050, D050, D051, D085, and D089) formulated at 10 mol% in LNPs after incubation in mouse plasma. The relative amount of intact prodrug in each LNP was measured by ultra-pressure liquid chromatography (UPLC) at 0 hours (left bar) and 2 hours (right bar) after incubation. Data were normalized to the content of each conjugate in the mixture before incubation. Error bars represent three separate experiments. [Figure 7B] FIG. 1 is a graph showing the amount of free dexamethasone released over time after incubation of various LNP-formulated ricinoleyl-dexamethasone prodrugs (INT-D034, INT-D035, INT-D045, INT-D046, INT-D047, INT-D048, INT-DQ49, INT-DO50, and INT-D051) in mouse plasma. Prodrugs were formulated into LNPs at 10 mol% and free drug was measured after 2 hours of incubation and reported as area under the curve (AU). Error bars represent three separate experiments. [Figure 7C] Figure 1 shows the degradation over time of various ricinoleyl-dexamethasone (INT-D034, INT-D045) or lidnoreyl-calcitriol (INT-D053, INT-D083) prodrugs formulated at 10-99 mol% in LNPs after incubation in mouse plasma. The relative amount of intact prodrug in each LNP was measured by ultra-pressure liquid chromatography (UPLC) at 0 hours (left bar) and 2 hours (right bar) after incubation. Data were normalized to the content of each conjugate in the mixture before incubation. Error bars represent three separate experiments. [Figure 8]Proinflammatory cytokine levels in cultured macrophage cell line J774.2 incubated with prodrugs INT-D034 and INT-D035 (D034 and D035), free dexamethasone (Dex-21-P), no prodrug (control) and untreated LNP formulations. Graphs show expression of cytokines IL-1β (top), TNFα (middle) and IL-6 (bottom) after 24 h incubation after cells were incubated with the above components at doses 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 data were normalized to cells treated with control LNPs without drug-lipid conjugates. [Figure 9A] Proinflammatory cytokine levels in Raw264.7 cells incubated with LNP formulations of prodrugs INT-D034 and INT-D035 (D034 and D035), free dexamethasone (Dex-21-P), LNPs without prodrug (control) and untreated. Graphs show expression of cytokines IL-1β (top), TNFα (middle) and IL-6 (bottom) after 24 h incubation after cells were incubated with the above components at doses equivalent to 1, 3 or 10 μM dexamethasone followed by overnight stimulation with 10 ng / mL LPS. Cytokine levels were measured by qRT-PCR and data were normalized to cells treated with control LNPs without drug-lipid conjugate. [Figure 9B]Figure 1 shows proinflammatory cytokine levels in Raw264.7 cells incubated with LNP formulations of 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), LNPs without prodrug (control) and untreated. The graph shows the expression of the cytokine IL-1β after 24 h incubation after incubating cells with the above components at doses equivalent to 1 or 10 μM dexamethasone followed by overnight stimulation with 10 ng / mL LPS. Cytokine levels were measured by qRT-PCR and data were normalized to cells treated with control LNPs without drug-lipid conjugate. [Figure 10] Figure 10 shows the percentage proliferation of CD4+ T cells in different LNP formulations of prodrugs of dexamethasone (INT-D034 and INT-D045) and calcitriol (INT-D053 and INT-D083) at various mol% from 10% to 99% as shown in a mixed lymphocyte reaction (MLR) assay. Bone marrow-derived dendritic cells (BMDCs) from C57Bl / 6 male mice were first treated with LNPs containing various mol% of dexamethasone or calcitriol conjugates for 48 h and then activated by incubating with LPS for 24 h. They were then harvested and mixed with CD4+ T cells isolated from Balb / cJ male mice (Jackson Laboratory) at a T-to-BMDC ratio of 5:1 or 10:1. [Figure 11] Figure 11 shows electron micrographs of LNPs loaded with two different prodrugs derived from different parent drug moieties, namely dexamethasone and calcitriol. The prodrugs included INT-D045 and INT-D053 (left), INT-D045 and INT-D068 (middle), and INT-D045 and INT-083 (right). Each prodrug was formulated in the LNPs at an equimolar concentration of 10 mol%. [Figure 12]Figure 1 shows the dissociation of ricinoleyl-dexamethasone (INT-D045) or ricinoleyl-calcitriol (INT-D053, INT-D068, or INT-D083) conjugates formulated at 10 mol% individually or in combination in LNPs. The remaining amount of lipid-drug conjugate in each LNP formulation was measured over time at 0 hours (left bar), 2 hours (middle bar), and 24 hours (right bar) after incubation in human plasma. The top graph shows the levels of dexamethasone conjugate in single or combined formulations. The bottom graph shows the levels of calcitriol conjugate in single or combined formulations. Data was normalized to the content of each conjugate in the mixture before incubation. [Figure 13] Graph showing dissolution of 10 mol% ricinoleyl-dexamethasone (INT-D045) or ricinoleyl-calcitriol (INT-D053, INT-D068 or INT-D083) conjugates formulated individually or in combination in LNPs. The relative amount of intact prodrug in each LNP was measured by UPLC at 0 hours (left bar), 2 hours (middle bar) and 24 hours (right bar) after incubation in mouse plasma. The top graph shows the levels of dexamethasone conjugate in single or combined formulations. The bottom graph shows the levels of calcitriol conjugate in single or combined formulations. Data was normalized to the content of each conjugate in the mixture before incubation. Error bars represent three separate experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Lipid conjugates of formula I The lipid conjugates described herein may be prodrugs, and in certain embodiments refer to compounds that can become active after administration to a subject. However, other molecules of interest M than drug moieties can be linked to the lipid moiety, such as polymers as described herein. Regardless of the molecule of interest, the lipid conjugates include a carbon chain scaffold L that is typically linear, although branched structures are also encompassed in the compositions described herein. The molecule of interest M is linked to L through chemical linkage X1, which in some embodiments may include a direct linkage or a linker. The R hydrocarbon is linked to L through chemical linkage X2. Optionally, a second R hydrocarbon is linked to L through X2 chemical linkage. Additionally, a third R hydrocarbon is optionally linked to L through a chemical linkage as described below.

[0069] In one embodiment, the lipid conjugate has Formula I, shown below: M-X1-[L]-X2-R It has the structure: During the ceremony, M is a molecule of interest, including a drug or a polymer; X1 is a chemical linkage or linkage connecting M to any carbon atom on L, including a covalent or ionic bond, or a hydrogen bond or bond; L is a scaffolding carbon chain having 5 to 40 carbon atoms, optionally containing one or more cis or trans C=C double bonds; X2 is a chemical linkage covalently attaching R to any carbon atom on L; R is a hydrocarbon having 1 to 40 carbon atoms, optionally having one or more cis or trans C=C double bonds; Optionally, a second R hydrocarbon having from 1 to 40 carbon atoms and optionally having one or more cis or trans C=C double bonds may be chemically bonded to L via an X2 chemical linkage. Additionally, optionally, a third R hydrocarbon having from 1 to 40 carbon atoms and optionally having one or more cis or trans C=C double bonds may be chemically bonded to L via an X2 chemical linkage.

[0070] Optionally, the side chain R' may be linked to any one of the hydrocarbons R through an X2 chemical linkage. Without limitation, a second R' side chain may be linked to the R hydrocarbon through an X2 linkage, and a third R' may be linked to any one of the hydrocarbons R through an X2 chemical linkage. Various other combinations can be readily envisioned by one of skill in the art. The X2 chemical linkage may include any suitable functional group and / or linker described below, as well as others known to those of skill in the art.

[0071] In further embodiments, R, and / or any additional R or R' groups are independently a hydrocarbon chain having 1 to 40 carbon atoms, 2 to 30 carbon atoms, or 5 to 25 carbon atoms. Similarly, the L scaffold (described below) may have 1 to 40 carbon atoms, 2 to 30 carbon atoms, or 5 to 25 carbon atoms.

[0072] The diagram in Figure 1 is provided to pictorially illustrate a variety of different lipid conjugates of formula I, Ia, II and IIa that can be made in alternative embodiments using the techniques of the invention described herein. As shown in the figure, the molecule of interest M or a molecular-linker, R hydrocarbon and an optional second R hydrocarbon, or an optional additional third R hydrocarbon, can occupy various positions on the scaffold L to provide a custom-made prodrug. As further described (and as described above), one or more of the hydrocarbons R linked to the scaffold L can be A carbon-based side chain may be further bonded to the alkyl group.

[0073] 1 utilizes a linker X1 (also referred to in the art as a "spacer") to chemically link the molecule of interest to the scaffold molecule L, although optionally, the molecule of interest M can be directly attached to L via the X1 functional group. Additionally, the chemical linkage X1 can include any combination of a linker and one or more functional groups as further described below.

[0074] In particular, structure A in Figure 1 shows a scaffold molecule L, in this non-limiting example having 5-30 carbon atoms, whose terminal carbon atom is linked to a molecule of interest M via a linker, X1 chemical linkage. A hydrocarbon R is linked to an interior carbon of the scaffold carbon chain L via an X2 linkage.

[0075] Structure B in Figure 1 shows a scaffold molecule L having 5-30 carbon atoms, in which a terminal carbon atom is linked to a hydrocarbon R via an X2 chemical linkage (rather than to a molecule of interest M and a linker). The molecule of interest M is linked to an interior carbon of the scaffold via an X1 chemical linkage, which is a linker.

[0076] Similar to structure A, the structure shown in structure C of Figure 1 depicts a scaffold molecule L in which a terminal carbon atom is linked to a molecule of interest M via a linker X1 and a hydrocarbon R is linked to an interior carbon of the scaffold via an X2 linkage. However, in this embodiment, a second hydrocarbon R is linked to another interior carbon of the scaffold via an X2 linkage.

[0077] Structure D shows a scaffold molecule L in which a molecule of interest M is linked to an interior carbon of the scaffold via a linker, the X1 chemical linkage. A hydrocarbon R is linked to an interior carbon of the scaffold via an X2 linkage. A second hydrocarbon R' is linked to a terminal carbon atom of scaffold L via an X3 chemical linkage.

[0078] Structure E in FIG. 1 shows a scaffold molecule L with a molecule of interest M linked to an interior carbon of the scaffold via a linker, X1 chemical linkage. A hydrocarbon R is linked to an interior carbon of the scaffold via an X2 linkage. A second hydrocarbon R is linked to a terminal carbon atom of scaffold L via an X2 chemical linkage. Structure E differs from structure D above in that the molecule of interest M is linked to a carbon atom on scaffold L closer to the terminal carbon than the second R hydrocarbon is linked to.

[0079] In another example, structure F in FIG. 1 shows a scaffold molecule L in which a molecule of interest M is linked to an internal carbon of the scaffold via a linker, X1 chemical linkage. A hydrocarbon R is linked to an internal carbon of the scaffold via an X2 linkage. A second hydrocarbon R is linked to a terminal carbon atom of the scaffold L via an X2 chemical linkage. Structure F differs from structure E above in that a third hydrocarbon R is attached to the scaffold L via an X2 chemical linkage. It can be easily imagined that other combinations include a drug-linker moiety at C1 and three hydrocarbon moieties linked to internal carbons of L via their respective X2 chemical linkages.

[0080] In yet another example, shown in structure G of Figure 1, the R hydrocarbon is linked to an R' hydrocarbon side chain linked via X2. The terminal carbon atom is linked to a molecule of interest M via a linker, X1 chemical linkage. The R hydrocarbon is linked to an internal carbon of a scaffold carbon chain L via an X2 linkage.

[0081] The above structures A-G are examples, and other permutations and embodiments that fall within the scope of the disclosure can be readily envisioned by those of skill in the art.

[0082] In some embodiments, the point on the scaffold L to which the R group is attached may be at least 3 carbon atoms from the terminal carbon on L (counting from the first carbon of L, designated C1). To depict such branching points in the prodrug formula (Formula I above), the scaffold molecule L may be referred to using the notation "L1-L2". According to such an embodiment, L1 is at least 3 carbon atoms and S is linked to a carbon atom of L2. In one particularly advantageous embodiment, L1 is at least 4 or 5 carbon atoms.

[0083] In these embodiments where the R group is linked to L at a position that is at least 3 carbon atoms from C1, formula I may be represented by the following formula Ia:

[0084] [ka]

[0085] It can take the form: where M is a molecule of interest; X1 is a chemical linkage that connects or links M to any carbon atom on L1-L2 via any suitable chemical linkage described herein; L1 is at least 3 carbon atoms; L1-L2 is 5-40 carbon atoms, and L2=L-L1. Chemical linkage X1 connects the molecule of interest M to any carbon atom on L1-L2, and chemical linkage X2 connects R to any carbon atom on L2. R is a hydrocarbon having 1-40 carbon atoms.

[0086] In one embodiment, L1 is 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 further embodiments, L1 can be 3-30 carbon atoms, 4-30 carbon atoms, 5-25 carbon atoms, or 6-25 carbon atoms, or 7-20 carbon atoms. Optionally, L1 has one or more cis or trans C=C double bonds. In another embodiment, L1 is a linear carbon chain.

[0087] L2 is typically a linear carbon chain, although branched structures are contemplated as well. As noted above, L2=L-L1. By way of example, in an embodiment where L is 20 carbon atoms and L1 is 11 carbon atoms, L2 is 9 carbon atoms.

[0088] In an alternative embodiment, the lipid conjugate has Formula II, as set forth below:

[0089] [ka]

[0090] It has a lipid portion having the following structure: wherein the L lipid scaffold backbone is represented by L1+L2+L3+L4+L5+L6, where L comprises 5-40 carbon atoms, or 5-30 carbon atoms, or 5-25 carbon atoms, or 5-20 carbon atoms and 0-2 cis or trans C=C double bonds; 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, optionally L1 having one or more cis or trans C=C double bonds, or 0-2 cis or trans C=C double bonds; L2 and L4 are each a carbon atom; L3 is 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L5 is 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L6 is -CH 3 , =CH 2 or H; 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, and according to one alternative embodiment, each R is independently branched at each branch point that includes a heteroatom-containing X2 functional group; In the formula, n is 0-8, p is 0-8, and n+p is ≧1 or 1-8, or n is 0-6, p is 0-6, and n+p is ≧1 or 1-6, or n is 0-4, p is 0-4, and n+p is ≧1 or 1-4; X1 and X2 are independently selected from the group consisting of esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thionocarbamate, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, phosphoramidates, phosphates, phosphonates, phosphodiesters, phosphonooxymethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azos, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH 2) or urea; or X1 contains one or more hydrogen bonds and has a structure of formula V, as defined below:

[0091] In one embodiment, at least one of X1 and X2 is biodegradable.

[0092] In one embodiment, X1 and / or X2 are independently selected from ester, ether or carbamate. Ester or carbamate can be in any orientation. For example, ester can be linked to the molecule of interest (M) through its carbonyl group or through its -O group. Similarly, carbamic acid can be linked to the molecule of interest (M) through its nitrogen atom or through its -O group.

[0093] In one embodiment, the lipid moiety of formula II as a whole has fewer than 300, fewer than 200, fewer than 150, fewer than 100 carbon atoms, fewer than 75 carbon atoms, or fewer than 50 carbon atoms (L+R).

[0094] Each of the R hydrocarbon chains of the lipid moiety can be substituted with a heteroatom at one of the internal carbon atoms of the chain, 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.

[0095] In another embodiment, the lipid conjugate is not an ionizable lipid.

[0096] In an alternative embodiment, the lipid conjugate has Formula IIa, shown below:

[0097] [ka]

[0098] It has a lipid portion having the following structure: In the formula, L is [CH 2 ] m -L2-L3-L4-[CH 2 ] q -CH3 It is represented by L The total number of carbon atoms is 5 to 30; L2 and L4 are carbon atoms; m is 0-20, n is 1-4, p is 0-4, n+p is 1-4; L3 is 0-10 carbon atoms and has 0-2 cis or trans C=C; X1 and X2 are independently selected from ether, ester, and carbamate groups; Each R is independent, (a) a linear or branched terminal hydrocarbon chain having 0 to 5 cis or trans C=C and 1 to 30 carbon atoms, in which each R is linked to one of the respective X2 at any carbon atom of the hydrocarbon chain; or (b) Formula IIb having a scaffold designated L':

[0099] [ka]

[0100] is a branched structure of; In the formula, L' is [CH 2 ] r -L2-G 3 -L4-[CH 2 ] u -CH 3 , and the total number of carbon atoms in L is 3 to 30; r is 0-20, 2-20, 3-20, or 4-20; s is 0-4, t is 0-4;s+t is >1 or 1-4; u is 1 to 20; G 3 has 0-10 carbon atoms and 0-2 cis or trans C=C; Each R' of formula IIb is a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms; The total number of R′ hydrocarbon chains in formula IIb is 1 to 16; each of the R and R' hydrocarbon chains in the lipid moiety may be optionally substituted with heteroatoms, provided that not 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; The lipid conjugate is not an ionizable lipid.

[0101] Non-limiting examples of prodrug lipid conjugates having the structures of Formula I, Formula Ia, Formula II, and Formula IIa are shown in Table 1 below, and their chemical structures are shown in Figure 3. In such embodiments, the lipid conjugates are derived from dexamethasone and are linked using a succinic acid linker (X1 chemical linkage). However, as discussed further herein, a wide range of drugs or other molecules of interest and linkers can be incorporated into the Lipid conjugates. Table 1: Non-limiting examples of prodrugs

[0102] [Table 1]

[0103] Scaffold L of formula I, Ia, II or IIa In one embodiment, L of formula I, Ia, II, or IIa is derived from a fatty acid having a functional group on its carbon chain for attachment to R.

[0104] For example, L in formula I, Ia, II or IIa can be derived from a hydroxy fatty acid (HFA), which is a fatty acid with an OH group attached at any position on its carbon chain. Without limitation, 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 is saturated or unsaturated. Two or more hydroxy functional groups can also be present on the carbon chain.

[0105] Non-limiting examples of HFAs from which fatty alcohols can be derived are shown in Table 2 below: Table 2: Examples of hydroxy fatty acids (HFAs) and corresponding fatty alcohols

[0106] [Table 2]

[0107] Examples of HFAs with two or more hydroxy functional groups present on the carbon chain include 9,10-dihydroxyoctadecanoic acid and ustylic acid (also known as 2,15,16-trihydroxypalmitic acid or 2,15,16-trihydroxy-hexadecanoic acid).

[0108] Alternatively, L in formula I, Ia, II or IIa is derived from a branched fatty acid ester of HFA, known as a fatty acid ester of hydroxy fatty acid (FAHFA). These fatty acid esters contain a branched ester bond between the fatty acid and the HFA. For example, 9-[(9Z)-octadecenoyloxy]octadecanoic acid is a fatty acid ester obtained by condensation of the carboxy group of oleic acid with the hydroxy group of 9-hydroxyoctadecanoic acid.

[0109] In an alternative embodiment, L is derived from a fatty acid amide, which may include ethanolamine as the amine component.

[0110] L in formula I, Ia, II or IIa may be derived from other fatty acids than those mentioned above. Furthermore, it will be appreciated that the fatty acids may be derived from their corresponding triglycerides.

[0111] L of formula I, Ia, II or IIa may contain an OH group introduced via oxidation of a double bond on the lipid carbon backbone. Thus, the precursor of L is unsaturated and introduces a reactive OH group. The fatty acid may be derived from any fatty acid, fatty alcohol or fatty amide precursor that is oxidized to obtain the desired fatty acid.

[0112] The lipid portion of the lipid conjugate, such as prodrug or lipid-polymer conjugate, can be compatible with lipid for incorporation into drug delivery vehicle.For example, this can include compatibility with vesicle-forming lipid, such as phospholipid, which forms part of lipid nanoparticle, such as liposome.The lipid portion can also be compatible with other drug delivery vehicles, such as polymer-based nanoparticles, emulsions, micelles and nanotubes.

[0113] In one embodiment, L can 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.

[0114] Lipid-based precursor P In an alternative embodiment, the lipid moiety of the lipid conjugate of formula I above has the following formula III: RG-[L]-X2-R The precursor may be derived from a precursor referred to herein as "P" defined by: where 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 a hydroxyl group, an amine, or a carboxyl group. In another embodiment, the reactive functional group is a hydroxyl group or a carboxyl group. In another embodiment, the RG functional group forms a biodegradable chemical linkage with a linker on a molecule of interest M, or directly with such a molecule. L is a scaffold carbon chain having 5-40 carbon atoms, optionally containing one or more cis or trans C=C double bonds; X2 is a chemical linkage covalently attaching R to any carbon atom on L; R is a hydrocarbon having 1 to 40 carbon atoms, optionally having one or more cis or trans C=C double bonds.

[0115] In another embodiment, the lipid moiety of formula Ia has formula IIIa:

[0116] [ka]

[0117] It can be derived from a precursor P having the structure: where 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 a hydroxyl group, an amine, or a carboxyl group. In another embodiment, the reactive functional group is a hydroxyl group, or a carboxyl group. In another embodiment, the RG functional group forms a biodegradable chemical linkage with a drug or a linker on the drug. In another alternative embodiment, the RG functional group is a hydrogen bond donor or acceptor. L1 is at least 3 carbon atoms, L1-L2 is 5-40 carbon atoms, and L2=L-L1. The chemical linkage X2 connects R to any carbon atom of L2. R is a hydrocarbon having 1-40 carbon atoms.

[0118] In one non-limiting embodiment, RG in formula III or IIIa is a hydroxyl group. RG can be bonded to a corresponding reactive group on a drug or linker, such as a carboxyl group. The bond formed in such a reaction (X1 in formula I or Ia) is selected from an ester bond or an amide bond, although other bonds can also be formed.

[0119] The carbon backbone of L in formula III or L1-L2 in formula IIIa may also contain an additional reactive group RG for the attachment of a second hydrocarbon R group. Additionally, a third hydrocarbon group R may 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 one non-limiting example, each RG is independently selected from a hydroxyl group, an amine group, or a carboxylic acid group, as well as other suitable groups known to those skilled in the art.

[0120] Additionally, more than one hydrocarbon moiety, R of formula III and IIIa, may be attached to a respective R' side chain. For example, an R' side chain may be linked to an R via an X2 linkage, a second R' side chain may be linked to another R via an X2 linkage, and / or a third R' may be linked to any R via an X2 linkage, as described above in relation to formulas I, Ia, II and IIa. However, various other combinations can be readily envisioned by one of skill in the art.

[0121] In another embodiment, the lipid moiety of formula II has formula IIIb:

[0122] [ka]

[0123] It can be derived from a precursor P having the structure: where RG is a reactive functional group comprising one or more reactive atoms selected from O, C, N, P, Si or B. In one embodiment, the reactive functional group is selected from a hydroxyl group, an amine or a carboxyl group. In another embodiment, the reactive functional group is a hydroxyl group or a carboxyl group. In another embodiment, the RG functional group forms a biodegradable chemical linkage with a drug or a linker on the drug. In a further embodiment, the RG functional group is a hydrogen bond donor or acceptor or atom for forming a hydrogen bond with a respective hydrogen bond acceptor or donor on the molecule of interest M; The L lipid scaffold backbone is represented by L1+L2+L3+L4+L5+L6, where L contains 5-40 carbon atoms, or 5-30 carbon atoms, or 5-25 carbon atoms, or 5-20 carbon atoms and 0-2 cis or trans C=C double bonds; 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, optionally L1 having one or more cis or trans C=C double bonds, or 0-2 cis or trans C=C double bonds; L2 and L4 are each carbon atoms; L3 is 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L5 is 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L6 is -CH 3 , =CH 2 or H; 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, and according to one alternative embodiment, each R is independently branched at each branch point containing a X2 functional group containing a heteroatom; n is 0 to 8, p is 0 to 8, and n+p is ≧1 or 1 to 8, or n is 0 to 6, p is 0 to 6, and n+p is ≧1 or 1 to 6, or n is 0 ~4, p is 0-4, and n+p is ≧1 or 1-4; X1 and X2 are independently selected from the group consisting of esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thionocarbamate, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, phosphoramidates, phosphates, phosphonates, phosphodiesters, phosphonooxymethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azos, carbon-based functional groups including alkanes, alkenes, or alkynes, methylene (CH 2 ) or urea; or X1 contains one or more hydrogen bonds and has a structure of formula V, as defined below:

[0124] In another embodiment, the lipid moiety of formula IIa has formula IIIc:

[0125] [ka]

[0126] It can be derived from a precursor P having the structure: where RG is a reactive functional group comprising one or more reactive atoms selected from O, C, N, P, Si, or B. In one embodiment, the reactive functional group is selected from a hydroxyl group, an amine, or a carboxyl group. In another embodiment, the reactive functional group is a hydroxyl group or a carboxyl group. In another embodiment, the RG functional group forms a biodegradable chemical linkage with a linker on a molecule of interest, such as a drug; L is [CH 2 ] m -L2-L3-L4-[CH 2 ] q -CH 3 The carbon atom of L is represented by The total number of offspring is between 5 and 30; L2 and L4 are carbon atoms; m is 0 to 20, n is 1 to 4, p is 0 to 4, and n+p is 1 to 4. L3 is 0-10 carbon atoms and has 0-2 cis or trans C=C; X1 and X2 are independently selected from ether, ester, and carbamate groups; Each R is independent, (a) a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms, each R being linked to one of the respective X2 at any carbon atom of the hydrocarbon chain; or (b) Formula IIb having a skeleton represented by L':

[0127] [ka]

[0128] It is a branched structure of L' is [CH 2 ] r -L2-G 3 -L4-[CH 2 ] u -CH 3 and the total number of carbon atoms in L is 3 to 30; r is 0-20, 2-20, 3-20, or 4-20; s is 0-4, t is 0-4; s+t is >1 or 1-4; u is 1 to 20; G 3 has 0-10 carbon atoms and 0-2 cis or trans C=C; Each R' of formula IIb is independently a linear or branched terminal hydrocarbon chain having 0 to 5 cis or trans C=C and 1 to 30 carbon atoms; The total number of R′ hydrocarbon chains in formula IIb is 1 to 16; Each of the R and R' hydrocarbon chains in the lipid moiety may be optionally substituted with heteroatoms, provided that no more than 8 heteroatoms are substituted in the R and R' hydrocarbon chains and the predicted or experimental logP of the conjugate is greater than 5; The lipid conjugate is not an ionizable lipid.

[0129] Molecules of interest A variety of molecules of interest can be attached to the lipid moiety. As mentioned above, the lipid conjugate can be a prodrug. The drug moiety of the prodrug conjugate can be derived from any class of drug, including any drug that is used, for example, after its activation, to treat, prevent, improve, alleviate symptoms, and / or diagnose a disease or other undesirable condition in a subject. The drug moiety can be an active agent, or an active agent that is activated after being released from the conjugate. However, other molecules of interest, including hydrophilic polymers, can also be attached to the lipid moiety.

[0130] The molecule of interest M can be characterized in some embodiments by the nature of the bond or association with the lipid moiety. For example, the drug moiety D in certain embodiments can be derived from a drug that loses one or more atoms upon binding to a reactive group or linker group on the scaffold L to form the chemical linkage X1. In one embodiment, the drug loses a hydroxyl group or a hydrogen atom upon binding to P or a linker to form a prodrug of formula I, Ia, IIa or IIb. However, the method of the invention described herein is applicable to the bond or association of a wide range of drugs to the lipid moiety, so the drug moiety D can be derived from any known drug. The drug D can be a small molecule or a polymeric structure. The moiety M (molecule of interest) can be, without limitation, the orientation of the atoms, -(C=O)O, -OH, -NH, among others known to those skilled in the art. 2 ,-NHR,-PO 3 H 2 It can be derived from a chemical structure that contains one or more reactive functional groups such as,.

[0131] For example, a prodrug or other lipid conjugate described herein, when RG is -OH, can be formed (directly or through one or more intermediates) by a bond between a (C=O)OH group on a molecule of interest and a hydroxyl group on the precursor scaffold P. The general reaction is shown below:

[0132] [ka]

[0133] In the above exemplary embodiment, the X1 chemical linkage of formula I, Ia, II or IIa is an ester and has the following structure:

[0134] [ka]

[0135] In another illustrative example, the molecule of interest M may have a hydroxyl group (-OH) that reacts with a carboxyl group ((C=O)OH) in the linker. A second carboxyl group ((C=O)OH) on the linker can react with a hydroxyl group on a carbon atom on the precursor scaffold P via a condensation reaction. The following reaction shows the use of succinic acid as a linker. The use of such a linker results in a prodrug with two ester groups according to the following reaction:

[0136] [ka]

[0137] In the above non-limiting example, the X1 chemical linkage has the following structure:

[0138] [ka]

[0139] It should be recognized that the above reaction can proceed in two steps: the drug may first be attached to the linker, and the resulting drug-linker conjugate may then be reacted with the precursor scaffold P to generate the prodrug reaction product.

[0140] As discussed below, the above is provided for illustrative purposes only, since a variety of different linkers other than succinic acid can be used to prepare prodrugs. In another example, the molecule of interest M or the linker may have a carboxyl group ((C=O)O) for coupling with an amine group of L to form an amide or amide-containing linkage X1 between the drug moiety and L. As discussed below, other reactions between functional groups on the drug or between the scaffold L and the linker to generate the X1 chemical linkage can be envisioned by one of skill in the art.

[0141] A particular molecule of interest may contain multiple reactive functional groups for attachment to the precursor scaffold P. In such an embodiment, a protecting group may be used during the synthesis of the drug-lipid conjugate, which will be recognized by one of skill in the art to selectively attach certain groups on the drug to the scaffold L while leaving other groups unattached. The drug may also be characterized by its biological effect, including its ability to treat, prevent and / or ameliorate a condition in a subject or cell in vitro. The drug moiety may be derived from an anti-cancer drug, such as an anti-tumor drug. In another embodiment, the drug moiety may be derived from an immunomodulatory drug, such as an immunosuppressant drug for treating an autoimmune disease, such as Crohn's disease, rheumatoid arthritis, psoriasis, ulcerative colitis or diabetes. In one embodiment, the immunomodulatory drug is an anti-inflammatory drug.

[0142] As used herein, drugs that function as anti-cancer agents may have a direct or indirect effect on the growth, proliferation, invasiveness and / or survival of neoplastic cells and / or tumors. Anti-tumor drugs include alkylating agents, antimetabolites, cytotoxic antibiotics, various plant alkaloids, and the like. These include amides and their derivatives, and immunomodulatory agents.

[0143] Examples of immunosuppressant classes include glucocorticoids, cytostatic drugs, antibodies, and drugs acting on immunophilins, among others known to those skilled in the art. Examples of glucocorticoids include prednisone, prednisolone, and dexamethasone. Methotrexate is an example of a cytostatic drug.

[0144] In one embodiment, the drug moiety is selected from the group consisting of docetaxel, dexamethasone, methotrexate, NPC1I, abiraterone, prednisone, prednisolone, ruxolitinib, tofacitinib, calcitriol, calcifediol, cholecalciferol, sirolimus, tacrolimus, acetylsalicylic acid, mycophenolic acid, cabazitaxel, betamethasone, and CY09 (4-[4-oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl)benzoic acid), INT - NLRP3 inhibitors, including MA014 or MCC950 (N-(1,2,3,5,6,7-hexahydro-s-indacen-4-ylcarbamoyl)-4-(2-hydroxy-2-propanyl)-2-furansulfonamide) and derivatives thereof; and cannabinoids, including cannabigerol, cannabichromene, cannabidiol, cannabidivarin, cannabicyclol, cannabicitran, cannabiersoin, cannabinol, tetrahydrocannabinol or tetrahydrocannabivarin and derivatives thereof.

[0145] In further embodiments, the drug has a free hydroxyl group for attachment to a linker or group on any carbon of L. However, other functional groups on the drug can also be used for such attachment.

[0146] Other molecules of interest M, other than drugs, can be linked to the lipid portion of the scaffold L through X1 using reactive groups similar to those described above. This includes small molecules and molecules that form macromolecular structures. For example, in some embodiments, the molecule of interest M is a polymer for forming lipid-polymer conjugates. The polymer may be a hydrophilic polymer suitable for use in biological systems. Examples of hydrophilic polymers include polyalkyl ethers such as polyethylene glycol (PEG), polymethylethylene glycol, polypropylene glycol, and polyhydroxypropylene glycol. Further suitable polymers include polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyvinylmethylether, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide, polydimethylacrylamide, polyhydroxypropylmethacrylate, polyhydroxyethylacrylate, hydroxymethylcellulose, hydroxyethylcellulose, or polyaspartamide. The polymer chains may have a molecular weight between about 300 and 10,000 daltons. The polymer may be a block copolymer in certain non-limiting embodiments.

[0147] In yet another embodiment, the molecule of interest M is an antibody, a peptide, genetic material such as siRNA.

[0148] In one embodiment, the molecule of interest M is genetic material, such as a nucleic acid. Nucleic acids include RNA, including but not limited to small interfering RNA (siRNA), small nuclear RNA (snRNA), microRNA (miRNA), or DNA, such as plasmid DNA or linear DNA. The length of the nucleic acid can vary and can include nucleic acids from 5 to 50,000 nucleotides in length. The 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.

[0149] In one particularly advantageous embodiment, the molecule of interest is siRNA.siRNA is incorporated into the internal cellular apparatus, causing the degradation of mRNA, thereby preventing transcription.Since RNA is easily degraded, by incorporating it into delivery vehicle as described herein, such degradation can be reduced or prevented, thereby facilitating delivery to target site.

[0150] Chemical Linkage X1 In one embodiment, the molecule of interest M is directly attached to the L scaffold carbon chain via the X1 functional group. In such an embodiment, X1 in formula I, Ia, II, or IIa is a carbon-based functional group such as an ester, amide, amidine, hydrazone, disulfide, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene, or alkyne, methylene (CH 2 ) or urea.

[0151] In one embodiment, the X1 group is not a disulfide or a thioether. In another embodiment, X1 does not contain a sulfur atom.

[0152] As mentioned above, the molecule of interest M can be attached to the L scaffold via the linker X1. The inclusion of a linker group in the lipid conjugate is particularly advantageous for molecules that are released from the lipid moiety after administration, such as prodrugs, because its inclusion can facilitate the cleavage of the molecule of interest M from the lipid moiety by enzymes. As described below, the linker molecule can include, but is not limited to, one or more of the functional groups exemplified in Table 3 below, most advantageously such functional groups can be cleaved under in vivo conditions.

[0153] Non-limiting examples of lipid conjugates having an X1 chemical linkage selected from a succinic acid linker, an ester, an amide, a hydrazone, an ether, a carbamate, a carbonate, or a phosphodiester group are shown in Table 3 below. The following chemical linkages are shown as part of Formula I or Formula Ia. As noted above, the linkages are depicted as resulting from a direct linkage between the drug and L (with the exception of succinic acid) for simplicity, however, it will be understood that the groups shown in the table can be incorporated within the linker group. Table 3: Examples of lipid conjugates with various functional groups forming X1 chemical linkages

[0154] [Table 3]

[0155] As mentioned above, in certain advantageous embodiments, the hydroxy group of the precursor scaffold P (RG=OH of formula III, IIIa, IIIb or IIIc) or the hydroxy group of the precursor scaffold P (RG=NH 2 ) reacts with a carboxyl group on the drug to undergo a condensation reaction to form a X1 chemical linkage, which is an ester or amide group, respectively.

[0156] In such embodiments, X1 of the lipid conjugate of formula I, Ia, II or IIa has the following structure:

[0157] [ka]

[0158] Here, X=-O, or -NH.

[0159] In such embodiments, the X1 chemical linkage forms part of the prodrug of formula I as follows:

[0160] [ka]

[0161] In an alternative embodiment, L is derived from the reaction of a carboxyl group of a fatty acid with a hydroxyl or amine group of a linker, or a molecule of interest. In this embodiment, X forms the following chemical linkage between the molecule of interest and P:

[0162] [ka]

[0163] Here, X=-O, or -NH.

[0164] In one particularly advantageous embodiment, X=-O in the above structure. In such an embodiment, X1 is an ester bond.

[0165] In one embodiment, the X1 linkage is biodegradable, meaning that it can be cleaved after administration to a patient. Without limitation, the ester bond can be hydrolyzed by esterase 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 linkages can be utilized for tailor-made drug release based on their release characteristics when exposed to the environment at the disease site. For example, a hydrazone bond disposed between the drug moiety D and the scaffold L can impart pH-sensitive release to the conjugate of formula I, Ia, II or IIa. At neutral pH, the hydrazone is little or does not decompose, but at low pH, the bond can break. Thus, the X1 chemical linkage consisting of or including one or more hydrazone bonds can provide drug release at low pH values ​​that are often present in tumor tissue.

[0166] In one embodiment, X1 may be cleavable by esterases, alkaline phosphatases, amidases, peptidases, or may be cleavable upon exposure to a reducing environment and / or high or low pH.

[0167] As mentioned above, when the lipid conjugate is a prodrug, the X1 chemical linkage in certain embodiments is most advantageously a linker. A wide variety of chemical linkers are known to those skilled in the art and can be used in certain 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 attaching one end of the linker to a molecule of interest M, and a second functional group for attaching the other end of the linker to a carbon atom on L. The two functional groups can be, respectively, ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, Carbon-based functional groups such as sulfonamide, imine, azo, alkane, alkene or alkyne, methylene (CH 2 ) or urea.

[0168] As will be appreciated by those of skill in the art, in some embodiments, when the molecule of interest is a drug D, the linker can enhance the release of drug D through the introduction of a biodegradable group. A linker having one or more ester bonds can be hydrolyzed by esterases after administration to a patient, thereby releasing the drug moiety D from the prodrug conjugate. Similar to linkages resulting from a direct reaction between D and L, a linker introducing a hydrazone bond between the drug moiety D and the scaffold L can impart pH-sensitive release to prodrugs of Formula I or Ia.

[0169] However, it will be understood that the above is merely exemplary. Further 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 aminohexanoic acid, polyglycine, polyamide, polyethylene, and short functionalized polymers having a carbon backbone that is 1-12 carbon atoms in length.

[0170] However, further examples of linkers suitable for use in the prodrugs described herein are provided in the following references: 1. Rautio et al., “The expanding role of prodrugs in contemporary drug design and development” Nature Reviews Drug Discovery 2018, 17, 559. 2. Irby et al., “Lipid-drug conjugate for enhancing drug delivery” Molecular Pharmaceutics 2017, 14, 1325. 3. Sun et al., “Chemotherapy agent-unsaturated fatty acid prodrugs and prodrug-nanoplatforms for cancer chemotherapy” Journal of Controlled Release 2017, 264, 145. 4. Walther et al., “Prodrugs in medicinal chemistry and enzyme prodrug therapies” Advanced Drug Delivery Reviews 2017, 118, 65. 5. Hu et al., “Glyceride-mimetic prodrugs incorporating self-immolative spacers “promote lymphatic transport, avoid first-pass metabolism and enhance oral bioavailability” Angewandte Chemie International Edition 2016, 55, 13700. 6. Blencowe et al., “Self-immolative linkers in polymeric delivery systems” Polymer Chemistry 2011, 2, 773.

[0171] Each of the above references is incorporated herein by reference in its entirety.In a further embodiment, X1 chemical linkage comprises both functional group and separate linker.Various combinations of linker and functional group (such as the combinations in Table 3 above) can be used to obtain the desired lipid conjugate of formula I, Ia, II or IIa.

[0172] In one embodiment, at least the second functional group joining one end of the linker to L1 is an ester linkage or an amide linkage. In another embodiment, the functional group on the linker can be hydrolyzed by an enzyme such as an esterase. In a further embodiment, both functional groups on the linker are ester linkages.

[0173] Although a wide range of known linkers can be utilized in the embodiments described herein, some non-limiting examples of formulas for X1 linkers are provided below.

[0174] In one embodiment, but not limited to, the molecule of interest M-linker X1 (D-X1) portion of formula I, Ia, II or IIa has the following formula IV:

[0175] [ka]

[0176] has. where M is a molecule of interest, X4 and X5 are independently selected from any of the functional groups described above, and M 1 is an optional spacer group linked to the functional groups of X4 and X5, having 0 to 12 carbon atoms or CH 2 ,CH 2 CH 2 , N-alkyl, N-acyl, O or S. X4 and X5 may be the same or different. In one embodiment, either or both of X4 and X5 can be cleaved in vivo. In another embodiment, X4 and / or X5 are ester groups.

[0177] X4, X5 or both functional groups in the above formula IV can independently be from 1 to about 20 repeat units. 1 -X5 units can be 1 to 20 repeat units, or M 1 If does not exist, X4-X5 can also be a repeating unit.

[0178] In another embodiment, X5 of formula IV is an ester group, in which case M-X1 of formula I, Ia, II or IIb is: Formula IVa:

[0179] [ka]

[0180] wherein M is a molecule of interest, X4 is a functional group that covalently bonds M to M1 and is selected from an ester, amide, hydrazone, ether, carbonate, carbamate, or phosphodiester group; and M1 has 0 to 12 carbon atoms or is CH 2 , C.H. 2 CH 2 , N-alkyl, N-acyl, or O.

[0181] In one embodiment, but not limited to, the linker X1 of formula I, Ia, II or IIa has the following structure: Formula IVb:

[0182] [ka]

[0183] wherein Z is selected from O or N and Y is CH 2 , C.H. 2 CH 2 or C=O, T is 0-6 carbon atoms, and W is O or N. In one embodiment, Z is O and Y is CH 2 , C.H. 2 CH 2 Or C=O, T is 0 to 6 carbon atoms and W is O. In another embodiment, the linker X1 is derived from succinic acid.

[0184] In such an embodiment, the linker of formula IVb can be represented by the formulas I, Ia and Ib as follows: Forms part of the lipid conjugate I: Formula I:

[0185] [ka]

[0186] Formula Ia:

[0187] [ka]

[0188] Formula II:

[0189] [ka]

[0190] Formula IIa:

[0191] [ka]

[0192] wherein Z is selected from O or N and Y is CH 2 , C.H. 2 CH 2 or C=O, T is 0-6 carbon atoms, and W is O or N. In one embodiment, Z is O and Y is CH 2 , C.H. 2 CH 2 Or C=O, T is 0 to 6 carbon atoms and W is O. In a further embodiment, the linker X1 is derived from succinic acid.

[0193] In one particularly advantageous embodiment, the X1 linker is a succinic acid group and the prodrugs of formulae I, Ia, II and IIa have the structure shown below: Formula I:

[0194] [ka]

[0195] Formula Ia:

[0196] [ka]

[0197] Formula II:

[0198] [ka]

[0199] Formula IIa:

[0200] [ka]

[0201] Non-limiting examples of X1 linkages other than succinic acid linkers include the following chemical structures:

[0202] [ka]

[0203] where M is a molecule of interest and L is a lipid scaffold. For simplicity, the remainder of the lipid moiety is not shown in the structure, but may include any of the lipid moieties of formulae I, Ia, II and IIb.

[0204] Needless to say, the reaction resulting in the X1 chemical linkage is not limited to reactions resulting from a direct reaction between the respective functional groups present on the molecule of interest, such as a drug, polymer or linker attached thereto, and the corresponding groups on the precursor scaffold P. Typically, such conjugates are produced by a synthetic scheme that is multi-step and proceeds through various intermediates. Furthermore, a precursor L, such as an aliphatic alcohol, can be modified to produce a derivative thereof, which can be reacted with a reactive functional group on the molecule of interest to produce a lipid conjugate, or vice versa. For example, US2002 / 0177609 (herein incorporated by reference) describes a method that includes derivatizing an aliphatic alcohol with suitable binding and leaving groups to form an intermediate, which is then reacted with a drug to form a conjugate compound. In this method, one or more carbonates, carbamates, ethers, phosphates, esters, etc. can be reacted with a reactive functional group on the molecule of interest to produce a lipid conjugate. Many different X1 linkages can be generated, including drugs attached to the scaffold L via guanidine, thionocarbamate, phosphonate, oxime, isourea, amide, phosphoramide, or phosphonamide groups. Similarly, it is possible to introduce reactive groups onto other molecules, other than aliphatic alcohols, that cannot be generated by reacting an existing functional group present on the drug with a fatty acid.

[0205] In a further embodiment, the molecule of interest M is linked to the scaffold L of the lipid moiety through an X1 linkage that includes one or more intermolecular hydrogen bonds. According to such an embodiment, the molecule of interest includes one or more electronegative atoms. The molecule of interest can include at least one hydrogen bond donor, which is a hydrogen atom covalently bonded to a relatively electronegative atom, and L can include at least one hydrogen bond acceptor, which is a relatively electronegative atom bonded to hydrogen by hydrogen bond. L can also include one or more hydrogen bond donors, and the molecule of interest M can include one or more hydrogen bond acceptors.

[0206] The hydrogen bond between L and M of the lipid conjugate can have the structure of formula V: Formula V:

[0207] [ka]

[0208] wherein E1, E2, E3, E4 and E5 are electronegative atoms selected from O, N and P; E1, E2 and E3 are hydrogen bond acceptors, E4 and E5 are hydrogen bond donors, dotted lines indicate hydrogen bonds and solid lines indicate covalent bonds, where L is a lipid scaffold of a lipid moiety according to formula I, Ia, II or IIa, n is 0 or 1, o is 0 or 1, p is 0 or 1, n+o+p≧2, q is 1-10 or 2-10 or 4-10, L is a lipid scaffold of a lipid moiety, and M is a molecule of interest, where E1 and E3 may optionally include a substituent linked thereto, such as alkyl, aryl, alkylene or H.

[0209] An example of a drug-lipid conjugate includes the following X1 hydrogen bond linkage: In this example, doxorubicin contains a hydrogen bond acceptor and a terminal group:

[0210] [ka]

[0211] The lipid moiety having the formula:

[0212] Example of a hydrogen bond X1 linkage in a drug-lipid conjugate:

[0213] [ka]

[0214] Chemical Linkage X2 Similarly, X2 is a chemical linkage that covalently bonds R to any carbon atom on L of formula I, Ia, II or IIa, and may be formed by reaction of a functional group on any carbon of L with a reactive group on R. However, like X1, X2 does not have to be the result of a direct reaction between functional groups on L, but rather can be formed by a multi-step synthetic scheme.

[0215] A variety of X2 functional groups can link R to L or L2. For example, X2 can be a carbon-based functional group such as an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene, or alkyne, methylene (CH 2 ) or urea. In one embodiment, the reactive group on L to form X2 with the reactive group on R is -OH, -NH 2 or -C=O(O). Most advantageously, X2 is -C=O(O) generated by reaction of a hydroxyl group on L with an acyl group. However, such groups are merely exemplary and other groups known to those skilled in the art can be used as well.

[0216] The X2 chemical linkage may be a linker. The linker may have 0-12 carbon atoms and at least one cleavable functional group to release R in formula I, Ib, II or IIa, if desired. In one embodiment, the linker has at least two functional groups, a first functional group that joins one end of the linker to the scaffold L and a second functional group that joins the other end of the linker to a carbon atom on R. The two functional groups may each be a carbon-based functional group such as an ester, amide, amidine, hydrazone, disulfide, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene or alkyne, methylene (CH 2 ) or urea. In one advantageous embodiment, at least one of the functional groups in the linker is an ester, an amide, a hydrazone, an ether, a carbonate, a carbamate or is a phosphodiester. In another embodiment, at least one of the functional groups of X2 can be cleaved in vivo to release R from the scaffold L. Such a latter embodiment may be desirable when R or L is a therapeutic lipid.

[0217] R or R' Group As stated above, in one embodiment, R or R' in formula I, Ia, II or IIa is a hydrocarbon group having 1-40 carbon atoms, optionally containing one or more cis or trans C=C double bonds. In another embodiment, R is an aliphatic hydrocarbon. In a further embodiment, R does not contain any heterocyclic structure. In another embodiment, R is not biotin.

[0218] In one embodiment, the number of carbon atoms in the R group is selected so that the lipid conjugate of formula I, Ia, II or IIa has the desired logP value. As can be seen from Table 1 above, in some embodiments, the logP of the lipid conjugate can generally be correlated with the number of carbon atoms on the hydrocarbon R. For example, in the example provided in Table 1 based on L (formula I) or L1-L2 (formula Ia) derived from ricinoleyl alcohol, when the R hydrocarbon is derived from an acyl group having two carbon atoms, such as INT-D047 (i.e., R is one carbon atom based on the above formula I or Ia nomenclature), the LogP is only 8.33. However, the LogP of INT-D048, derived from an acyl chain of five carbon atoms, is 10.13 (i.e., S is four carbon atoms based on formula I or IaS nomenclature). The LogP of INT-D035 increases to 15.34 when oleoyl having 18 carbon atoms is attached to L (i.e., R is 17 carbon atoms based on formula I or IaR nomenclature). When the acyl chain linked to L has 20 carbon atoms, as in INT-D051, the LogP is 15.14 (i.e., S is equal to 19 carbon atoms). As mentioned above, by designing prodrugs with desired hydrophobicity, the drug loading and retention properties after administration can be more easily controlled.

[0219] Thus, in one embodiment, R in Formula I, Ia, II or IIa has 1-40 carbon atoms, is linear or branched, and is selected to provide a lipid conjugate having a desired logP falling within the range of 5-25, or 5-18, or 6-16.

[0220] As discussed above, optionally, a second R hydrocarbon having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds may be chemically bonded to L via an X2 chemical linkage. Additionally, optionally, a third R hydrocarbon having 1-40 carbon atoms and optionally having one or more cis or trans C=C double bonds may be chemically bonded to L via an X2 chemical linkage.

[0221] Additionally, one or more of the R hydrocarbon moieties attached to L may be attached to a respective R' side chain. For example, the R' side chain may be linked to the first, second or third R via an X2 linkage, the second R' side chain may be linked to any R via an X2 linkage, and / or the third R' may be linked to any R via an X2 linkage. Numerous other combinations can be readily envisioned by one of skill in the art.

[0222] The R hydrocarbon does not have to be derived from an acyl group or a fatty acid. For example, R can be a cholesterol moiety or other hydrocarbon group. The R hydrocarbon can also be a therapeutic or prophylactic moiety that is released upon cleavage from the prodrug, such as a lipid or sterol that has therapeutic activity.

[0223] As already mentioned, the molecule of interest M can be L, one or more R hydrocarbons can be L, or one or more A variety of chemical linkages can be utilized to connect the R' group above to R. Those skilled in the art will appreciate that a variety of functional groups or combinations thereof can be used in these linkages. That is, in the various embodiments described above in connection with the lipid conjugates of formula I, Ia, II, and IIa and the precursors P of formula III, IIIa, IIIb, and IIIc, X1 and X2 can be independently selected from carbon-based functional groups such as esters, amides, amidines, hydrazones, ethers, carbonates, carbamates, thionocarbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, phosphonamidates, phosphates, phosphonates, phosphodiesters, phosphonooxymethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azos, alkanes, alkenes, or alkynes, methylene (CH 2 ) or urea. In another embodiment, any one of the linkages X1 and X2 is biodegradable.

[0224] In another embodiment, any X2 is a linkage that includes one or more hydrogen bonds. According to such an embodiment, X2 has the structure of a linking moiety of formula VI:

[0225] [ka]

[0226] wherein E1, E2, E3, E4 and E5 are electronegative atoms selected from O, N and P; E1, E2 and E3 are hydrogen bond acceptors, and E4 and E5 are hydrogen bond donors; Dotted lines indicate hydrogen bonds, solid lines indicate covalent bonds; L is a lipid scaffold of the lipid moiety shown in formula I, Ia, II or IIa; R and R' are hydrocarbon chains as shown in formula IIa; n is 0 or 1, o is 0 or 1, p is 0 or 1, and n+o+p≧2; q is 1 to 10 or 2 to 10 or 4 to 10; L is the lipid scaffold of the lipid moiety; M is the molecule of interest; E1 and E3 may optionally include a substituent attached thereto, such as alkyl, aryl, alkylene, or H.

[0227] Products, compositions and formulations The lipid conjugates described herein can be administered in free form, as a component of a pharmaceutical product or composition, or as part of a delivery vehicle. Such products or compositions typically include known pharma- ceutically acceptable salts and / or excipients.

[0228] A variety of delivery systems can be used to prepare pharmaceutical formulations. These include lipid-based, polymer-based nanoparticles, emulsions, micelles, and lipid-based vesicles with one or more bilayers, such as liposomes or polymeric nanoparticles, including carbon nanotubes. Examples of suitable lysosome-derived nanoparticles include, but are not limited to, lipid nanoparticles (LNPs).

[0229] The lipid conjugates of the present disclosure are particularly amenable to incorporation into nanoparticles, such as liposomes or polymer-based systems that contain lipids or other hydrophobic components. The lipid-like properties of the lipid conjugates in certain embodiments can facilitate their loading into these or other delivery vehicles. For example, in some embodiments, the loading efficiency into a given nanoparticle is 75%-100%, 80%-100%, or in the most advantageous cases, 90%-100%.

[0230] In one embodiment, the lipid conjugates are loaded into lipid nanoparticles, such as liposomes, by mixing with lipid formulation components including vesicle-forming lipids and, optionally, sterols. As a result, lipid nanoparticles incorporating these drug-lipid conjugates can be prepared using a wide variety of well-described formulation methodologies known to those skilled in the art, including, but not limited to, extrusion, ethanol injection, and in-line mixing. Such methods are described in Maclachlan, I. and P. Cullis, "Diffusible-PEG-lipid Stabilized Plasmid Lipid Particles", Adv. Genet., 2005. 53PA: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, which are incorporated herein by reference in their entireties.

[0231] Liposomes contain an aqueous inner solution surrounded by a phospholipid bilayer, whereas lipid nanoparticles may alternatively contain a lipophilic core. Such lipophilic cores can serve as reservoirs for prodrugs. Both solid and liquid lipid nanoparticles can be used for delivery of prodrugs as described herein.

[0232] In one embodiment, lipid nanoparticles are provided that comprise a phospholipid bilayer, and the lipid conjugate forms a hydrophobic oil phase within the bilayer. Such delivery vehicles are described in Example 3 and Example 4 herein. The hydrophobic oil phase can be visualized by electron microscopy. In one embodiment, the lipid conjugate has the structure of formula I, Ia, II or IIa. In another embodiment, the lipid nanoparticle is a particle with one or more bilayers, such as a liposome.

[0233] The delivery vehicle can also be a nanoparticle that comprises a lipid core stabilized by surfactant. Vesicle-forming lipids can be utilized as stabilizers. In another embodiment, the lipid nanoparticle is a polymer-lipid hybrid system that comprises a polymer nanoparticle core surrounded by stabilizing lipids. In such an embodiment, the lipid conjugate of the present disclosure can be a lipid-polymer conjugate.

[0234] Alternatively, nanoparticles can be prepared from lipid-free polymers. Such nanoparticles can contain a concentrated core of drug, which can be surrounded by a polymer shell or have a solid or liquid dispersed throughout the polymer matrix.

[0235] The lipid conjugates described herein can also be incorporated into emulsions, which are drug delivery vehicles that contain oil droplets or oil cores.Emulsions can be lipid-stabilized.For example, emulsions can contain an oil-filled core stabilized by emulsifying components such as lipid monolayers or bilayers.

[0236] Micelles are self-assembled particles consisting of amphiphilic lipid or polymeric components with a hydrophobic core. The present invention is applied to the delivery of drugs. Conjugating drugs to a scaffold molecule L and a hydrophobic group R as described herein can improve drug loading into micelles.

[0237] A further class of drug delivery vehicles known to those of skill in the art that can be used to encapsulate the lipid conjugates herein are carbon nanotubes.

[0238] A variety of methods for preparing the delivery vehicles and incorporating prodrugs therein are available and may be readily performed by one of ordinary skill in the art.

[0239] Certain lipid conjugates encompassed in the disclosure can form part of carrier-free systems.In such embodiments, lipid conjugates can self-assemble into particles.Without limitation, if drug moiety D or polymer is hydrophilic, amphiphilic prodrugs can assemble into nanoparticles with or without stabilizers.

[0240] Although pharmaceutical compositions are described above, the lipid conjugates can be ingredients of any nutritional, cosmetic, cleaning or food product.

[0241] Administration In certain embodiments, the lipid conjugate is a prodrug, either free or formulated in a drug delivery vehicle, that is administered to treat, prevent and / or improve a patient's condition. That is, the prodrug in free form or formulated in a delivery vehicle may provide a preventative, ameliorative or therapeutic benefit. The pharmaceutical composition containing the prodrug is administered in any suitable dosage. In one embodiment, the prodrug in free form or formulated in a drug delivery vehicle is administered parenterally, i.e., intraarterially, intravenously, subcutaneously or intramuscularly. In other embodiments, the prodrug in free form or formulated in a delivery vehicle described herein may be administered topically. In yet another embodiment, the prodrug in free form or formulated in a delivery vehicle described herein may be administered orally. In yet another embodiment, the prodrug is formulated in free form or in a delivery vehicle for pulmonary administration by aerosol or powder dispersion.

[0242] In a further embodiment, the molecule of interest is a hydrophilic polymer and the conjugate is a lipid-polymer conjugate. The lipid-polymer conjugate may be incorporated into a delivery vehicle along with one or more drugs and administered to treat, prevent and / or ameliorate a patient's condition.

[0243] As used herein, the term "patient" includes a human or non-human subject.

[0244] The following examples are given for illustrative purposes, but not to limit the scope of the invention.

[0245] Working Example Example 1: Ricinoleyl Alcohol as an Exemplary Scaffold Molecule L An example of a lipid conjugate is shown in FIG. 2, which shows a variety of conjugates that can be formed using ricinoleic acid or ricinoleyl alcohol as a precursor of scaffold L in formula I, Ia, II or IIa above. In the figure, the chemical structure of the X1 and X2 linkages of formula I is not depicted. Rather, the figure shows the hydroxyl groups at C1 and C12 of the fatty acid or alcohol (as well as the Z, Y atoms at positions 9 and 10 in the oxidized form of the molecule) that can react with complementary functional groups on the drug-linker and / or acyl group, such as carboxylic acid. In this example, the X1 and X2 linkages can include ester functional groups based on condensation reactions of carboxyl groups with hydroxyl groups, although other functional groups can be formed as well by specific functional groups present on the drug, molecular backbone, side group R or linker group that react to form X1 or X2.

[0246] The following examples also use a linker group to link the molecule of interest M to the scaffold molecule L. However, such a linker is optional in that the molecule of interest M may instead be directly attached to the scaffold molecule L itself.

[0247] Ricinoleyl alcohol is an unsaturated aliphatic alcohol derived from ricinoleic acid, an 18-carbon hydroxyl fatty acid (HFA) with a hydroxyl group substituted at C12. In the structure of Figure 2, ricinoleic acid or ricinoleyl alcohol is formed from the precursor scaffold P (X is C=O or CH 2 ), other molecules can be used as well, 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 ricinoleyl alcohol does not have to be prepared from a fatty acid or fatty alcohol with hydroxyl groups at both C12 and C1. For example, the precursor of L can be prepared from a corresponding molecule with a hydroxyl group at C1 and an ether substituent at C12 (such as the silyl ether I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol (2) intermediate described in Example 2).

[0248] In some embodiments, the double bond in the backbone of ricinoleic acid or ricinoleyl alcohol is partially or fully oxidized to provide additional reactive groups that can be used to attach a second acyl chain R'. Such groups are depicted in the figures as Y and Z.

[0249] In this example, the scaffold molecule L is described as the L1-L2 chain of formula Ia. L1 is the carbon chain from C1 to the carbon preceding the first branch point to which the side chain (e.g., acyl chain) or molecule of interest or M-linker is attached. L2 is the carbon chain from the carbon at the branch point including to the end of the backbone.

[0250] In the structure of Figure 2A, X1 is either ricinoleic acid (X=C=O) or ricinoleyl alcohol L (X=CH 2 ) is a linker that covalently attaches a molecule of interest to the ricinoleic acid or ricinoleyl alcohol. The linker is attached to C1 of the ricinoleic acid or ricinoleyl alcohol through a reactive group, which is the hydroxyl group at C1. At C12 of the ricinoleic acid or ricinoleyl alcohol, the side chain R from the acyl group is attached to L2 through a hydroxyl group. In this example, L1 is a linear 11 carbon chain with a cis double bond at C9 and C10 as shown in Figure 2, and L2 is a 7 carbon saturated carbon chain from C12 to C18. X2, not shown here, connects C12 of the ricinoleic acid or ricinoleyl alcohol to the R side chain from the acyl group. As mentioned above, the carboxylic acid of the acyl group reacts with the hydroxyl group at C12 of the ricinoleic acid or ricinoleyl alcohol to form an -O(C=O) ester bond. Similarly, in this example, the C1 OH of ricinoleic acid or ricinoleyl alcohol reacts with a carboxylic acid group on one end of the linker to form an X1-O(C=O) ester bond. Alternatively, the C1 OH of L reacts directly with a free carboxylic acid on the molecule of interest M to form an -O(C=O) linkage (X1).

[0251] In structure B of Figure 2, a linker X1 covalently attaches a molecule of interest M to the hydroxyl group at C12 of ricinoleic acid or ricinoleyl alcohol. At C1, the R hydrocarbon from the acyl side group is attached to L1 via the terminal hydroxyl group of C1. In this example, L1 of the molecular scaffold is 11 carbon atoms and L2 is 7 carbon atoms. Alternatively, the OH at C1 of L can react directly with a carboxylic acid on the molecule of interest M to form a -O (C=O) bond.

[0252] In Figure 2C, partially oxidized ricinoleic acid or ricinoleyl alcohol is used as the precursor scaffold for P. The double bonds of ricinoleic acid or ricinoleyl alcohol at C9 and C10 are oxidized to form a saturated hydrocarbon chain substituted at C10 with a Y reactive group and a C 12 is replaced with a hydroxyl group. A side chain R from an acyl group is linked to C12 of L1-L2 through a hydroxyl group, and a second side chain R' from another acyl chain is linked to the C10 position through Y. In this example, Y is a reactive group, including N, O, S, or P as the first atom of the group. Without being limited thereto, if Y is N, the reactive group can be an amine, and if Y is O, the reactive group can be a hydroxyl group. Similarly, if Y is P, the reactive group can be a phosphate. As stated above, these reactive groups are merely exemplary, and other groups can be easily imagined by one skilled in the art.

[0253] The molecule of interest M-linker X1 is attached to C1 through the terminal hydroxyl group of ricinoleic acid or ricinoleyl alcohol. Alternatively, the OH of C1 of L1-L2 reacts with a carboxylic acid on the molecule of interest M itself to form a -O (C=O) bond. In this example, L1 is 9 carbon atoms and L2 is 9 carbon atoms.

[0254] In structure D of FIG. 2, partially oxidized ricinoleic acid or ricinoleyl alcohol is again used as a scaffold precursor and contains the molecule of interest M via the linker X1 on C1. Alternatively, the OH on C1 of L1-L2 reacts directly with a carboxylic acid on the molecule of interest M to form a -O (C=O) bond, rather than utilizing the linker shown. A first side chain R from the acyl chain is attached at the C12 position via a hydroxyl reactive group, and a second side chain R' from the acyl chain is attached at the C9 position of the ricinoleyl alcohol via a Y group, the first atom of which is N, O, S or P as described in connection with structure C. In this example, L1 is 8 carbon atoms and L2 is 10 carbon atoms.

[0255] In structure E of Figure 2, oxidized ricinoleic acid or ricinoleyl alcohol is used as a precursor to a scaffold L with a side chain derived from an acyl chain R attached at the C12 position via a hydroxyl group and a second side chain R' derived from an acyl chain attached at the C9 position via a Z group. Like Y, the Z group is a reactive group, and the first atom of the group is N, O, S or P, as described in connection with structures C and D above. The drug moiety D is attached via a linker X1 at C1 by chemical linkage with the reactive hydroxyl group of C1. Alternatively, the OH at C1 of L1-L2 reacts directly with a carboxylic acid on the drug D to form a -O (C=O) bond, rather than utilizing a linker.

[0256] Example 2: Synthesis of lipid conjugates material and method: A variety of prodrugs were prepared using synthetic procedures A through E shown below.

[0257] All reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise noted. The following were used: tetrahydrofuran (freshly distilled from Na / benzophenone under nitrogen), Et 3 N, DMF and CH 2 Cl 2 (CaH under nitrogen 2 Freshly distilled from Life Sciences. TMAll 42111-10 ...

[0258] The general steps of the synthesis of lipid conjugates based on hydroxy and carboxy derivatives of castor oil (ricinolein) are shown below in Scheme 1. Schemes 2A to 2V are then described, designated General Procedures A to E, which describe the steps for preparing the prodrugs of Examples 2A to 2V below. Continued in 1.

[0259] Scheme 1: General synthesis of lipid conjugates based on hydroxy and carboxy derivatives of castor oil (ricinolein).

[0260] [ka]

[0261] According to the synthetic reactions depicted in Scheme I, castor oil, also known as ricinolein (the glyceride of ricinoleic acid), is the starting material for the synthesis of the prodrug shown in FIG.

[0262] In step 1) above, sodium methoxide (2.0 mL of a 3.0 M solution in MeOH, 6.00 mmol, 0.20 equiv) was added to a stirred solution of castor oil (28.0 g, 30.0 mmol, 1.00 equiv) in 1:1 THF / MeOH (30 mL) under argon at room temperature in a round bottom flask. After 14 h, the reaction was diluted with saturated NH 4 Quench with aqueous Cl and Et 2The combined organic layers were washed with water (1×150 mL), brine (1×150 mL), and extracted with Na 2 SO 4 The mixture was dried over water and concentrated to produce a clear, colorless oil, methyl (12R)-hydroxyoleate 1 (28.0 g, quantitative yield), which was used without further purification. The structure of methyl (12R)-hydroxyoleate and its physical properties are shown below: Methyl (12R)-hydroxyoleate (1):

[0263] [ka]

[0264] R f =0.50(SiO 2 , 70:30 hexane / EtOAc); 1 HNMR (300MHz, CDCl 3 ):δ5.64-5.50(m,1H),5.49-5.35(m,1H),3.68(s,3H),3.63(quint,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)

[0265] According to scheme 2 above, LiAlH 4 To a stirred suspension of 1.25 g (33.0 mmol, 1.10 equiv) in ice-cold THF (90 mL), a solution of methyl (12R)-hydroxyoleate (9.37 g, 30.0 mmol) in room temperature THF (15 mL) was added via addition funnel over 20-30 min. After addition was complete, the cold bath was removed. After 14 h, the reaction mixture was cooled in an ice bath and diluted with Et 2 Dilute with HO (150 mL) and add quenching solution (1.25 mL HO). 2 O, 1.25mL 1M NaOH aqueous solution, 3.75mL H 2 0), stirred at room temperature for 1 h, and then added Et 2The mixture was filtered through Celite, washing thoroughly with O. The filtrate was concentrated on a rotary evaporator to give the crude diol as a pale yellow oil (quantitative yield), which was used without further purification.

[0266] According to 3) of the above reaction scheme, to a solution of the above diol (8.21 g, 28.9 mmol, 1.10 equiv) and i-Pr2Net (5.73 mL, 32.8 mmol, 1.25 equiv) in DMF (25 mL) at 10-15 °C in a round bottom flask under argon, a solution of tert-butyldimethylsilyl chloride (3.96 g, 26.2 mmol, 1.00 equiv) in room temperature DMF (20 mL) was added via addition funnel over 30 min. The reaction was allowed to warm over 14 h, after which it was diluted with saturated NH 4 Quench with aqueous Cl and 1:1 Et 2 O / Hexane (3×1 The combined organic layers were extracted with H 2 Wash with 2×300 mL of HO and 1×100 mL of brine. 2 SO 4 The mixture was dried over ice and concentrated on a rotary evaporator to give the crude primary silyl ether as a slightly yellow oil. 2 The crude was purified by filtration using 220 mL of 99:1 to 95:5 hexanes / EtOAc) to give a clear, colorless oil consisting of the silyl ether 2 (8.38 g, 80% yield). The structure of silyl ether 2, as well as its physical properties, are shown below: I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol (2):

[0267] [ka]

[0268] R f =0.16(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.64-5.50(m,1H), 5. 49-5.35(m,1H), 3.68(s,3H), 3.63(quint,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)

[0269] According to reaction scheme 4), RCO was added to a round-bottom flask under argon. 2 H(279m g, 2.40 mmol, 1.20 equiv) in ice-cold CH 2 Cl 2 To the (6 mL) solution was added N,N'-dicyclohexylcarbodiimide (DCC) (495 mg, 2.40 mmol, 1.20 equiv.), the ice bath was then removed, and the resulting mixture was stirred for 15 min. 2 Although H was hexanoic acid, other acyl groups can be utilized to generate the desired hydrocarbon side chain S. The reaction mixture was cooled again in an ice bath and the silyl ether CH 2 Cl 2 (2 mL) solution of I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol 2 (797 mg, 2.00 mmol) was added followed by DMAP (366 mg, 3.00 mmol, 1.50 equiv) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was cooled to rt in Et 2 The mixture was diluted with O, stirred for 10 min, and then filtered through Celite. The filtrate was concentrated on a rotary evaporator to give the crude ester as a white semi-solid. The crude ester was poured onto a plug of silica gel (SiO 2 20 mL, 95:5 hexane / EtOAc) and filtered. f =0.53(SiO 2 , 90:10 hexanes / EtOAc) to yield a clear, colorless oil as the intermediate ester (quantitative yield).

[0270] According to Scheme 5 above, to a stirred ice-cold solution of pyridine (0.48 mL, 6.00 mmol, 3.00 equiv) and the above silyl ether (2.00 mmol) in THF (6 mL) in a round-bottom flask under argon, a solution of pure HF·pyridine (0.74 mL of 70% HF in pyridine, 6.00 mmol, 3.00 equiv) was added. After 2 h, saturated NaHCO 3 The reaction was quenched with aqueous solution of Et 2 After extraction with 2×10 mL of H2O, the combined organic extracts were washed with H2O. 2 20 mL, washed with brine, and then washed with Na 2 SO 4 The mixture was dried over ice and concentrated on a rotary evaporator to give the crude primary alcohol, which was purified by filtration through a plug of silica gel (20 mL, 90:10 hexanes / EtOAc) to give primary alcohol 3 (quantitative yield) as a clear, colorless oil with the following structure and physical properties: (12R)-Hexanoyloxyoleyl alcohol (3):

[0271] [ka]

[0272] According to Reaction Scheme 6 above, (12R)-hexanoyloxyoleyl alcohol (3) (765 mg, 2.00 mmol, 1.00 equiv.) was stirred in a round-bottom flask under argon at room temperature in CH 2 Cl 2 To the (6 mL) solution was added solid succinic anhydride (400 mg, 4.00 mmol, 2.00 equiv.) and DMAP (611 mg, 5.00 mmol, 2.50 equiv.). After 14 h, the reaction was quenched with 1 M aqueous hydrochloric acid and CH 2 Cl 2 (2×15 mL). The combined organic extracts were then washed with 1 M aqueous hydrochloric acid (1×15 mL), H 2 Wash with O (2 × 15 mL) and add Na 2 SO 4The mixture was dried over ice and concentrated on a rotary evaporator to give the intermediate hemisuccinate as a pale yellow oil (quantitative yield) which was used without further purification. f =0.32(SiO 2 , 50:50 hexane / EtOAc).

[0273] According to reaction scheme 7), solid DCC (99 mg, 0.48 mmol, 1.20 equiv.) was added to the above hemisuccinate (232 mg, 0.48 mmol, 1.20 equiv.) in a stirred ice-cold CH4Cl2 flask under argon. 2 Cl 2 (2 mL) was added, the ice bath was removed, and the mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid dexamethasone (157 mg, 0.40 mmol) and DMAP (73 mg, 0.60 mmol, 1.50 equiv.) were added. The mixture was allowed to warm for 14 h and then diluted with Et 2 The mixture was diluted with 200 mL of ethyl acetate, stirred for 10 min, and then filtered through Celite. The filtrate was concentrated to give the crude product, which was a slightly yellow oil. The crude product was purified by flash column chromatography (50 mL SiO 2 ,80:20→50:50Hexane / Et OAc) to give the desired prodrug 4 (328mM) having the following structure and properties: g, 95% yield) of a clear, colorless oil:

[0274] 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-dodecahydro-3H-cyclopenta[a]57ctadic57ren-17-yl)-2-oxoethyl(((R,Z)-12(hexanoyloxy)57ctadic-9-en-1-yl)succinate (4):

[0275] [ka]

[0276] Rf =0.38(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0277] The prodrug is based on a ricinoleyl scaffold L with a hexanoyl (C6:0) side chain attached to dexamethasone by a succinate linker (INT-D034).

[0278] In the above example, RCO was added in 4) of the above reaction. 2 H is hexanoic acid, generating a hexanoyl side chain (C6:0), although other fatty acids can be utilized to generate the desired hydrocarbon side chain R on the ricinoleyl scaffold.

[0279] General operations A Acylation of (R)-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol 3 (4a-h): Stir the desired carboxylic acid (1.20 equiv.) in a round-bottom flask under argon in ice-cold CH 2 Cl 2 DCC (1.20 equiv.) was added to the solution, then the ice bath was removed and the resultant mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath and the alcohol 3 was added in CH 2 Cl 2 Liquid (CH 2 Cl 2In Et (1.00 eq., 0.25 M) was added, followed by DMAP (1.50 eq.) and the reaction mixture was allowed to warm to room temperature over 14 h. 2 The mixture was diluted with 200 ml of ethyl acetate, stirred for 10 minutes, and then filtered through Celite®. The filtrate was concentrated on a rotary evaporator to give the crude ester as a white semi-solid. The crude material was purified by filtration through a plug of silica gel (95:5 hexanes / EtOAc) to give the pure ester.

[0280] General operation B Desilylation-succinylation of (12R)-acyloxyoleyl alcohols 4a-h (5a-h): Under argon, stir pyridine (3.00 equiv.) and 12-acyl ricinol alcohol silyl ether (1.00 equiv.) in ice-cold tetrahydrofuran (starting silyl ether) in a round-bottom flask. To a solution of HF in pyridine (0.30 M relative to diethyl ether), a solution of HF and pyridine (70% HF in pyridine, 3.00 equiv.) was added. When TLC showed uptake of starting material (2–8 h), the reaction was diluted with saturated NaHCO 3 The mixture was quenched with Et 2 After extraction with 2×10 mL of H2O, the combined organic extracts were washed with H2O. 2 20 mL, washed with brine, and then washed with Na 2 SO 4 The mixture was dried over ice and concentrated on a rotary evaporator to give the crude primary alcohol. The crude material was purified by filtration through a plug of silica gel (90:10 hexanes / EtOAc), concentrated on a rotary evaporator, and dried under high vacuum to give the primary alcohol as a clear, colorless oil, which was used in the succinylation without further purification.

[0281] Solid succinic anhydride (2.00 equiv.) and DMAP (2.50 equiv.) were added to the CH solution of 12-acylricinol alcohol (1.00 equiv.) stirred at room temperature in a round-bottom flask under argon. 2 Cl 2 (0.30 M relative to the starting primary alcohol) solution. After 14 h, the reaction was quenched with 1 M aqueous hydrochloric acid and CH 2 Cl2 (2×15 mL). The combined organic extracts were then extracted with 1 M hydrochloric acid (1×15 mL), H 2 Wash with aqueous 2×O (2×15 mL) and 2 SO 4 The residue was redissolved in hexane, treated with activated charcoal, filtered through Celite®, and the filtrate was concentrated to give a colorless to slightly yellow oil. The intermediate hemisuccinate was obtained as a crude product and used without further purification.

[0282] General operations C Acylation of methyl (12R)-ricinoleate 2 (6a-c): Stir the desired carboxylic acid (1.20 equiv.) in a round-bottom flask under argon in ice-cold CH 2 Cl 2 To the solution was added DCC (1.20 equiv.), then the ice bath was removed and the resultant was stirred for 15 min. The reaction mixture was cooled again in an ice bath and then methyl (12R)-ricinoleate (CH 2 Cl 2 0.30M, 1.00 equiv.) 2 Cl 2 The reaction mixture was then warmed to room temperature over 14 hours. The reaction mixture was diluted with hexanes, stirred for 10 minutes, and then filtered through Celite®. The filtrate was concentrated on a rotary evaporator. This afforded the crude diester as a white semi-solid which was purified by filtration through a plug of silica gel (95:5 hexanes / EtOAc) to give the pure ester.

[0283] General Procedure D Binding of dexamethasone to hemisuccinates 5a-h: DCC (1.20 equiv.) was stirred in 12-acylricinoleyl hemisuccinate (1.20 equiv.) in a round-bottom flask under argon and cooled in CH 2 Cl 2(0.2M in dexamethasone), the ice bath was removed and the resultant mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid dexamethasone (1.00 equiv.) and DMAP (1.50 equiv.) were added. The reaction was allowed to warm over 14 h and then diluted with Et 2 The mixture was diluted with 200 ml of ethyl acetate, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to give a pale yellow oil, which was then purified by flash column chromatography. Chromatography (SiO 2 , 80:20→50:50 hexanes / EtOAc) to give a clear colorless oil as the desired dexamethasone conjugate.

[0284] General Procedure E Dexamethasone binding to ricinoleic acid 12a-b,13: Stir acyloxystearic acid (1.10 equiv.) in a round-bottom flask under argon in ice-cold CH 2 Cl 2 To a solution (0.1 M in dexamethasone) was added DCC (1.10 equiv), the ice bath was removed, and the resultant was stirred for 15 min. The reaction mixture was cooled again in an ice bath, and solid dexamethasone (1.00 equiv) and DMAP (1.50 equiv) were added. The reaction was allowed to warm over 14 h and then diluted with Et 2 The mixture was diluted with O, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to give the crude product as a slightly yellow oil, which was purified by flash column chromatography to give a clear colorless oil as the desired conjugate.

[0285] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl acetate (4a): Acetyl chloride (0.43 mL, 6.00 mmol, 1.20 equiv.) was added to a stirred mixture of silyl ether 3 (2.00 g, 5.00 mmol, 1.00 equiv.), acetyl chloride (0.43 mL, 6.00 mmol, 1.20 equiv.), triethylamine (0.83 mL, 6.00 mmol, 1.2 equiv.) and DMAP (733 mg, 6.00 mmol, 1.20 equiv.) in a round-bottom flask in ice-cold CH4. 2 Cl 2(10 mL) solution under argon and allowed to warm to room temperature. After 14 h, the reaction was diluted with CH 2 Cl 2 Dilute with 1×15 mL of saturated NH4Cl2 solution, wash with 2×15 mL of water, and add Na 2 SO 4 The residue was redissolved in the eluent and poured onto a plug of silica gel (SiO 2 Purification with 30 mL of 97:3 hexanes / EtOAc gave the ester 4a (1.83 g, 83%) as a pale yellow oil.

[0286] R f =0.45(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0287] [ka]

[0288] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-ylhexanoate (4b) According to general procedure A, 2 Cl 2 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) in (15 mL) gave 2.37 g (2.39 g, quantitative yield) of ester 4b as a clear, colorless oil.

[0289] R f =0.43(SiO 2, 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.56-5.42(m,1H), 5. 41-5.27(m,1H), 4.90(quint,J=6.3Hz,1H), 3.61(t,J=6.6Hz,2H), 2.37-2.2 2(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)

[0290] [ka]

[0291] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl laurate (4c): According to general procedure A, 2 Cl 2 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 (8 mL) gave ester 4c (1.38 g, quantitative yield) as a clear, colorless oil.

[0292] R f =0.56(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.56-5.41(m,1H), 5. 41-5.26(m,1H), 4.90(quint,J=6.2Hz,1H), 3.61(t,J=6.6Hz,2H), 2.37-2.2 1(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)

[0293] [ka]

[0294] (R,Z)-18-(tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl stearate (4d): According to general procedure A, 2:1 THF / CH 2 Cl 2 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 (6 mL) gave ester 4d (1.56 g, 94%) as a clear, colorless oil.

[0295] R f =0.48(SiO 2 , 90:10 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.57-5.41(m,1H),5. 41-5.25(m,1H),4.90(quint,J=6.3Hz,1H),3.61(t,J=6.5Hz,2H),2.39-2.2 0(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)

[0296] [ka]

[0297] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl oleate (4e): According to general procedure A, 2 Cl 2Silyl 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 (10 mL) gave ester 4e (1.64 g, quant.) as a clear, colorless oil.

[0298] R f =0.41(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.56-5.25(m,4H), 4. 90(quint,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)

[0299] [ka]

[0300] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl linoleate (4f): According to General Procedure A, 2 Cl 2 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 (7 mL) gave ester 4f (1.06 g, 76%) as a clear, colorless oil.

[0301] R f =0.46(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.67-5.24(m,6H), 4. 90(quint,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)

[0302] [ka]

[0303] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl linolenate (4g): According to General Procedure A, 2 Cl 2 Silyl ether 3 (997 mg, 2.50 mmol), linoleic acid (835 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in (8 mL) gave the ester 4g (1.52 g, 92%) as a clear, colorless oil.

[0304] R f =0.34(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.58-5.26(m,8H), 4. 90(quint,J=6.2Hz,1H), 3.61(t,J=6.5Hz,2H), 2.83(t,J=5.8Hz,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=7.5Hz,3H), 0.91(br s,12H), 0. 07(s,6H)

[0305] [ka]

[0306] (R,Z)-18-(tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl arachidonate (4h): According to General Procedure A, 2 Cl 2 Silyl ether 3 (797 mg, 2 0.00 mmol), arachidonic acid (670 mg, 2.20 mmol), DCC (227 mg, 2.20 mmol) and DMAP (366 mg, 3.00 mmol) afforded ester 4h (730 mg, 53%) as a clear, colorless oil after flash column chromatography (99:1→95:5 hexanes / EtOAc).

[0307] R f =0.57(SiO 2 , 95:5 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.57-5.26(m,10H),4 .91(quint,J=6.3Hz,1H),3.61(t,J=6.5Hz,2H),2.94-2.84(m,6H),2.38-2.22(m,4H),2.2 0-1.96(m,6H),1.71(quint,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)

[0308] [ka]

[0309] (R,Z)-4-((12-acetoxyoctadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5a): Following general procedure B, silyl ether 4a (1.79 g, 4.07 mmol) was desilylated with HF·pyridine (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 reacted with succinic anhydride (814 mg, 8.14 mmol), DMAP (1.24 g, 10.2 mmol), and CH 2 Cl 2 (10 mL) to give the carboxylic acid 5a (1.72 g, quantitative yield).

[0310] R f =0.23(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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,2H), 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)

[0311] [ka]

[0312] (R,Z)-4-(12-(hexanoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5b): Following general procedure B, silyl ether 4b (2.35 g, 5.00 mmol) was desilylated with HF·pyridine (1.86 mL, 15.0 mmol), pyridine (1.21 mL, 15.0 mmol), and THF (13 mL) to give the intermediate primary alcohol (2.01 g), which was then reacted with succinic anhydride (1.00 g, 10.0 mmol), DMAP (1.53 g, 12.5 mmol), and CH 2 Cl 2(13 mL) to give the carboxylic acid 5b (2.20 g, 92% yield).

[0313] R f =0.32(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.56-5.42(m,1H), 5. 41-5.27(m,1H), 4.90(quint,J=6.4Hz,1H), 4.11(t,J=6.5Hz,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)

[0314] [ka]

[0315] (R,Z)-4-((12-(lauroyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5c): Following general procedure B, silyl ether 4c (1.38 g, 2.50 mmol) was desilylated with HF·pyridine (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 reacted with succinic anhydride (500 mg, 5.00 mmol), DMAP (764 mg, 6.25 mmol), and CH 2 Cl 2 (8 mL) to give the carboxylic acid 5c (1.33 g, 94%).

[0316] R f =0.44(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.57-5.42(m,1H), 5. 41-5.26(m,1H), 4.90(quint,J=6.2Hz,1H), 4.12(t,J=6.6Hz,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)

[0317] [ka]

[0318] (R,Z)-4-oxo-4-((12-(stearoyloxy)octadec-9-en-1-yl)oxy)butanoic acid (5d): Following general procedure B, silyl ether 4d (1.66 g, 2.50 mmol) was desilylated with HF·pyridine (0.93 mL, 7.50 mmol), pyridine (0.60 mL, 7.50 mmol), and tetrahydrofuran (8 mL) to give the intermediate primary alcohol (1.30 g), which was then reacted with succinic anhydride (500 mg, 5.00 mmol), DMAP (764 mg, 6.25 mmol), and CH 2 Cl 2 (8 mL) to give the carboxylic acid 5d (1.29 g, 79% yield).

[0319] R f =0.35(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.56-5.42(m,1H), 5. 41-5.27(m,1H), 4.90(quint,J=6.3Hz,1H), 4.11(t,J=6.5Hz,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)

[0320] [ka]

[0321] (R,Z)-4-oxo-4-((12-(oleoyloxy)octadec-9-en-1-yl)oxy)butanoic acid (5e): Following general procedure B, silyl ether 4e (663 mg, 1.00 mmol) was desilylated with HF·pyridine (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 reacted with succinic anhydride (200 mg, 2.00 mmol), DMAP (305 mg, 2.50 mmol), and CH 2 Cl 2 (5 mL) to give the carboxylic acid 5e (630 mg, 97% yield).

[0322] R f =0.42(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.57-5.25(m,4H), 4. 90(quint,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)

[0323] [ka]

[0324] 4-(((R,Z)-12-(linoleoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5f): Following general procedure B, silyl ether 4f (1.06 g, 1.60 mmol) was desilylated with HF·pyridine (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 reacted with succinic anhydride (320 mg, 3.20 mmol), DMAP (489 mg, 4.00 mmol), and CH 2 Cl 2 (8 mL) to give the carboxylic acid 5f (1.04 g, quantitative yield).

[0325] R f =0.35(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.57-5.26(m,6H), 4. 90(quint,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)

[0326] [ka]

[0327] 4-(((R,Z)-12-(linolenoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5g): Following general procedure B, the silyl ether 4g (1.54 g, 2.34 mmol) was desilylated with HF·pyridine (0.87 mL, 7.01 mmol), pyridine (0.57 mL, 7.01 mmol), and THF (6 mL) to give the intermediate primary alcohol (1.31 g), which was then reacted with succinic anhydride (468 mg, 4.68 mmol), DMAP (714 mg, 5.84 mmol), and CH 2 Cl 2(6 mL) to give the carboxylic acid 5g (1.47 g, quantitative yield).

[0328] R f =0.35(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.56-5.25(m,8H), 4. 90(quint,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)

[0329] [ka]

[0330] 4-(((R,Z)-12-(arachidonoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5h): Following general procedure B, silyl ether 4h (711 mg, 1.04 mmol) was desilylated with HF·pyridine (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 reacted with succinic anhydride (201 mg, 2.01 mmol), DMAP (306 mg, 2.51 mmol) and CH 2 Cl 2 (5 mL) to give the carboxylic acid 5h (582 mg, 87% yield).

[0331] R f =0.31(SiO 2 , 50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ5.58-5.24(m,10H), 4 .90(quint,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(quint,J=7.4Hz,2H), 1.69-1.47(m,4H), 1.46-1.13(m,26H), 0.99-0.80(m,6H)

[0332] [ka]

[0333] Methyl (12R)-hexanoyloxyoleate (6a): According to general procedure C, 2 Cl 2 The silyl group was synthesized by 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 10 mL of ethyl acetate. After filtration through silica gel (95:5 hexane / EtOAc), ricinoleate 6a was obtained as a clear, colorless oil (2.52 g, 96% yield).

[0334] R f : 0.62(SiO 2 , 70:30 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):δ5.54-5.42(m,1H), 5.40-5.28(m,1H), 4.90(quint,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)

[0335] [ka]

[0336] Methyl (12R)-linoleoyloxyoleate (6b): According to general procedure C, 2 Cl 2 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 (5 mL) gave ricinoleate 6c (875 g, 93% yield) as a pale yellow oil after filtration through silica gel (95:5 hexane / EtOAc).

[0337] R f : 0.67(SiO 2 , 80:20 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):δ5.54-5.42(m,1H), 5.40-5.28(m,1H), 4.90(quint,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)

[0338] [ka]

[0339] (12R)-Hexanoyloxyoleic acid (7a): An argon flushed round bottom flask was charged with the methyl ester 6a (1.97 g, 4.79 mmol, 1.00 equiv) and t-BuOH (12 mL), followed by 2.0 M aqueous NaOH. Solution (1.80 mL, 3.60 mmol, 0.75 equiv.) was charged. After 17 h, the pH of the reaction was adjusted to 2 with 1 M aqueous hydrochloric acid and Et 2 The combined organics were washed with water (1×30 mL), brine (1×30 mL) and extracted with Na 2 SO 4The mixture was dried over hexanes and concentrated under reduced pressure on a rotary evaporator. The residue was filtered through a plug of silica (98:2:0→50:45:5 hexanes:EtOAc:MeOH) to give carboxylic acid 7a (1.30 g, 92% yield) as a pale yellow oil.

[0340] R f =0.24(SiO 2 ,75:20:5 Hexane / EtOAc / MeOH); 1 H NMR (300 MHz, CDCl 3 ): δ5.55-5.28(m,6H), 4. 90(quint,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,6 H), 1.72-1.46(m,8H), 1.46-1.18(m,32H), 1.00-0.80(m,6H)

[0341] [ka]

[0342] (12R)-Linoleoyloxyoleic acid (7b): To an argon flushed round bottom flask was added the methyl ester 6b (5.97 g, 10.4 mmol, 1.00 equiv) and t-BuOH (26 mL), followed by 2.0 M aqueous NaOH. Solution (4.70 mL, 9.30 mmol, 0.90 equiv.) was charged. After 17 h, the pH of the reaction was adjusted to 2 with 1 M aqueous hydrochloric acid and Et 2 The combined organics were washed with water (1×30 mL), brine (1×30 mL) and extracted with Na 2 SO 4 The mixture was dried over silica gel and concentrated under reduced pressure on a rotary evaporator. The residue was purified by flash column chromatography (SiO 2 , 95:5:0→80:15:5 hexanes:EtOAc:MeOH) to give carboxylic acid 7b (4.48 g, 85% yield) as a pale yellow oil.

[0343] R f : 0.35(SiO 2 , 75:20:5 hexane / EtOAc / MeOH); 1 H(CDCl 3 , 300MHz): δ5.55-5.28(m,6H), 4.90(quint,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)

[0344] [ka]

[0345] Methyl 9,10-dihydroxystearate (8): To a rapidly stirred room temperature mixture of oleic acid (7.06 g, 25.0 mmol) and water (175 mL) in a 500 mL Erlenmeyer flask was added KOH (7.01 g, 125 mmol, 5.00 equiv.) and cooled to approximately 10°C. 4 (7.11g, 45.0mmol A solution of 1.80 equiv. (NaHSO 4 , 1.80 equiv.) in water (75 mL) was added dropwise over 10 min. After stirring for an additional 10-15 min, saturated NaHSO 4 3 The reaction was quenched by addition of aqueous solution and adjusted to pH ≦2 with concentrated hydrochloric acid in a cold bath. The white fluffy mixture was stirred at room temperature for 1 h, then the solid was collected by suction filtration and dried in air overnight. The resulting white solid was hot gravity filtered and recrystallized from EtOH to give (±)-syn-9,10-dihydroxystearic acid as white crystals (5.86 g, 74% yield).

[0346] A suspension of the above dihydroxy acid (6.33 g, 20.0 mmol) in MeOH (50 mL) was added to concentrated H 2 SO 4(0.06 mL, 1.00 mmol, 0.05 equiv) was added and heated to reflux. After 14 h, the mixture was cooled to room temperature, concentrated under reduced pressure on a rotary evaporator, and the residue was purified by EtOAc and saturated NaHCO 3 The organic layer was washed with water (1×75 mL), brine, and then with Na 2 SO 4 The mixture was dried over water and concentrated under reduced pressure on a rotary evaporator to give the methyl ester 8 (6.44 g, 97% yield) as a white solid.

[0347] R f =0.45(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ3.68(s,3H),3.61(ap p 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)

[0348] [ka]

[0349] Methyl 9,10,12R-trihydroxystearate (9): To a rapidly stirred room temperature mixture of ricinoleic acid (14.9 g, 50.0 mmol) and water (500 mL) in a 1 L Erlenmeyer flask was added KOH (5.61 g, 100 mmol, 2.00 equiv.) and cooled to approximately 10°C. 4 (13.4g, 85.0mmol, 1 A solution of 0.70 equivalents of NaCl in water (250 mL) was added dropwise over 15 minutes. After stirring for an additional 10-15 minutes, saturated NaCl was added. 2 SO 3The reaction was quenched by addition of aqueous solution and the pH was adjusted to ≦2 with concentrated hydrochloric acid in a cold bath. The white fluffy mixture was stirred at room temperature for 4 hours, then the solid was collected by suction filtration and dried in air overnight. The resulting white solid was hot gravity filtered with EtOH to give crude 9,10,12-trihydroxystearic acid, which was used without further purification.

[0350] A suspension of the above dihydroxy acid (6.33 g, 20.0 mmol) in MeOH (120 mL) was added to concentrated H 2 SO4 (0.13 mL, 2.50 mmol, 0.05 equiv) was added and heated to reflux. After 14 h, the mixture was cooled to ambient temperature, concentrated under reduced pressure on a rotary evaporator, and the resulting residue was purified by dilution with hot EtOAc and saturated NaHCO 3 The organic layer was washed with water (1×75 mL), brine, and then with Na 2 SO 4 The mixture was dried over silica gel and concentrated under reduced pressure on a rotary evaporator. The resulting pale yellow solid was dissolved in hot Et 2 Trituration with O four times gave the methyl ester 9 (9.52 g, 55% yield) as a white solid.

[0351] R f =0.33(SiO 2 ,50:50 hexane / EtOAc); 1 HNMR (300MHz, CDCl 3 ):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)

[0352] [ka]

[0353] Methyl 9,10-dihexanoyloxystearate (10a): In a round-bottom flask under argon, DCC (2.27 g, 11.0 mmol, 2.20 equiv.) was stirred while ice-cold hexanoic acid (1.28 g, 11.0 mmol, 2.20 equiv.) was dissolved in CH 2 Cl2 (13 mL) solution, the ice bath was removed, and the mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath and diol 8 (1.65 g, 5.00 mmol) was added, followed by DMAP (1.53 g, 12.5 mmol, 2.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was cooled in Et 2 The mixture was diluted with 2×200 mL of HCl, stirred for 10 minutes, and then filtered through Celite®. The filtrate was diluted with 1M aqueous hydrochloric acid (2×30 mL), 1M aqueous NaOH ( 2 x 30 mL), H 2 20 (1 × 30 mL), washed with saline, and then washed with Na 2 SO 4 After drying over 50° C., the mixture was concentrated under reduced pressure on a rotary evaporator to give triester 10a (2.61 g, quantitative yield) as a clear, colorless oil.

[0354] R f =0.66(SiO 2 ,70:30 hexane / EtOAc); 1 HNMR (300MHz, CDCl 3 ):δ5.08-4.92(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)

[0355] [ka]

[0356] Methyl 9,10-dilinoleoyloxystearate (10b): In a round-bottom flask under argon, DCC (4.33 g, 21.0 mmol, 2.10 equiv.) was stirred and cooled on ice. 2 Cl 2(25 mL) solution, the ice bath was removed, and the mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath and diol 8 (3.30 g, 10.0 mmol) was added, followed by DMAP (3.05 g, 25.0 mmol, 2.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with hexanes and stirred for 10 min, then washed with Celite® The filtrate was concentrated on a rotary evaporator to a white semi-solid which was purified by filtration through a plug of silica gel (95:5 hexanes / EtOAc) to give triester 10b (7.24 g, 85% yield) as a clear, colorless oil.

[0357] R f =0.57(SiO 2 ,70:30 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0358] [ka]

[0359] Methyl 9,10,12R-trihexanoyloxystearate (11): In a round-bottom flask under argon, DCC (2.64 g, 12.8 mmol, 3.20 equiv.) was stirred while ice-cold hexanoic acid (1.49 g, 12.8 mmol, 3.20 equiv.) was dissolved in CH 2 Cl 2 (13 mL) solution, the ice bath was removed, and the mixture was stirred for 15 min. The reaction mixture was cooled again 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 equiv.) and the reaction mixture was allowed to warm to room temperature. The reaction mixture was heated for 14 hours. The reaction mixture was diluted with hexane, stirred for 10 minutes, and then filtered through Celite®. The filtrate was diluted with 1M aqueous HCl (2×30 mL), 1M aqueous NaOH ( 2 x 30 mL), H 2 20 mL, washed with brine, and then washed with Na 2 SO 4 The mixture was dried over ice and concentrated on a rotary evaporator under reduced pressure to give triester 11 (1.99 g, 78% yield) as a clear, colorless oil.

[0360] R f =0.77(SiO 2 ,70:30 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0361] [ka]

[0362] 9,10-Dihexanoyloxystearic acid (12a): A solution of triester 10a (1.05 g, 2.00 mmol, 1.10 equiv) in t-BuOH (7 mL) at room temperature in a round-bottom flask under argon was diluted with 2.0 M aqueous KOH (0.9 mL). After stirring for 20 hours, the mixture was acidified to pH ≦2 with 3M aqueous hydrochloric acid, and Et 2 The combined organic layers were washed with brine and extracted with NaO (3×20 mL). 2 SO 4 The mixture was dried over ice and concentrated under reduced pressure on a rotary evaporator. The crude residue was purified by flash column chromatography (90:5:5 to 85:10:5 hexanes / EtOAc / MeOH) to give carboxylic acid 12a (802 mg, 86% yield) as a clear, colorless oil.

[0363] R f =0.22(SiO 2 ,85:10:5 Hexane / EtOAc / MeOH); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0364] [ka]

[0365] 9,10-Dilinoleoyloxystearic acid (12b): A solution of triester 10b (5.64 g, 6.60 mmol, 1.10 equiv) in t-BuOH (7 mL) at room temperature in a round-bottom flask under argon was diluted with 2.0 M aqueous KOH (3.0 After stirring for 20 h, the reaction mixture was acidified to pH ≦2 by addition of 3 M aqueous HCl and diluted with hexane (3×75 mL). The combined organic layers were washed with brine and extracted with Na 2 SO 4 Dry on a rotary evaporator under reduced pressure. The crude residue was purified by flash column chromatography (90:10:0 to 85:10:5 hexanes / EtOAc / MeOH) to give carboxylic acid 12b (2.39 g, 68% yield) as a clear, colorless oil.

[0366] R f =0.33(SiO 2 ,85:10:5 Hexane / EtOAc / MeOH); 1 H NMR (300 MHz, CDCl 3 ): 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,4 H), 2.13-2.00(m,8H), 1.72-1.45(m,10H), 1.45-1.15(m,50H), 0.98-0.81(m,9H)

[0367] [ka]

[0368] 9,10,12R-trihexanoyloxystearic acid (13): A solution of tetraester 11 (1.98 g, 3.10 mmol, 1.10 equiv) in t-BuOH (10 mL) at room temperature in a round-bottom flask under argon was treated with 2.0 M aqueous KOH (1. After stirring for 20 h, the reaction mixture was acidified to pH ≦2 by addition of 3 M aqueous HCl and hexanes (3×30 mL) were added. The combined organic layers were washed with brine and extracted with Na 2 SO 4 The mixture was dried over ice and concentrated under reduced pressure on a rotary evaporator. The crude residue was purified by flash column chromatography (90:10:0→85:10:5→75:20:5 hexanes / EtOAc / MeOH) to give carboxylic acid 13 (1.40 g, 78% yield) as a clear, colorless oil.

[0369] R f =0.32(SiO 2 ,80:15:5 Hexane / EtOAc / MeOH); 1 H NMR (300 MHz, CDCl 3 ): δ5.13-4.82(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)

[0370] Example 2A: Synthesis of INT-D047

[0371] [ka]

[0372] (R,Z)-12-acetoxyoctadec-9-en-1-yl(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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl)succinate (INT-D047):

[0373] According to general procedure 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 CH 2 Cl 2 (4 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D047 (541 mg, 90% yield) was obtained as a clear, colorless oil.

[0374] R f =0.36(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.21(d, J=10.1Hz), 6 .36(dd, J=10.2, 1.7Hz), 6.13(s,1H), 5.58-5.43(m,1H), 5.43-5.28(m,1H), 4.92(s ,2H), 4.89(quint,J=6.4Hz), 4.45-4.34(m,1H), 4.11(t,J=6.7Hz,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),2.11-1.99(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)

[0375] Example 2B: Synthesis of INT-D046

[0376] [ka]

[0377] (R,Z)-12-(dodecanoyloxy)octadec-9-en-1-yl(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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl)succinate (INT-D046):

[0378] According to general procedure 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 CH 2 Cl 2 (2 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D046 (363 mg, 96% yield) was obtained as a clear colorless oil.

[0379] R f=0.48(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.21(d,J=10.1Hz),6 .36(dd,J=10.2,1.7Hz),6.13(s,1H),5.57-5.42(m,1H),5.40-5.28(m,1H),4.92(s,2H),4.90(quint,J=6.4Hz),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)

[0380] Example 2C: Synthesis of INT-D050

[0381] [ka]

[0382] 2-((8S,9R,10S,11S,13S,14S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,10,11,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl((R,Z)-12-(stearoyloxy)octadec-9-en-1-yl)succinate (INT-D050): JZ-25-009

[0383] According to general procedure 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 CH 2 Cl 2 (5 mL), and then subjected to flash column chromatography (SiO 2After purification (80:20→50:50 hexanes / EtOAc), INT-D050 (933 mg, 91% yield) was obtained as a clear colorless oil.

[0384] R f =0.42(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.22(d,J=10.1Hz),6 .36(dd,J=10.1,1.5Hz),6.13(s,1H),5.55-5.41(m,1H),5.40-5.27(m,1H),4.93(s,2H),4.89(quint,J=6.2Hz),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)

[0385] Example 2D: Synthesis of INT-D035

[0386] [ka]

[0387] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl(((R,Z)-12-(oleoyloxy)octadec-9-en-1-yl)succinate (INT-D035):

[0388] According to general procedure 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 CH 2 Cl 2 (2 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D035 (330 mg, 95% yield) was obtained as a clear colorless oil.

[0389] R f =0.49(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.22(d,J=10.1Hz),6 .36(dd,J=10.2,1.8Hz),6.13(s,1H),5.55-5.26(m,4H),4.92(s,2H),4.89(quint,J=6.3Hz),4.44-4.34(m,1H),4.11(t,J=6.8Hz,2 H),3.20-3.04(m,1H),2.85-2.54(m,5H),2.54-1.94(m,13H),1.92-1.46(m,16H),1.44-1.16(m,36H),1.06(s,3H),0.99-0.81(m,9H)

[0390] Example 2E: Synthesis of INT-D045

[0391] [ka]

[0392] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl(((R,Z)-12-linoleoyloxy)octadec-9-en-1-yl)succinate (INT-D045):

[0393] According to general procedure 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 CH 2 Cl 2 (2 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D045 (278 mg, 68% yield) was obtained as a clear colorless oil.

[0394] R f =0.50(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.22(d, J=10.1Hz), 6 .36(dd, J=10.2, 1.8Hz), 6.13(s,1H), 5.56-5.25(m,6H), 4.93(s,2H), 4.89(quint,J=6.3Hz), 4.46-4.31(m,1H), 4.10(t,J=6.8Hz,2 H), 3.20-3.04(m,1H), 2.88-2.54(m,7H), 2.53-1.91(m,15H), 1.90-1.46(m,14H),1.47-1.12(m,34H),1.06(s,3H),0.99-0.81(m,9H)

[0395] Example 2F: Synthesis of INT-D049

[0396] [ka]

[0397] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl(((R,Z)-12-(linolenoyloxy)octadec-9-en-1-yl)succinate (INT-D049):

[0398] According to general procedure 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 CH 2 Cl 2 (4 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D049 (740 mg, 96% yield) was obtained as a clear colorless oil.

[0399] R f =0.42(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.21(d,J=10.1Hz),6 .36(dd,J=10.2,1.8Hz),6.13(s,1H),5.56-5.25(m,8H),4.93(s,2H),4.89(quint,J=6.3Hz),4.46-4.32(m,1H),4.10(t,J=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)

[0400] Example 2G: Synthesis of INT-D051

[0401] [ka]

[0402] 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl((R,Z)-12-(arachidonoyloxy)octadec-9-en-1-yl)succinate (INT-D051):

[0403] According to general procedure 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 CH 2 Cl 2 (5 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D051 (758 mg, 94% yield) was obtained as a clear colorless oil.

[0404] R f =0.29(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.22(d,J=10.2Hz),6 .36(dd,J=10.2,1.8Hz),6.13(s,1H),5.56-5.26(m,10H),4.93(s,2H),4.88(quint,J=6.3Hz),4.43-4.33(m,1H),4.10(t,J=6.9Hz,2 H),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)

[0405] Example 2H: Synthesis of INT-D055

[0406] [ka]

[0407] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl((R,Z)-12-hexanoyloxyoleate (INT-D055):

[0408] In an argon flushed round bottom flask, carboxylic acid 7a (114 mg, 0.30 mmol, 1.20 equiv) and CH 2 Cl 2 (1.2 mL) was filled and cooled on ice. To this flask was added DCC (63 mg, 0.30 mmol, 1.2 equiv.) and left to stir at ambient room temperature for 15 min. The flask was cooled again in an ice bath and diluted with CH, which had been prepared in a separate argon-flushed round-bottom flask. 2 Cl 2 A mixture of dexamethasone (99 mg, 0.25 mmol, 1.00 equiv) and DMAP (47 mg, 0.38 mmol, 1.50 equiv) in Et (1.3 mL) was added via syringe. After 17 h, the reaction was diluted with Et 2 The mixture was diluted with 200 mL of ethyl acetate, filtered through Celite®, and concentrated under reduced pressure on a rotary evaporator. The crude residue was purified using hexanes / EtOAc (80:20→50:50 hexanes / EtOAc) to give INT-D055 as a slightly yellow viscous oil (185 mg, 96% yield).

[0409] R f : 0.15(SiO 2 ,70:30 hexane:EtOAc); 1H (300MHz, CDCl 3 ):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)

[0410] Example 2I: Synthesis of INT-D089

[0411] [ka]

[0412] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl (RZ)-12-linoleoyloxyoleate (INT-D089):

[0413] In an argon flushed round bottom flask, carboxylic acid 7b (600 mg, 1.07 mmol, 1.20 equiv) and CH 2 Cl 2 (4.4 mL) and cooled in an ice bath. To this flask was added DCC (221 mg, 1.07 mmol, 1.20 equiv) and left to stir at ambient room temperature for 15 min. The flask was cooled again in an ice bath and another argon flushed round bottom flask was charged with CH 2 Cl 2A mixture of dexamethasone (350 mg, 0.89 mmol, 1.00 equiv) and DMAP (163 mg, 1.34 mmol, 1.50 equiv) in 4.5 mL was added via syringe. After 17 h, the reaction was diluted with hexanes, filtered through Celite®, and concentrated under reduced pressure on a rotary evaporator. Flash column The crude residue was purified using (80:20→50:50 hexanes / EtOAc) to afford INT-D089 as a slightly yellow viscous oil (750 mg, 90% yield).

[0414] R f : 0.46 (silica, 50:50 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):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)

[0415] Example 2J: Synthesis of INT-D085

[0416] [ka]

[0417] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-1-oxooctadecane-9,10-diyl dihexanoate (INT-D085):

[0418] According to general procedure E, dexamethasone (235 mg, 0.60 mmol), carboxylic acid 1 2a (338mg, 0.66mmol), DCC (136mg, 0.66mmol), DMAP (110mg, 0.90mmol) and CH 2 Cl 2 (6 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D085 (336 mg, 63% yield) was obtained as a clear, colorless oil.

[0419] R f =0.52(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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),4.44-4.32(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)

[0420] Example 2K: Synthesis of INT-D086

[0421] [ka]

[0422] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-1-oxooctadecane-9,10-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (INT-D086):

[0423] According to general procedure 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 CH 2 Cl 2 (6 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D086 (584 mg, 80% yield) was obtained as a clear, colorless oil.

[0424] R f =0.28(SiO 2 ,70:30 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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,2 H),4.90(s,2H),4.44-4.31(m,1H),3.21-3.02(m,1H),2.78(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)

[0425] Example 2L: Synthesis of INT-D056

[0426] [ka]

[0427] 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-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 9,10,12R-trihexanoyloxystearate (INT-D056):

[0428] According to general procedure E, dexamethasone (175 mg, 0.44 mmol), carboxylic acid 13 (307 mg, 0.49 mmol), DCC (101 mg, 0.49 mmol), DMAP (82 mg, 0.67 mmol), CH 2 Cl 2 (5 mL), and then subjected to flash column chromatography (SiO 2 After purification (80:20→50:50 hexanes / EtOAc), INT-D056 (318 mg, 90% yield) was obtained as a clear, colorless oil.

[0429] R f =0.50(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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),2.6 1(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)

[0430] Example 2M: Synthesis of INT-D059

[0431] [ka]

[0432] 2-((8S,9R,10S,11S,13S,14S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl((12S,Z)-12-(((12S)-9,10,12-tris(hexanoyloxy)octadecanoyl)oxy)octadec-9-en-1-yl)succinate (INT-D059):

[0433] According to general procedure E, dexamethasone (137 mg, 0.35 mmol), the hemisuccinate derived from ricinoleyl alcohol 2 and carboxylic acid 13 (382 mg, 0.38 mmol), DCC (79 mg, 0.38 mmol), DMAP (64 mg, 0. 52 mmol) and CH 2 Cl 2 (3.5 mL), and then subjected to flash column chromatography (SiO 2 After purification (70:30→50:50 hexanes / EtOAc), INT-D059 (336 mg, 66% yield) was obtained as a clear, colorless oil.

[0434] R f =0.50(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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,1H),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,2 H),1.93-1.43(m,32H),1.43-1.16(m,62H),1.05(s,3H),0.99-0.81(m,21H)

[0435] Example 2N: Synthesis of INT-D060

[0436] [ka]

[0437] (R,Z)-12-Hexanoyloxyoctadec-9-en-1-yl 2-acetoxybenzoate (INT-D060): A solution of HF·pyridine (0.53 mL, 4.20 mmol, 3.00 equiv. of 70% HF in pyridine) was added to a stirred solution of pyridine (0.34 mL, 4.20 mmol, 3.00 equiv.) and silyl ether 4b (700 mg, 1.41 mmol) in ice-cold THF (7 mL) in a round-bottom flask under argon. When TLC indicated uptake of starting material (2–8 h), the reaction was diluted with saturated NaHCO 3 The mixture was quenched with Et 2 After extraction with 2×10 mL of H2O, the combined organic extracts were washed with H2O. 2 20 mL, washed with brine, and then washed with Na 2 SO 4 The crude was purified by filtration through a plug of silica gel (90:10 hexanes / EtOAc) and concentrated on a rotary evaporator to give the intermediate primary alcohol (518 mg) as a clear, colorless oil, which was used without further purification.

[0438] In a flame-dried, argon-flushed round-bottom flask, add the above alcohol, pyridine (0.22 mL, 2.70 mmol, 2.00 equiv.) and CH 2 Cl 2 (6 mL) and cooled in an ice bath. The round-bottom flask was equipped with an addition funnel and charged with acetylsalicyloyl chloride (541 mg, 2.73 mmol, 2.00 equiv), which had been prepared in a separate argon-flushed round-bottom flask, in CH 2 Cl 2 (7.5 mL) solution was charged dropwise over 15 min. After 16.5 h, the reaction solvent was removed under reduced pressure on a rotary evaporator. The crude residue was purified by two successive flash column chromatography runs (first 90:10 hexanes / EtOAc, then 95:5 hexanes / EtOAc) to give INT-D060 as a pale yellow oil (557 mg, 76% yield).

[0439] R f : 0.60(SiO 2 ,70:30 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):δ8.02(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(quint,J=6.2Hz,1H),4.27(t,J=6.7Hz,2H),2.35(s,3H),2.33-2.21(m,4H),2.09-1.96(m, 2H),1.74(quint,J=7.1Hz,2H),1.62(quint,J=7.3Hz,2H),1.58-1.48(m,2H),1.48-1.16(m,22H),0.97-0.80(m,6H)

[0440] Example 2O: Synthesis of INT-D061

[0441] [ka]

[0442] (R,Z)-12-(linoleoyloxyoctadec-9-en-1-yl 2-acetoxybenzoate (INT-D061): A solution of HF·pyridine (0.39 mL, 3.20 mmol, 3.00 equiv. of 70% HF in pyridine) was added to a stirred solution of pyridine (0.26 mL, 3.20 mmol, 3.00 equiv.) and silyl ether 4f (700 mg, 1.06 mmol) in ice-cold THF (5 mL) in a round-bottom flask under argon. When TLC indicated uptake of starting material (2–8 h), the reaction was diluted with saturated NaHCO 3 The mixture was quenched with Et 2 After extraction with 2×10 mL of H2O, the combined organic extracts were washed with H2O. 2 20 mL, washed with brine, and then washed with Na 2 SO 4 The crude was purified by filtration through a plug of silica gel (90:10 hexanes / EtOAc) and concentrated on a rotary evaporator to give the intermediate primary alcohol (553 mg) as a clear, colorless oil, which was used without further purification.

[0443] In an argon flushed round bottom flask, add the above alcohol (553 mg, 1.01 mmol, 1.00 equiv), pyridine (0.13 mL, 1.7 mmol, 1.60 equiv), and CH 2 Cl 2 (4 mL) and cooled in an ice bath. In a separate argon flushed round bottom flask, acetylsalicyloyl chloride (327 mg, 1.65 mmol, 1.63 equiv) in CH 2 Cl 2 A (6 mL) solution was prepared and slowly transferred in portions via syringe to the other round bottom flask over 15 min. After 18 h, the reaction solvent was removed on a rotary evaporator under reduced pressure. The crude residue was purified by two successive flash column chromatography runs (95:5 hexanes / EtOAc) to give INT-D061 as a pale yellow oil (516 mg, 72% yield).

[0444] R f : 0.56(SiO 2 ,70:30 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):δ8.03(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(quint,J=6.2Hz,1H),4.28(t,J= 6.7Hz,2H),2.79(t,J=5.9Hz,2H),2.36(s,3H),2.34-2.21(m,4H),2.12-1.96(m,6H) ,1.75(quint,J=7.1Hz,2H),1.69-1.49(m,4H),1.49-1.18(m,32H),0.98-0.81(m,6H)

[0445] [ka]

[0446] (E)-6-(4-tert-butyldimethylsilyloxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoic acid (14): A flame-dried, argon-flushed round-bottom flask was charged with DMF (0.98 mL), imidazole (159 mg, 2.34 mmol, 7.50 equiv), and mycophenolic acid (100 mg, 0.312 mmol, 1.00 equiv), and to this mixture was added TBSCl (282 mg, 1.87 mmol, 6.00 equiv). After 1 h, the reaction mixture (10 mL) was diluted with Et 2 The combined organics were washed with water (3 × 10 mL), brine (1 × 10 mL), and extracted with Na 2 SO 4 The crude residue was taken up in tetrahydrofuran (0.60 mL) and stirred with water (0.60 mL) and acetic acid (0.60 mL) for 1 h. It was then dissolved in water (1×10 mL) and Et 2The combined organics were washed with water (5 × 10 mL), brine (1 × 10 mL), and extracted with Na 2 SO 4 The mixture was dried over hexanes and concentrated under reduced pressure on a rotary evaporator. The crude residue was purified by flash column chromatography (80:20 to 20:80 hexanes / EtOAc) to give mycophenolic acid silyl ether (14) (121 mg, 89% yield) as a white solid.

[0447] R f : 0.36(SiO 2 ,50:50 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):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)

[0448] Example 2P: Synthesis of INT-D062

[0449] [ka]

[0450] (12R) Hexanoyloxyoleyl (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (INT-D062):

[0451] A solution of HF·pyridine (0.11 mL, 0.91 mmol, 3.00 equiv. of 70% HF in pyridine) was added to a stirred solution of pyridine (0.07 mL, 0.91 mmol, 3.00 equiv.) and silyl ether 4b (150 mg, 0.30 mmol) in ice-cold THF (1.5 mL) in a round-bottom flask under argon. When TLC indicated uptake of starting material (2–8 h), the reaction was diluted with saturated NaHCO 3The mixture was quenched with Et 2 After extraction with 2×5 mL of H2O, the combined organic extracts were washed with H2O. 2 2H2O (1 × 5 mL), washed with brine, and then washed with Na 2 SO 4 The crude was purified by filtration through a plug of silica gel (90:10 hexanes / EtOAc) and concentrated on a rotary evaporator to give the crude primary alcohol. Concentration at rt gave the intermediate primary alcohol (102 mg) as a clear colorless oil which was used without further purification.

[0452] In an argon-flushed round-bottom flask cooled in an ice bath, add CH 2 Cl 2 The flask was charged with 1.2 mL of DCC and mycophenolic acid silyl ether (14) (105 mg, 0.24 mmol, 1.00 equiv). DCC (50 mg, 0.24 mmol, 1.00 equiv) was added to the flask and the ice bath was removed. After 15 min, the ice bath was replaced under the flask and the above alcohol (102 mg, 0.267 mmol, 1.10 equiv) and DMAP (44 mg, 0.36 mmol, 1.50 equiv) were added to CH 2 Cl 2 (1.2 mL) solution was added. After 15.5 h, the reaction mixture was concentrated under reduced pressure. The crude residue was diluted with hexane (4 volumes), filtered through Celite®, and then concentrated under reduced pressure on a rotary evaporator. The residue was purified by flash column chromatography. Chromatograph (85:15 hexanes / EtOAc) and combine and concentrate the product containing fractions.

[0453] The residue was transferred to a round-bottom flask and flushed with argon. 2 Cl 2 (1.5 mL) and pyridine (0.06 mL, 0.7 mmol, 2.88 equiv.) were added and cooled in an ice-water bath. Benzoyl chloride (0.05 mL, 0.50 mmol, 2.06 equiv.) was then added to the flask. After 18 h, the reaction mixture was concentrated under reduced pressure on a rotary evaporator. The crude residue was purified by evaporation of Et 2The combined organics were extracted with 1M HCl (1×10 mL), 1M NaOH (1×10 mL), water (1×10 mL), and then washed with 1M HCl (1×10 mL). Wash with brine (1 × 10 mL) and add Na 2 SO 4 It was dried over silica gel and concentrated under reduced pressure on a rotary evaporator.

[0454] The crude residue was flushed with argon and taken up in tetrahydrofuran (1.4 mL) in a round-bottom flask cooled in an ice-water bath. Pyridine (0.07 mL, 0.80 mmol, 3.29 equiv.) and HF·pyridine (0.10 mL of 70% HF, 0.83 mmol, 3.42 equiv.) were added to the flask and the ice bath was removed. After 2 h, saturated NaHCO was added until bubbling ceased. 3 The aqueous solution was slowly added to the reaction mixture. 2 The mixture was extracted with 2×O (1×10 mL) and the combined organic layers were washed with 1 M aqueous hydrochloric acid (1×10 mL), water (1×10 mL), and brine (1×10 mL), and then with Na 2 SO 4 The mixture was dried at 40° C. and concentrated under reduced pressure on a rotary evaporator. The resulting residue was purified by flash column chromatography (90:10→80:20 hexanes / EtOAc) to afford INT-D062 as a clear, colorless oil (100 mg, 60% yield over three steps).

[0455] R f : 0.22 (silica, 80:20 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):δ7.69(s,1H),5.55-5.41(m,1H),5.41-5.17(m,4H),4.90(quint,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)

[0456] Example 2Q: Synthesis of INT-D063

[0457] [ka]

[0458] (12R) Linoleoyloxyoleyl (E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydroisobenzofuran-5-yl)-4-methylhex-4-enoate (INT-D063): To a stirred, ice-cold solution of pyridine (0.05 mL, 0.60 mmol, 3.00 equiv) and silyl ether 4f (125 mg, 0.19 mmol) in THF (1.5 mL) in a round-bottom flask under argon, HF·pyridine solution (70% HF in pyridine, 0.07 mL, 0.60 mmol, 3.00 equiv) was added. When TLC indicated uptake of starting material (2–8 h), the reaction was diluted with saturated NaHCO 3 The mixture was quenched with Et 2 After extraction with 2×10 mL of H2O, the combined organic extracts were washed with H2O. 2 20 mL, washed with brine, and then washed with Na 2 SO 4 The crude was purified by filtration through a plug of silica gel (90:10 hexanes / EtOAc) and concentrated on a rotary evaporator to give the intermediate primary alcohol (95 mg) as a clear, colorless oil, which was used without further purification.

[0459] In an argon-flushed round-bottom flask cooled in an ice bath, add CH 2 Cl 2 The flask was charged with 1,000 mL of 1,000 sulphate (0.5 mL) and mycophenolic acid silyl ether (14) (68 mg, 0.16 mmol, 1.00 equiv). DCC (32 mg, 0.16 mmol, 1.00 equiv) was added to the flask and the ice bath was removed. After 15 min, the ice bath was replaced under the flask and the above alcohol and DMAP (29 mg, 0.24 mmol, 1.50 equiv) were added in CH 2 Cl2 (1 mL) solution was added. After 19 h, the reaction mixture was concentrated under reduced pressure. The crude residue was diluted with hexanes (4 volumes), filtered through Celite®, and then concentrated under reduced pressure on a rotary evaporator. was subjected to flash column chromatography (85:15 hexanes / EtOAc) and the product containing fractions were combined and concentrated.

[0460] The residue was transferred to a round-bottom flask and flushed with argon. 2 Cl 2 (1 mL) and pyridine (0.03 mL, 0.40 mmol, 2.50 equiv.) were added and cooled in an ice-water bath. Benzoyl chloride (0.03 mL, 0.20 mmol, 1.25 equiv.) was then added to the flask. After 18 h, the reaction mixture was concentrated under reduced pressure on a rotary evaporator. The crude residue was purified by evaporation of Et 2 The combined organics were extracted with 1M aqueous hydrochloric acid (1×10 mL), 1M NaOH (1×10 mL), and water (1×10 mL). ), washed with brine (1 × 10 mL), and then with Na 2 SO 4 It was dried over silica gel and concentrated under reduced pressure on a rotary evaporator.

[0461] The crude residue was taken up in tetrahydrofuran (1 mL) in a round-bottom flask that had been flushed with argon and cooled in an ice-water bath. Pyridine (0.04 mL, 0.50 mmol, 3.13 equiv.) and HF·pyridine (0.06 mL of 70% HF, 0.50 mmol, 3.13 equiv.) were added to the flask and the ice bath was removed. After 2 h, saturated NaHCO was added until bubbling ceased. 3 The reaction mixture was diluted with Et 2 The mixture was extracted with 2×O (1×10 mL) and the combined organic layers were washed with 1 M aqueous hydrochloric acid (1×10 mL), water (1×10 mL), and brine (1×10 mL), and then with Na 2 SO 4The mixture was dried at 4° C. and concentrated under reduced pressure on a rotary evaporator. The resulting residue was purified by flash column chromatography (90:10 hexanes / EtOAc) to give INT-D063 as a clear, colorless oil (66 mg, 50% yield over three steps).

[0462] R f : 0.17(SiO 2 ,85:15 hexane:EtOAc); 1 H (300MHz, CDCl 3 ):δ7.69(s,1H),5.55-5.17(m,9H),4.90(quint,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),2.36-2.2 3(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)

[0463] Example 2R: Synthesis of INT-D065

[0464] [ka]

[0465] (4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)amino)-2-(((R,Z)-12-(((9Z,12Z)-octadec-9,12-dienoyl)oxy)octadec-9-enoyl)oxy)-3-phenylpropionate lopanoyl)oxy)-4,6,11-trihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxeth-12-yl benzoate (INT-D065):

[0466] Et 3 N (0.10 mL, 0.75 mmol, 2.50 equiv.) followed by Mukaiyama's reagent (100 mg, 0.39 mmol, 1.30 equiv.) was added to a round-bottom flask under argon to dissolve docetaxel (242 mg, 0.30 mmol) and 12R-linoleoyloxyoleic acid 7b (202 mg, 0.36 mmol, 1.20 equiv.) in CH at room temperature. 2 Cl 2 (3 mL) was added to the solution. After stirring for 14 h, the reaction mixture was diluted with EtOAc, filtered through Celite®, and concentrated under reduced pressure on a rotary evaporator. Flash column chromatography (SiO 2 The crude residue was purified by elution with hexanes / EtOAc (80:20→50:50 hexanes / EtOAc) to give INT-D065 as a clear, colorless oil (243 mg, 60% yield).

[0467] R f =0.45(SiO 2 ,50:50 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ8.12(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(quint,J=6.2Hz,1H),4.39-4.16(m,4H),3.95(d,J=6.9Hz,1H),2.79(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)

[0468] Example 2S: Synthesis of INT-D053

[0469] [ka]

[0470] (1R,3S,Z)-3-Hydroxy-5-(2-((1R,3aS,7aR,E))-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexyl (R,Z)-12-acetoxyoctadec-9-enoate and (1S,5R,Z)- 5-Hydroxy-3-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-2-methylenecyclohexyl (R,Z)-12-acetoxyoctadec-9-enoate (INT-D053): JZ-25-057,029

[0471] DCC (50 mg, 0.24 mmol, 1.20 equiv.) was stirred in a round-bottom flask with (12R)-acetoxyoleic acid (82 mg, 0.24 mmol, 1.20 equiv.) under argon and cooled in ice in a 1:1 mixture with CH 2 Cl 2 The reaction mixture was cooled again in an ice bath and solid calcitriol (83 mg, 0.20 mmol) and DMAP (29 mg, 0.24 mmol, 1.20 equiv.) were added. The reaction mixture was warmed for 14 h, diluted with EtOAc, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to a pale yellow oil and purified by flash column chromatography. Topography (SiO 2 , 80:20→65:35 hexanes / EtOAc) to afford a .about.1:1 mixture of 1- and 3-acylated conjugates (61 mg, 41% yield) as a clear, colorless oil.

[0472] R f =0.33(SiO 2,60:40 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ6.44-6.25(m,2H),6. 02(d,J=11.2Hz,1H),5.92(d,J=11.2Hz,1H),5.56-5.40(m,3H),5.40-5.27(m,4H),5.2 6-5.16(m,1H),5.07-4.97(m,2H),4.87(quint,J=6.2Hz,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,25 H),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)

[0473] Example 2T: Synthesis of INT-D068

[0474] [ka]

[0475] (R,Z)-18-(((1R,3S,Z)-3-hydroxy-5-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexyl)oxy)-18-oxooctadec-9-en-7-yl linoleate and (R,Z )-18-(((1S,5R,Z)-5-hydroxy-3-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-2-methylenecyclohexyl)oxy)-18-oxooctadec-9-en-7-yl linoleate (INT-D068):

[0476] DCC (50 mg, 0.24 mmol, 1.20 equiv.) was stirred in a round-bottom flask with (12R)-linoleoyloxyoleic acid (135 mg, 0.24 mmol, 1.20 equiv.) under argon and cooled in ice in a 1:1 CH 2 Cl 2 The reaction mixture was cooled again in an ice bath and solid calcitriol (83 mg, 0.20 mmol) and DMAP (29 mg, 0.24 mmol, 1.20 equiv.) were added. The reaction mixture was warmed for 14 h, diluted with EtOAc, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to a pale yellow oil and then fractionated. Powder column chromatography (SiO 2 , 95:5→90:10→70:30 hexanes / EtOAc) to afford a ∼1:1 mixture of 1- and 3-acylated conjugates (75 mg, 39% yield) as a clear, colorless oil.

[0477] R f =0.26(SiO 2 ,70:30 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ6.43-6.26(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(quint,J=6.2Hz,2H),4.47-4.34(m,1H),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)

[0478] Example 2U: Synthesis of INT-D070

[0479] [ka]

[0480] 3-Fluorobenzyl isothiocyanate (15):

[0481] To a solution of 3-fluorobenzylamine (750 mg, 6.00 mmol) in ice-cold THF (10 mL) in a round-bottom flask under argon, 3 N (2.75 mL, 3.30 mmol, 3.30 equiv) was added. A solution of carbon disulfide (0.45 mL, 7.20 mmol, 1.20 equiv) in tetrahydrofuran (10 mL) was then added via syringe pump over 30 min. The reaction mixture was allowed to warm to room temperature and after 3 h the mixture was cooled again in an ice bath and tosyl chloride (1.26 g, 7.20 mmol, 1.20 equiv) was added. After an additional 3 h, 1M Aqueous HCl (10 mL) was added and the reaction mixture was extracted with ethyl acetate (3×10 mL). The mixed organic matter was washed with salt water and 2 SO 4 The mixture was dried over ice and concentrated under reduced pressure on a rotary evaporator. The crude product was purified by flash column chromatography (98:2 to 96:4 hexanes / EtOAc) to give isothiocyanate 15 (846 mg, 84% yield) as a clear, colorless oil. R f =0.45(SiO 2 ,80:20 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): δ7.42-7.35(m,1H),7. 13-7.04(m,3H),7.74(s,2H)

[0482] [ka]

[0483] 3-(3-fluorobenzyl)-2-thioxothiazolidin-4-one (16): Et in a round-bottom flask under argon 3 To an ice-cold mixture of N (0.90 mmol, 6.40 mmol, 2.00 equiv) and water (10 mL) was added thioglycolic acid (0.17 mL, 2.40 mmol, 0.75 equiv) and a solution of isothiocyanate 15 (539 mg, 3.20 mmol) in THF (5 mL) was added over 5 min. The reaction mixture was allowed to warm to room temperature until it turned pale orange in color. The pH was adjusted to ≤ 2 by the addition of 6 M aqueous HCl. The reaction mixture was heated under reflux for 14 h, then cooled to room temperature and extracted with EtOAc (3×10 mL). The combined organics were washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure on a rotary evaporator. The crude product was purified by filtration through silica gel (50:50 hexane / EtOAc) to give rhodanine 16 (466 mg, 80% yield) as a yellow solid.

[0484] R f =0.52(SiO 2 ,70:30 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0485] [ka]

[0486] (Z)-4-((3-(3-fluorobenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)benzoic acid (INT-MA014): To a solution of rhodanine 16 (221 mg, 0.92 mmol) and piperidine (0.01 mL, 0.14 mmol, 0.15 equiv) in EtOH (4 mL) in a round-bottom flask was added 4-carboxybenzaldehyde (151 mg, 1.01 mmol, 1.10 equiv), The mixture was heated at reflux. After 1.5 h, the reaction mixture was concentrated under reduced pressure on a rotary evaporator. The crude liquor was then purified by column chromatography on silica gel (90:8:2 CH 2 Cl 2 The mixture was filtered through a 100% aqueous hexane / MeOH / HOAc column and concentrated under reduced pressure. Precipitation from hot EtOH afforded rhodanine carboxylic acid INT-MA014 (179 mg, 52% yield) as a yellow solid.

[0487] R f =0.26(SiO 2 ,50:40:10 Hexane / EtOAc / MeOH); 1 H NMR (300 MHz, CDCl 3 ): 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)

[0488] [ka]

[0489] 12R-Linoleyloxyoleyl 4-((Z)-(3-(3-fluorobenzyl)-4-oxo-2-thioxothiazolidin-5-ylidene)methyl)benzoate (INT-D070)-JZ-25-169

[0490] i-Pr 2 NEt (0.37 mL, 2.10 mmol, 1.50 equiv) followed by BOP reagent (682 mg, 1.54 mmol, 1.10 equiv) were added to a room temperature solution of the carboxylic acid INT-MA014 (523 mg, 1.40 mmol) and 12R-linoleoyloxyoleyl alcohol (843 mg, 1.54 mmol, 1.10 equiv) in DMF (3.5 mL) in a round bottom flask under argon. After stirring for 14 h, the reaction mixture was diluted with water and extracted with t-BuOMe (3×15 mL). The organic layers were combined, washed with water (4×10 mL), brine (1×10 mL), and concentrated to 100% by weight with NaCl. 2 SO 4The extract was dried over silica gel and concentrated under reduced pressure on a rotary evaporator. 2 The crude residue was purified by elution with hexanes / EtOAc (99:1→95:5 hexanes / EtOAc) to give INT-D070 as a yellow oil (1.18 g, 93% yield).

[0491] R f =0.47(SiO 2 ,90:10 hexane / EtOAc); 1 H NMR (300 MHz, CDCl 3 ): 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),4.90(quint,J=6.2Hz,1 H),4.36(t,J=6.7Hz,2H),2.79(t,J=5.9Hz,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)

[0492] Example 2V: Synthesis of INT-H001

[0493] [ka]

[0494] 1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-hydroxypropyl)thiourea (15): JZ-25-145 3-Aminopropanol (0.33 mL, 4.40 mmol, 1.10 equiv.) was dissolved in 3,5-bis(trifluoromethyl)phenylisothiocyanate (1.08 g, 4.00 mmol) and Et 3 N (0.61 mL, 4.40 equiv, 1.10 equiv) in MeCN (8 mL) at room temperature. After 14 h, the reaction was diluted with H 2The mixture was diluted with 20 mL of EtOAc and extracted with EtOAc (3×15 mL). The combined organic layers were washed with H 2 HO (1 × 15 mL), washed with brine, and then with Na 2 SO 4 The crude semi-solid was filtered through a plug of silica gel (75:25 EtOAc / Hexanes) and the filtrate was concentrated under reduced pressure and then recrystallized from t-BuOMe / Hexanes to give thiourea 15 (1.18 g, 85% yield) as a white solid.

[0495] 1 H NMR (300 MHz, DMSO-d 6 ):δ10.1(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(quint,J=6.4Hz,2H); 13 C NMR (75.5MHz, DMSO-d 6 ): δ180.4,142.0,13 0.1(q,J=34Hz),123.3(q,J=273Hz),121.7(br),115.9(br),58.7,41.6,31.3.

[0496] [ka]

[0497] 1-(3,5-bis(trifluoromethyl)phenyl)-3-(3-hydroxypropyl)thiourea (INT-H001):

[0498] Carboxylic acid 12b (252 mg, 0.30 mmol, 1.10 equiv.) was stirred in a round-bottom flask under argon and cooled in CH 2 Cl 2To the (3 mL) solution was added DCC (68 mg, 0.33 mmol, 1.10 equiv), the ice bath was removed, and stirred for 15 min. The reaction mixture was cooled again in an ice bath, and thiourea 15 (114 mg, 0.33 mmol) and DMAP (44 mg, 0.36 mmol, 1.20 equiv) were added. The reaction mixture was warmed for 14 h, diluted with t-BuOMe, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to a slightly yellow oil and purified by flash column chromatography (SiO 2 , 80:18:2 hexanes / EtOAc / MeOH) to give INT-H001 (222 mg, 63% yield) as a clear, colorless oil.

[0499] R f =0.35(SiO 2 ,80:18:2 Hexane / EtOAc / MeOH); 1 H NMR (300 MHz, CDCl 3 ):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=5.9Hz,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)

[0500] Example 2W: Synthesis of Disubstituted Calcitriol, INT-D087 A synthetic scheme for preparing calcitriol lipid conjugates disubstituted with two lipid moieties. Here is an example:

[0501] [ka]

[0502] INT-D087 Example 3: Formulation of prodrugs in lipid nanoparticles (LNPs) The lipid-like properties of the prodrugs allow them to be easily loaded in LNP systems by simply mixing with lipid formulation components. That is, in some embodiments, loading can be achieved without further modification of known formulation processes. As a result, LNPs incorporating these drug-lipid conjugates can be made using widely described formulation methodologies, including but not limited to extrusion, ethanol injection, and in-line mixing.

[0503] LNPs were prepared by dissolving 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-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).

[0504] 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, drug-lipid conjugates, and PEG-DSPE (molar ratio 49 / 40 / 10 / 1) with phosphate buffered saline (PBS) using a cross-section junction mixer. The formulations were dialyzed against PBS to remove residual ethanol. In formulations with greater than 10 mol % drug-lipid conjugate, the amount of phospholipid or cholesterol was reduced accordingly.

[0505] The physicochemical properties of the LNPs prepared as above were subsequently characterized. After buffer exchange into phosphate-buffered saline, particle size was measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK). Number-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 LNP formulations containing LD-DEX was examined by cryo-transmission electron microscopy (cryoT The results were analyzed using EM.

[0506] Table 4 below shows that the prodrugs described herein can be formulated into LNPs with high encapsulation efficiency and low polydispersity, both of which are desirable physicochemical properties for a drug delivery vehicle.

[0507] Table 4. Physicochemical parameters of LNPs containing 10 mol% ricinoleyl-dexamethasone conjugate.

[0508] [Table 4-1]

[0509] [Table 4-2]

[0510] [Table 4-3]

[0511] Example 4: Prodrugs forming monodisperse LNPs of novel polymeric structures A prodrug bearing a hexanoyl group, INT-D034, (Figure 3A), was mixed with neutral phospholipids and cholesterol at 0-99 mol% prodrug concentrations using the rapid mixing technique set out in Example 3 to produce monodisperse LNP formulations (Figure 4). All INT-D034 formulations showed high encapsulation efficiency, with particle sizes ranging from about 29-87 nm and polydispersity index (PDI) of 0.1 or less (Table 5 below). Electron micrographs of the LNP formulations show that as the amount of INT-D034 increases, a spherical electron-dense region immediately beneath the membrane expands, suggesting that the prodrug INT-D034 exists as a hydrophobic oil phase in the LNP lipid bilayer (Figure 4).

[0512] Table 5: Particle size and polydispersity index of LNPs containing various amounts of prodrug.

[0513] [Table 5-1]

[0514] [Table 5-2]

[0515] [Table 5-3]

[0516] [Table 5-4]

[0517] To determine whether this new superstructure is consistent with other ricinoleyl-based conjugates, INT-D035 (with the R hydrocarbon derived from an oleoyl group instead of the hexanoyl group in INT-D034 as in Figure 3B) was incorporated into LNPs as described in Example 3 with an equal amount of prodrug (10 mol%). Similar to the INT-D034 formulation, the INT-D035 formulation was also observed to exhibit a globular electron-dense region just beneath the membrane (Figure 5). These results indicate that ricinoleyl-based conjugates have the appropriate properties to exist as a hydrophobic oil phase in the LNP lipid bilayer.

[0518] Other prodrugs containing various 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 efficiently incorporated into the LNPs up to 99 mol% (Table 5).

[0519] Example 5: Dissociation of Prodrug from LNP as a Function of Hydrophobicity (LogP) of S Group The release of ricinoleyl-dexamethasone conjugates from LNPs was examined using an assay that uses human plasma, which contains lipoproteins, as a lipid sink where lipid exchange occurs. Plasma lacks active esterases that could digest ricinoleyl-dexamethasone conjugates, which would prevent detection and monitoring of the intact conjugate.

[0520] LNP formulations containing 10-99 mol% of 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 (see Figure 3 and Example 7 for structures) were incubated in human plasma at 1.2 mM total lipid for 0, 2 or 24 h at 37 °C. Size exclusion chromatography was performed to separate LNPs from lipoproteins (1.5 x 27 cm Sepharose CL-4B size exclusion column). Thirty fractions of 2 mL were collected. Then, three volumes of isopropanol / methanol (1:1 v / v) were added to each fraction.

[0521] The drug-lipid conjugates were analyzed using a Waters® Acquity equipped with a photodiode array detector (PDA). TM Ultra-high pressure liquid chromatography (UPLC) was used Quantification was performed using Empower TM Data acquisition software version 2.0 was used (Waters, USA). Separation was performed using Waters® Acquity TM BEHC A 18 column (1.7 μm, 2.1 × 100 mm) was used with a flow rate of 0.5 ml / min and a linear gradient from 80 / 20 (%A / B) to 0 / 100 (%A / B). Mobile phase A consisted of water and mobile phase B was methanol / acetonitrile (1:1, v / v). The method was performed at a column temperature of 55 °C for 6 min and the analytes were measured using a PDA detector monitored at a wavelength of 239 nm.

[0522] The amount of each ricinoleyl-dexamethasone conjugate that remained bound to the LNP in each fraction, as quantified by UPLC, is shown in Figure 6. Ricinoleyl-dexamethasone conjugates with various logP values ​​showed different levels of dissociation (Figure 6). Conjugates with higher predicted LogP values ​​(i.e., more hydrophobic) dissociated less from the LNP than those with lower predicted LogP values. For example, more than 90% of INT-D086 (LogP 21.2) remained in the LNP, compared to ~40% of INT-D047 (LogP 8.33) (Figure 6A and Table 6 below). These results indicate that the design of prodrugs based on the scaffolds described here is a reliable method to control drug release from LNPs. In situations where LNPs are required to circulate in the body for extended periods of time to reach disease sites (e.g., distant tumors), it is desirable for the drug to remain bound to the LNP and not exhibit premature drug leakage, which may directly correlate with poor therapeutic activity.

[0523] Table 6: Biophysical parameters of LNP formulations containing prodrugs.

[0524] [Table 6]

[0525] Example 6: Biodegradable and in vitro active prodrugs To provide therapeutic activity, the active drug must eventually be released from the conjugate. The exemplified ricinoleyl-based conjugates contain a biodegradable, esterase-sensitive linker between the active drug and the ricinoleyl scaffold. Mouse plasma was used to investigate the biodegradability of the ricinoleyl-based conjugates, as they contain active esterases that can cleave the linker.

[0526] LNP formulations 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 were incubated with mouse plasma for 0 or 2 hours, after which intact conjugate and released dexamethasone or calcitriol were quantified using UPLC as described above (Figure 7A, Figure 7B and Figure 7C). Varying levels of intact tricinooleyl-drug conjugates were detected, indicating differential levels of degradation by plasma esterases (Figure 7A).

[0527] Notably, the various amounts of free dexamethasone detected in mouse plasma corresponded to the degradation levels exhibited by the prodrugs in FIG. 7B.

[0528] Dexamethasone is known to suppress unwanted immune responses. We next demonstrate the activity of ricinoleyl-based conjugates in a cellular model of immunostimulation mediated by lipopolysaccharide (LPS).

[0529] Cultured macrophage cell lines J774.2 (Figure 8) and Raw264.7 (Figure 9A) were incubated with immune stimulators LPS and LNP with or without ricinoleyl-dexamethasone conjugates INT-D034 / INT-D035. After 24 hours, cells were harvested and analyzed for expression of proinflammatory cytokines IL1β, TNFα and IL-6. RNA was isolated from cells and levels of proinflammatory cytokines IL1β, TNFα and IL6 were measured by qRT-PCR.

[0530] Cells incubated with the control formulation (i.e., without ricinoleyl-dexamethasone conjugate) showed increased levels of all three cytokines suggestive of an inflammatory response. In contrast, cells treated with LNP formulations containing either INT-D034 or INT-D035 showed reduced levels of proinflammatory cytokines in a dose-dependent manner. A similar reduction in IL1β concentrations was observed for INT-D045, INT-D046, INT-D047, INT-D048, and INT-D049 in Raw264.7 (Figure 9B). These results suggest that ricinoleyl-dexamethasone conjugates can be processed intracellularly to release the active drug (dexamethasone) to suppress unwanted immune responses.

[0531] Dexamethasone and calcitriol can tolerize antigen-presenting cells (APCs). The activity of ricinoleyl-based dexamethasone and calcitriol conjugates is next demonstrated in a mixed lymphocyte reaction (MLR) assay for the assessment of immune tolerance. Bone marrow-derived dendritic cells (BMDCs) from C57Bl / 6 male mice (Charles River) were first treated with LNPs containing various mol% of dexamethasone or calcitriol conjugates for 48 h, followed by activation 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. The level of T-cell proliferation after 3 days was quantified using flow cytometry. As shown in Figure 10 , LNPs containing 10–99 mol% dexamethasone conjugates (INT-D034 or INT-D045) or calcitriol conjugates (INT-D053 or INT-D083) were able to suppress allogeneic T cell proliferation, indicating that these ricinoleyl-based conjugates could be processed intracellularly to release dexamethasone or calcitriol to tolerize BMDCs.

[0532] Thus, the prodrugs described herein can not only be efficiently loaded in large quantities into LNPs to enable controlled drug release, but are also active, as shown in in vitro models of immune stimulation and ex vivo models of immune tolerance.

[0533] Example 7: Additional Prodrug Examples Various classes of drugs can be used as prodrugs as described herein.Selected examples of such compounds are shown below, including acetylsalicylic acid, MCC950, INT-MA014, calcitriol, ruxolitinib, tofacitinib, sirolimus, docetaxel, mycophenolic acid, cannabidiol and tetrahydrocannabinol.Exemplary prodrugs of such compounds are also shown below:

[0534] [ka]

[0535] [ka]

[0536] [ka]

[0537] [ka]

[0538] [ka]

[0539] [ka]

[0540] These prodrugs can be synthesized with ester or carbonate X1 linker groups as shown in the reaction scheme below. The mechanism of biodegradation of ruxolitinib prodrugs with ester X1 linkages is also shown below. In the first step, esterases cleave the ester group on the prodrug. This is followed by spontaneous decomposition of the resulting hemiaminal to liberate the free drug.

[0541] Example of Ruxolitinib Prodrug Synthesis Using Esters or Carbonates

[0542] [ka]

[0543] Example 8 - Multiple prodrugs can be formulated within the same LNP As mentioned above, the lipid-like properties of the prodrugs allow for easy loading into the LNP system by simply mixing them with the lipid formulation components. It was confirmed that more than one prodrug from each of the different parent drugs can be loaded in the same LNP system because these prodrugs have lipid-like properties. Table 7 shows LNP formulations made by mixing two different prodrugs in equimolar ratios (i.e., 10 mol% of each). In particular, it was demonstrated that the dexamethasone and calcitriol prodrugs can be encapsulated together at very high levels (close to 100%) to produce monodisperse nanoparticle formulations of 44-50 nm diameter with PDI<0.1. Electron micrographs in Figure 11 show that these combined formulations exhibit spherical electron-dense regions just beneath the membrane, similar to those observed for formulations containing a single prodrug, as seen in Figures 4 and 5. Without being limiting, these morphological data suggest that prodrugs of different parent drugs may coexist as hydrophobic oil phases in the LNP lipid bilayer. Furthermore, it was confirmed that various ratios of dexamethasone and calcitriol conjugates (ranging from 1 to 10 mol % each) could be formulated together with high encapsulation efficiency to form monodisperse nanoparticles with diameters of approximately 50 to 60 nm (Table 8).

[0544] LNP formulations containing 10 mol% dexamethasone conjugate (INT-D045) with or without 10 mol% calcitriol conjugate (INT-D053, INT-D068, or INT-D083) were incubated in human or mouse plasma at 37° C. for 0, 2, or 24 hours, and lipid conjugate dissociation and biodegradation were measured as described in Example 5 (FIGS. 12 and 13). Combinations (i.e., formulations containing multiple lipid-drug conjugates) showed similar lipid dissociation or biodegradation compared to formulations containing only one lipid-drug conjugate. These data indicate that certain lipid-drug conjugates may remain functional when encapsulated with another lipid-drug conjugate in the same lipid nanoparticle.

[0545] Table 7. Particle size and polydispersity index of LNPs containing prodrug combinations

[0546] [Table 7]

[0547] Table 8. Particle size and polydispersity index of LNPs containing different molar ratios of prodrug combinations.

[0548] [Table 8]

[0549] The above examples are illustrative only: various modifications can be made without departing from the scope of the specific aspects of the invention described herein.

Claims

1. A lipid conjugate comprising a branched lipid moiety having a scaffold backbone L for attaching one or more R hydrocarbon chains, The lipid moiety has the structure of Formula IId: Formula IId: 【Chemistry 1】 wherein the L lipid scaffold backbone is represented as L1+L2+L3+L4+L5+L6, where L contains 5-40 carbon atoms and 0-2 cis or trans C═C double bonds; L1 is a carbon chain having 3 to 30 carbon atoms, L1 may have 1 or more cis or trans C═C double bonds, or 0 to 2 cis or trans C═C double bonds; L2 and L4 are each a carbon atom; L3 is 0 to 20 carbon atoms and contains 0 to 2 cis or trans C═C double bonds; L5 is 0 to 20 carbon atoms and contains 0 to 2 cis or trans C═C double bonds; L6 is -CH ３ , =CH ２ or H; 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, and when one or more of R is branched, each branch point contains an X2 functional group; n is 0 to 8, p is 0 to 8, and n+p is 1 to 8 or n+p≧1; wherein each X2 is independently a carbon-based functional group including an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene, or alkyne, methylene (CH ２ ) or urea; and The conjugate is not an ionizable lipid.

2. The conjugate of claim 1, wherein X2 is independently a group that exhibits biodegradability after administration to a patient.

3. The conjugate of claim 1, wherein X2 is independently a carbamate, ether or ester linkage.

4. L is attached at X1 to a molecule of interest M in the conjugate at L1 to form M-X1-L, where X1 is a carbon-based functional group including an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo, alkane, alkene or alkyne, methylene (CH ２ ) or urea or L is linked to the molecule of interest by a hydrogen bond between L and M of the lipid conjugate.

5. The conjugate of claim 4, wherein X1 is an ester, ether or carbamate.

6. The conjugate of any one of claims 1 to 5, wherein L1 has from 3 to 30 carbon atoms.

7. The conjugate of any one of claims 1 to 6, wherein L1 has from 4 to 30 carbon atoms.

8. 5. The conjugate of claim 4, wherein L is linked to a molecule of interest M by a hydrogen bond between L and M of the lipid conjugate, and L-X1-M has the structure of formula V: Formula V: 【Chemistry 2】 wherein E1, E2, E3, E4 and E5 are independently electronegative atoms selected from O, N and P; E1, E2 and E3 are hydrogen bond acceptors, and E4 and E5 are hydrogen bond donors; Dotted lines indicate hydrogen bonds, solid lines indicate covalent bonds; L is the lipid scaffold of the lipid moiety; n is 0 or 1, o is 0 or 1, p is 0 or 1, and n+o+p≧2; q is 1 to 10 or 2 to 10 or 4 to 10; L is the lipid scaffold of the lipid moiety; M is a molecule of interest; wherein E1 and E3 are independently selected from alkyl, aryl, alkylene, or H, and optionally include substituents attached thereto.

9. 9. The conjugate of any one of claims 1 to 8, wherein R is branched and each branch point is independently selected from an ester, an ether, or a carbamate.

10. The conjugate according to any one of claims 1 to 9, wherein the lipid moiety is non-cylindrical and flared or frusto-conical in the direction of L1 to L6.

11. The conjugate of any one of claims 1 to 10, wherein X2 of the lipid moiety is not a disulfide or thioether group.

12. 12. The conjugate of any one of claims 1 to 11, wherein the lipid moiety is derived from a lipid having one or more reactive groups selected from hydroxyl, amino, and / or amide linked to an internal carbon atom that serves as a scaffold carbon chain in the lipid moiety, and at least one other hydrocarbon chain in the hydrocarbon structure is derived from an acyl lipid, and the X1 linkage is formed by reaction of a reactive group on the scaffold carbon chain with a carboxylic acid of the acyl chain.

13. The conjugate of claim 4 or 5, wherein the second L is linked to the molecule of interest by X1.

14. The conjugate of claim 13 , wherein the second L has the structure of formula IId:

15. A lipid conjugate comprising a branched lipid moiety having a scaffold backbone L for attaching one or more R hydrocarbon chains, The lipid moiety has the structure of Formula IIe: Formula IIe: 【Chemistry 3】 In the formula, L is [CH ２ ] ｍ -L2-L3-L4-[CH ２ ] ｑ -CH ３ and L the total number of carbon atoms is 5 to 30; L2 and L4 are carbon atoms; m is 0 to 20, n is 1 to 4, p is 0 to 4, and n+p is 1 to 4; L3 is 0-10 carbon atoms and has 0-2 cis or trans C=C; X2 is independently selected from an ether group, an ester group, and a carbamate group; Each R is independently: (c) a linear or branched terminal hydrocarbon chain having 0-5 cis or trans C=C and 1-30 carbon atoms, each R being bonded to one of the respective X2 at any carbon atom of the hydrocarbon chain; or (d) A branched structure of formula IIb having a scaffold designated L': Formula IIb: 【Chemistry 4】 In the formula, L′ is [CH ２ ] ｒ -L2-G ３ -L4-[CH ２ ] ｕ -CH ３ where the total number of carbon atoms in L is 3 to 30; r is 0 to 20, 2 to 20, 3 to 20, or 4 to 20; s is 0 to 4, t is 0 to 4; s+t is 1 to 4 or s+t>1; u is 1 to 20; G ３ has 0-10 carbon atoms and 0-2 cis or trans C=C; Each R' of formula IIb is independently a linear or branched terminal hydrocarbon chain having 0 to 5 cis or trans C=C and 1 to 30 carbon atoms; The total number of R′ hydrocarbon chains in formula IIb is 1 to 16; Each of the R and R′ hydrocarbon chains in the lipid moiety may be substituted with a heteroatom, provided that no more than 8 heteroatoms are substituted in the R and R′ hydrocarbon chains and the predicted or experimental log P of the conjugate is greater than 5; and The lipid conjugate is not an ionizable lipid.

16. A pharmaceutical, nutritional, cosmetic, cleaning or food product comprising a conjugate according to any one of claims 1 to 15.

17. A nanoparticle comprising the conjugate according to any one of claims 1 to 15.

18. The nanoparticle of claim 17 , wherein the nanoparticle is a lipid nanoparticle.

19. The nanoparticle of claim 17 , wherein the nanoparticle comprises one or more bilayers.

20. The nanoparticles according to any one of claims 17 to 19, wherein the encapsulation efficiency of the lipid conjugate is at least 80% and the polydispersity index (PDI) of the nanoparticles is less than 0.

15.

21. The nanoparticle according to any one of claims 17 to 20, wherein the lipid conjugate is present in the hydrophobic oil phase in the nanoparticle or in the membrane of the nanoparticle as globular electron density.

22. The nanoparticles according to any one of claims 17 to 21, which have a percentage of nanoparticles and lipid conjugate remaining after 2 hours of in vitro incubation in human serum of 30 to 100 mol %.

23. The nanoparticles according to any one of claims 17 to 21, wherein the percentage of lipid conjugate remaining on the nanoparticles after 2 hours of in vitro incubation in human serum is between 60 and 100 mol %.

24. The nanoparticles according to any one of claims 17 to 21, which have a percentage of nanoparticles and lipid conjugate remaining after 2 hours of in vitro incubation in human serum of 80 to 100 mol %.

25. The nanoparticle of any one of claims 17 to 21, wherein the nanoparticle further comprises an additional lipid conjugate comprising a second molecule of interest M.

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