ANTIBODY DRUG CONJUGATE FOR ANTI-INFLAMMATORY APPLICATIONS

- Merck Sharp & Dohme LLC

Antibody drug conjugates (ADCs) comprising an antibody conjugated to an anti-inflammatory therapeutic agent via a phosphate-based linker with tunable extracellular and intracellular stability are described.

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Description
CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent Application No. 62/237,668 filed Oct. 6, 2015 and which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file name “23597WOPCTSEQ.txt”, creation date of Aug. 31, 2016, and a size of 68 Kb. This sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION (1) Field of the Invention

The present invention relates to antibody drug conjugates (ADCs) comprising an antibody conjugated to an anti-inflammatory therapeutic agent via a phosphate-based linker having tunable extracellular and intracellular stability.

(2) Description of Related Art

Antibody drug conjugates (ADC) are targeted chemotherapeutic molecules combining the ideal properties of both antibodies and cytotoxic drugs by targeting potent cytotoxic drugs to antigen-expressing tumor cells. The antigen-expressing tumor cells internalize the ADC, which then releases the drug from the ADC, thereby enhancing the drug's anti-tumor activity. This strategy has met limited success in part because many cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands. Promising advancements with immunoconjugates have seen cytotoxic drugs linked to antibodies through a linker that is cleaved at the tumor site or inside tumor cells.

The successful ADC development for a given target antigen depends on optimization of antibody selection, linker design and stability, drug potency and mode of drug and linker conjugation to the antibody. Linker properties such as pH and redox sensitivities and protease susceptibility influence circulatory stability and release of the drug moiety. The intracellular cleavage of disulfide containing linkers of an ADC is limited by the oxidizing potential of endosomes and lysosomes and are probably not released by reductive cleavage within the endocytic pathway (Austin et al., Proc. Natl. Acad. Sci. USA 102: 17987-17992 (2005)). Reductive cleavage may occur at the cell membrane and impart a bystander killing effect of tumor and susceptible normal cells by free drug. Inappropriate release of drug likely contributes to toxicity. Linker stability plays an important role in both the efficacy and toxicity of ADC (Alley et al., Bioconjugate Chem. 19:759-765 (2008)). Stable, non-cleavable linkers such as mcc are more efficacious and safer than unstable, disulfide linkers, widening the therapeutic window. However, while mcc linkers are more stable than disulfides, they can only be used for drugs that can tolerate residual linker on it and still be potent. Thus, self-immolative linkers are needed for drugs that do not have this flexible structure activity relationship (SAR).

A chemical solution to targeted delivery of cytotoxic or cytostatic drugs conjugated to cell-specific ligands is the “self-immolative linker”, PABC or PAB (para-aminobenzyloxycarbonyl) linker, attaching the drug moiety to the ligand in the conjugate (Carl et al., J. Med. Chem. 24: 479-480 (1981); Chakravarty et al., J. Med. Chem. 26: 638-644 (1983)). The PAB linker unit is also referred to as an electronic cascade spacer. The amide bond linking the carboxy terminus of a peptide unit and the para-aminobenzyl of PAB may be a substrate and cleavable by certain proteases. Following cleavage, the aromatic amine becomes electron-donating and initiates an electronic cascade that leads to the expulsion of the leaving group, which releases the free drug after elimination of carbon dioxide (de Groot, et al. Journal of Organic Chemistry 66: 8815-8830 (2001)). Cathepsin B is a ubiquitous cysteine protease with increasing activity within low pH environments (i.e. lysosomes). It is an intracellular enzyme, except in pathological conditions, such as metastatic tumors (Sinha et al., Prostate 49: 172-184 (2001)) or rheumatoid arthritis (Hashimoto et al., Biochem. Biophys. Res. Commun. 283: 334-339 (2001)). Therefore, conjugates produced with cathepsin B-cleavable linkers are likely to be stable in circulation. Upon cleavage of a peptide bond adjacent to the PABC, i.e. by an intracellular enzyme, the drug is released from the ligand whereby no remaining portion of the linker is bound (de Groot et al., Molecular Cancer Therapeutics 1: 901-911 (2002); de Groot et al., J. Med. Chem. 42: 5277-5283 (1999)).

Linkers containing the para-aminobenzyloxycarbonyl (PAB or PABC) unit, in conjunction with a peptide unit, have been developed with a “self-immolating” or “self-immolative” mechanism of 1,6 elimination and fragmentation under enzymatic, hydrolytic, or other metabolic conditions to release a drug moiety from a targeting ligand, such as an antibody (U.S. Pat. Nos. 6,214,345; 6,677,435 5,621,002; 6,218,519; 6,835,807; 6,268,488; and 6,759,509; US Pat. Pub. Nos. 20030130189; 20030096743; 20040052793; 20040018194; 20040052793; and 20040121940; PCT Pub. Nos. WO 98/13059 and WO2004/032828).

Limitations of the PAB type self-immolating linkers are the propensity to cause poor solubility and aggregation of the conjugates. In addition, some PAB-containing conjugates may not be suitable substrates for certain cleaving enzymes or cleave too slowly to achieve efficacy. While the PAB/PABC linkers have been exemplified for amine-terminus payloads that form stable carbamate bonds, for payloads that do not contain a linkable amine, the carbonate that is formed may not be stable and so there is a need for self-immolative linkers that can handle payloads with an oxygen terminus, for example, dexamethasone.

In light of the above, there is a need for linkers for constructing drug-ligand conjugates with improved therapeutic efficacy.

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibody-drug conjugates (ADCs) for use in treatments where it is desirable that the treatment include an anti-inflammatory component. The antibody-drug conjugates comprise an antibody that targets a CD74 or CD163 protein conjugated to an anti-inflammatory therapeutic agent via a phosphate-based linker with tunable stability for intracellular delivery of the therapeutic agent. The phosphate-based linker comprises a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm comprising from the distal to the proximal direction a tuning element, optionally a spacer element, and a reactive functional group. The phosphate group of the phosphate-based linker is capable of being conjugated to the therapeutic agent and the reactive functional group is capable of being conjugated to the side chain of an amino acid (natural or non-natural amino acid) comprising the antibody. The phosphate-based linker has a differentiated and tunable stability in blood vs. an intracellular environment (e.g., lysosomal compartment). The inventors have discovered that the rate at which the phosphate group is cleaved in the intracellular environment to release the anti-inflammatory agent in its native or active form may be affected by the structure of the tuning element with further effects mediated by substitutions of the phosphate group as well as whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate.

Therefore, the present invention provides composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier.

In a further embodiment, the present invention provides a method for treating an inflammatory disease or disorder by providing to a subject having the disease or disorder a composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier to treat the inflammatory disease or disorder.

In a further embodiment, the present invention provides for the use of a composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier for the treatment of an inflammatory disease or disorder.

The present invention provides composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD74 protein (an anti-CD74 antibody); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier.

In a further embodiment, the present invention provides a method for treating an inflammatory disease or disorder by providing to a subject having the disease or disorder a composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD74 protein (an anti-CD74 antibody); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier to treat the inflammatory disease or disorder.

In a further embodiment, the present invention provides for the use of a composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD74 protein (an anti-CD74 antibody); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier for the treatment of an inflammatory disease or disorder.

The present invention provides composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD163 protein (an anti-CD163 antibody); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier.

In a further embodiment, the present invention provides a method for treating an inflammatory disease or disorder by providing to a subject having the disease or disorder a composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD163 protein (an anti-CD163 antibody); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier to treat the inflammatory disease or disorder.

In a further embodiment, the present invention provides for the use of a composition comprising a compound having formula (I)

wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory agent; antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD163 protein (an anti-CD163 antibody); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and, a pharmaceutically acceptable carrier for the treatment of an inflammatory disease or disorder.

In particular aspects, the anti-inflammatory agent comprises a glucocorticoid receptor agonist. In particular aspects, the anti-inflammatory agent comprises Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, or mometasone.

In particular aspects, the antibody comprises a substitution of an amino acid in the heavy chain or light chain of the antibody with pAzF and the reactive functional group comprises a strained cycloalkyne.

In particular aspects, the inflammatory disease or disorder comprises Alzheimer's disease, ankylosing spondylitis arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, or ulcerative colitis.

In further aspects of the compound of formula I, the m comprises 2. In further still aspects of the compound of formula I, n comprises 2. In further still aspects, n comprises 3.

In a further embodiment, the present invention provides an anti-CD74 antibody (antibody that binds CD74) comprising a para-azidophenylalanine (pAzF) residue conjugated to a molecule selected from the group of molecules consisting of 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-1, 10-1, 11-5, 21-1, 13-1, 14-5, 15-1, 16-5, and 17-2.

In particular aspects, the anti-CD74 antibody comprises light chain complementarity-determining region (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:44), CDR2 (TVSNRFS; SEQ ID NO:45), and CDR3 (SQSSHVPPT; SEQ ID NO:46) and heavy chain CDR sequences CDR1 (NYGVN; SEQ ID NO:47), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:48), and CDR3 (SRGKNEAWFAY; SEQ ID NO:49). In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises at least one, two, three, four, five, or six CDR(s) selected from SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49. In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73. In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises light chain CDR sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ASGRYYGSGSYSSYFD; SEQ ID NO:54). In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises light chain CDR sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSIK; SEQ ID NO:55), and CDR3 (ARGREYTSQNIVILLD; SEQ ID NO:56). In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises light chain CDR sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ARGREITSQNIVILLD; SEQ ID NO:57). In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises light chain CDR sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (IWYDGSNK; SEQ ID NO:58), and CDR3 (ARGGTLVRGAMYGTDV; SEQ ID NO:59). In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from AAS, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59. In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78. In particular aspects, the anti-CD74 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In a further embodiment, the present invention provides an anti-CD163 antibody (antibody that binds CD163) comprising a para-azidophenylalanine (pAzF) residue conjugated to a molecule selected from the group of molecules consisting of 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-1, 10-1, 11-5, 21-1, 13-1, 14-5, 15-5, 16-5, and 17-2.

In particular aspects, the anti-CD163 antibody comprises light chain CDR sequences CDR1 (ASQSVSSDV; SEQ ID NO:60), CDR2 (YAS), and CDR3 (QDYTSPRT; SEQ ID NO:61) and heavy chain complementarity-determining region (CDR) sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63). In particular aspects, the anti-CD163 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the anti-CD163 antibody comprises light chain CDR sequences CDR1 (ASQSVSHDV; SEQ ID NO:54), CDR2 (YTS), and CDR3 (QDYSSPRT; SEQ ID NO:65) and heavy chain CDR sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63).

In particular aspects, the anti-CD163 antibody comprises at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from YYAS, YSG, YTS, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65. In particular aspects, the anti-CD163 antibody comprises a non-natural amino acid, which is a further aspect, may be pAzF.

In particular aspects, the antibody is selected from the group consisting of ADC 12-1, ADC 12-2, ADC 12-3, ADC 12-4, ADC 12-5, ADC 12-6, ADC 12-7, ADC 12-8, ADC 12-9, ADC 12-10, ADC 12-13, ADC 12-14, and ADC 12-15.

Definitions

Definitions of specific functional groups, chemical terms, and general terms used throughout the specification are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modem Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

Acyl—As used herein, the term “acyl,” refers to a group having the general formula —C(═O)RX1, —C(═O)ORX1, —C(═O)—O—C(═O)RX1, —C(═O)SRX1, —C(═O)N(RX1)2, —C(═S)RX1, —C(═S)N(RX1)2, and —C(═S)S(RX1), —C(═NRX1)RX1, —C(═NRX1)ORX1, —C(═NRX1)SRX1, and —C(═NRX1)N(RX1)2, wherein RX1 is hydrogen; halogen; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; substituted or unsubstituted acyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl; cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl; substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, or mono- or di-heteroarylamino; or two RX1 groups taken together form a 5- to 6-membered heterocyclic ring. Exemplary acyl groups include aldehydes (—CHO), carboxylic acids (—CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

Aliphatic—As used herein, the term “aliphatic” or “aliphatic group” denotes an optionally substituted hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (“carbocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-12 carbon atoms. In some embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

Alkenyl—As used herein, the term “alkenyl” denotes an optionally substituted monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. In certain embodiments, the alkenyl group employed in the invention contains 2-6 carbon atoms. In certain embodiments, the alkenyl group employed in the invention contains 2-5 carbon atoms. In some embodiments, the alkenyl group employed in the invention contains 2-4 carbon atoms. In another embodiment, the alkenyl group employed contains 2-3 carbon atoms. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

Alkyl—As used herein, the term “alkyl” refers to optionally substituted saturated, straight- or branched-chain hydrocarbon radicals derived from an aliphatic moiety containing between 1-6 carbon atoms by removal of a single hydrogen atom. In some embodiments, the alkyl group employed in the invention contains 1-5 carbon atoms. In another embodiment, the alkyl group employed contains 1-4 carbon atoms. In still other embodiments, the alkyl group contains 1-3 carbon atoms. In yet another embodiment, the alkyl group contains 1-2 carbons. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.

Alkynyl—As used herein, the term “alkynyl” refers to an optionally substituted monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. In certain embodiments, the alkynyl group employed in the invention contains 2-6 carbon atoms. In certain embodiments, the alkynyl group employed in the invention contains 2-5 carbon atoms. In some embodiments, the alkynyl group employed in the invention contains 2-4 carbon atoms. In another embodiment, the alkynyl group employed contains 2-3 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

Aryl—As used herein, the term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to an optionally substituted monocyclic and bicyclic ring systems having a total of five to 10 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.

Arylalkyl—As used herein, the term “arylalkyl” refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group). Bivalent hydrocarbon chain—As used herein, the term “bivalent hydrocarbon chain” (also referred to as a “bivalent alkylene group”) is a polymethylene group, i.e., —(CH2)z—, wherein z is a positive integer from 1 to 30, from 1 to 20, from 1 to 12, from 1 to 8, from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 30, from 2 to 20, from 2 to 10, from 2 to 8, from 2 to 6, from 2 to 4, or from 2 to 3. A substituted bivalent hydrocarbon chain is a polymethylene group in which one or more methylene hydrogen atoms are replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group.

Carbonyl—As used herein, the term “carbonyl” refers to a monovalent or bivalent moiety containing a carbon-oxygen double bond. Non-limiting examples of carbonyl groups include aldehydes, ketones, carboxylic acids, ester, amide, enones, acyl halides, anhydrides, ureas, carbamates, carbonates, thioesters, lactones, lactams, hydroxamates, isocyanates, and chloroformates.

Cycloaliphatic—As used herein, the terms “cycloaliphatic”, “carbocycle”, or “carbocyclic”, used alone or as part of a larger moiety, refer to an optionally substituted saturated or partially unsaturated cyclic aliphatic monocyclic or bicyclic ring systems, as described herein, having from 3 to 10 members. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons.

Halogen—As used herein, the terms “halo” and “halogen” refer to an atom selected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), and iodine (iodo, —I).

Heteroaliphatic—As used herein, the terms “heteroaliphatic” or “heteroaliphatic group”, denote an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, heteroaliphatic groups contain 1-6 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

Heteroaralkyl—As used herein, the term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

Heteroaryl—As used herein, the term “heteroaryl” used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refers to an optionally substituted group having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 71 electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, carbocyclic, or heterocyclic rings, where the radical or point of attachment is on the heteroaromatic ring. Non limiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, and tetrahydroisoquinolinyl. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.

Heteroatom—As used herein, the term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen.

Heterocyclic—As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable optionally substituted 5- to 7-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more heteroatoms, as defined above. A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclic moiety”, and “heterocyclic radical”, are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or carbocyclic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

Unsaturated—As used herein, the term “unsaturated”, means that a moiety has one or more double or triple bonds.

Partially unsaturated—As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

Optionally substituted—As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH2)0-4R∘; —(CH2)0-4OR∘; —O—(CH2)0-4C(O)OR∘; —(CH2)0-4CH(OR∘)2; —(CH2)0-4SR∘; —(CH2)0-4Ph, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1Ph which may be substituted with R∘; —CH═CHPh, which may be substituted with R∘; —NO2; —CN; —N3; —(CH2)0-4N(R∘)2; —(CH2)0-4N(R∘)C(O)R∘; —N(R∘)C(S)R∘; —(CH2)0-4N(R∘)C(O)NR∘2; —N(R∘)C(S)NR∘2; —(CH2)0-4N(R∘)C(O)OR∘; —N(R∘)N(R∘)C(O)R∘; —N(R∘)N(R∘)C(O)NR∘2; —N(R∘)N(R∘)C(O)OR∘; —(CH2)0-4C(O)R∘; —C(S)R∘; —(CH2)0-4C(O)OR∘; —(CH2)0-4C(O)SR∘; —(CH2)0-4C(O)OSiR∘3; —(CH2)0-4OC(O)R∘; —OC(O)(CH2)0-4SR—, SC(S)SR∘; —(CH2)0-4SC(O)R∘; —(CH2)0-4C(O)NR∘2; —C(S)NR∘2; —C(S)SR∘; —SC(S)SR∘, —(CH2)0-4OC(O)NR∘2; —C(O)N(OR∘)R∘; —C(O)C(O)R∘; —C(O)CH2C(O)R∘; —C(NOR∘)R∘; —(CH2)0-4SSR∘; —(CH2)0-4S(O)2R∘; —(CH2)0-4S(O)2OR∘; —(CH2)0-40S(O)2R∘; —S(O)2NR∘2; —(CH2)0-4S(O)R∘; —N(R∘)S(O)2NR∘2; —N(R∘)S(O)2R∘; —N(OR∘)R∘; —C(NH)NR∘2; —P(O)2R∘; —P(O)R∘2; —OP(O)R∘2; —OP(O)(OR∘)2; SiR∘3; —(C1-4 straight or branched alkylene)O—N(R∘)2; or —(C1-4 straight or branched alkylene)C(O)O—N(R∘)2, wherein each R∘ may be substituted as defined below and is independently hydrogen, C1-6 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R∘, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R∘ (or the ring formed by taking two independent occurrences of R∘ together with their intervening atoms), are independently halogen, —(CH2)0-2R●, -(haloR●), —(CH2)0-2OH, —(CH2)0-2OR●, —(CH2)0-2CH(OR●)2; —O(haloR●), —CN, —N3, —(CH2)0-2C(O)R●, —(CH2)0-2C(O)OH, —(CH2)0-2C(O)OR●, —(CH2)0-2SR●, —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR●, —(CH2)0-2NR●2, —NO2, —SiR●3, —OSiR●3, —C(O)SR●, —(C1-4 straight or branched alkylene)C(O)OR●, or —SSR● wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R∘ include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2))2-3O—, or —S(C(R*2))2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*2)2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R● include halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R, —NR†2, —C(O)R†, —C(O)OR†, —C(O)C(O)R, —C(O)CH2C(O)R†, —S(O)2R†, —S(O)2NR†2, —C(S)NR†2, —C(NH)NR†2, or —N(RT)S(O)2R; wherein each R is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R†, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on the aliphatic group of R† are independently halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

In any case where a chemical variable (e.g., an R group) is shown attached to a bond that crosses a bond of ring, this means that one or more such variables are optionally attached to the ring having the crossed bond. Each R group on such a ring can be attached at any suitable position, this is generally understood to mean that the group is attached in place of a hydrogen atom on the parent ring. This includes the possibility that two R groups can be attached to the same ring atom. Furthermore, when more than one R group is present on a ring, each may be the same or different than other R groups attached thereto, and each group is defined independently of other groups that may be attached elsewhere on the same molecule, even though they may be represented by the same identifier.

Antibody—As used herein the term “antibody” includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies) and the term “antibody” is used interchangeably with the terms “immunoglobulin,” “immunoglobulins” and “immunoglobulin molecule”. Each antibody molecule has a unique structure that allows it to bind its specific antigen, but all antibodies have the same overall structure as described herein. The basic immunoglobulin structural unit is known to comprise a tetramer of subunits. Each tetramer has two identical pairs of polypeptide chains, each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Thus, an antibody as defined herein can be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

The light and heavy chains are subdivided into variable regions and constant regions (See generally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7. The variable regions of each light/heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are the same. The chains all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. The terms include naturally occurring forms, as well as fragments and derivatives. Included within the scope of the term are classes of immunoglobulins (Igs), namely, IgG, IgA, IgE, IgM, and IgD. Also included within the scope of the terms are the subtypes of IgGs, namely, IgG1, IgG2, IgG3 and IgG4. The term is used in the broadest sense and includes single monoclonal antibodies (including agonist and antagonist antibodies) as well as antibody compositions which will bind to multiple epitopes or antigens. The terms specifically cover monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies), and antibody fragments so long as they contain or are modified to contain at least the portion of the CH2 domain of the heavy chain immunoglobulin constant region which comprises an N-linked glycosylation site of the CH2 domain, or a variant thereof. Included within the terms are molecules comprising only the Fc region, such as immunoadhesins (U.S. Published Patent Application No. 20040136986), Fc fusions, and antibody-like molecules. Alternatively, these terms can refer to an antibody fragment of at least the Fab region that at least contains an N-linked glycosylation site.

The term “Fc” fragment refers to the ‘fragment crystallized’ C-terminal region of the antibody containing the CH2 and CH3 domains. The term “Fab” fragment refers to the ‘fragment antigen binding’ region of the antibody containing the VH, CH1, VL and CL domains.

The term “antibodies” further includes chemical analogues and derivatives of antibodies and antibody fragments, provided that the antibody or antibody fragment maintains its ability to bind specifically to its target antigen. Thus, for example, chemical modifications are possible (e.g., glycosylation, acetylation, PEGylation and other modifications without limitation) provided specific binding ability of the antibody is retained. An antibody may be, for example, human, humanized, or chimeric

A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies (mAbs) are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The term “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., (1975) Nature, 256:495, or may be made by recombinant DNA methods (See, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.).

Monoclonal antibodies further include chimeric antibodies in which a portion of the heavy and/or light chain is identical to or homologous with the corresponding s of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

An “intact antibody” is one that comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, CH3 and CH4, as appropriate for the antibody class. The constant domains may be native sequence constant domains such as human native sequence constant domains or amino acid sequence variants thereof. An intact antibody may or may not have one or more “effector functions”, which refers to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include complement dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis.

An “antibody fragment” comprises a portion of an intact antibody, preferably comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), a fragment(s) produced by a Fab expression library, camelids, or an epitope-binding fragments of any of the above which immunospecifically bind to a target antigen (e.g., a cancer cell antigen).

“Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. The term “capable of specific binding” refers to protein or peptide (e.g., antibody) binding to a predetermined target substance (e.g., an antigen and/or groups of antigens), e.g. a target substance that is expressed on the surface of a cell; thus the term “binding to a target cell” or “binding to a cancer cell” is to be understand as referring to protein or peptide (e.g., antibody) binding to a predetermined target substance (e.g. antigen or antigens) that is expressed on such a cell.

Typically, the protein or peptide (e.g., antibody) binds with an affinity of at least about 1×107 M1, and/or binds to the predetermined target substance (e.g., antigen, antigens or cell) with an affinity that is at least two-fold greater than its affinity for binding to a non-specific control substance (e.g., BSA, casein, non-cancer cells) other than the predetermined target substance or a closely-related target substance.

Drug—As used herein, the term “drug” refers to small molecules or biomolecules that alter, inhibit, activate, or otherwise affect a biological event. For example, drugs may include, but are not limited to, anti-AIDS substances, anti-cancer substances, antibiotics, anti-diabetic substances, immunosuppressants, anti-viral substances, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, anti-parasite and/or anti-protozoal compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, anti-hypertensives, analgesics, anti-pyretics, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-secretory factors, anticoagulants and/or anti-thrombotic agents, local anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, and imaging agents. A more complete listing of exemplary drugs suitable for use in the present invention may be found in “Pharmaceutical Substances: Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”, edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25/National Formulary-20, published by the United States Pharmcopeial Convention, Inc., Rockville Md., 2001.

Polymer—As used herein, a “polymer” or “polymeric structure” is a structure that includes a string of covalently bound monomers. A polymer can be made from one type of monomer or more than one type of monomer. The term “polymer” therefore encompasses copolymers, including block-copolymers in which different types of monomer are grouped separately within the overall polymer. A polymer can be linear or branched.

Treat—As used herein, the term “treat” (or “treating”, “treated”, “treatment”, etc.) refers to the administration of a conjugate of the present disclosure to a subject in need thereof with the purpose to alleviate, relieve, alter, ameliorate, improve or affect a condition (e.g., diabetes, cancer, inflammatory disease), a symptom or symptoms of a condition (e.g., hyperglycemia), or the predisposition toward a condition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a scheme for synthesis of ADC 12-4.

FIG. 2 is a graph showing stability of ADC 12-1 in mouse serum. (aDEX is ADC 12-1).

FIG. 3 is a graph showing deconvoluted intact mass spectrum for ADC 12-1 stock solution.

FIG. 4A is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 1 hour.

FIG. 4B is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 8 hours.

FIG. 4C is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 14 days.

FIG. 4D is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37° C. for 21 days.

FIG. 5 is a graph showing In vivo stability of ADC 12-1 versus “naked” antibody (non-conjugated) following IV dosing to DBA1 mice.

FIG. 6A is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample B1 at 1 hour.

FIG. 6B is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample H3 at 5 days.

FIG. 6C is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample B7 at 1 hour.

FIG. 6D is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: sample H9 at 5 days.

FIG. 7 is a graph showing In vitro activity of ADC 12-2 versus “naked” antibody (non-conjugated) in 786-0 cells.

FIG. 8 shows the activity of anti-hC74 (LL1) ADCs in Hut78 cells.

FIG. 9 shows the activity of anti-hC74 (LL1) ADCs in SUDHL-6 cells.

FIG. 10 shows the activity of anti-hC74 (LL1) ADCs in 786-0 cells.

 DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibody-drug conjugates (ADCs) for use in treatments where it is desirable that the treatment include an anti-inflammatory therapeutic component. The antibody-drug conjugates comprise an antibody that targets or binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively) conjugated to an anti-inflammatory therapeutic agent via a phosphate-based linker with tunable stability for intracellular delivery of the therapeutic agent. The phosphate-based linker comprises a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) covalently linked to the distal end of a linker arm comprising from the distal to the proximal direction a tuning element, optionally a spacer element, and a reactive functional group. The phosphate group of the phosphate-based linker is capable of being conjugated to the therapeutic agent and the reactive functional group is capable of being conjugated the antibody. The phosphate-based linker has a differentiated and tunable stability in blood vs. an intracellular environment (e.g., lysosomal compartment). The inventors have discovered that the rate at which the phosphate group is cleaved in the intracellular environment to release the payload in its native or active form may be affected by the structure of the tuning element with further effects mediated by substitutions of the phosphate group as well as whether the phosphate group is a monophosphate, diphosphate, triphosphate, or tetraphosphate.

The phosphate-based linkers comprise a monophosphate, diphosphate, triphosphate, or tetraphosphate group and a linker arm comprising a tuning element, an optional spacer element, and a reactive functional group. The phosphate-based linkers have a distal end and a proximal end. The distal end of the phosphate-based linker comprises a monophosphate, diphosphate, or triphosphate group (phosphate group) linked to the distal end of the tuning element comprising the linker arm. The phosphate group is covalently linked to an anti-inflammatory therapeutic agent. The proximal end of the linker arm comprises a reactive functional group capable of reacting with a group (side chain of an amino acid) on an antibody to covalently link the phosphate-based linker to the antibody. Interspersed between the tuning element and the reactive functional group of the linker arm may be an optional spacer element. Such a compound comprises formula (I)

Wherein V is selected from O and S; W is selected from O, N, and CH2; X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom; an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin K sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR2, O, or S; D is an anti-inflammatory therapeutic agent; wherein the antibody binds the human CD25, human CD70, human CD74 protein, or human CD163 protein (anti-CD25 antibody, anti-CD70 antibody, anti-CD74, or anti-CD163 antibody, respectively); Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) a side chain of an amino acid of the antibody; each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; n is 1, 2, 3, or 4; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The phosphate-therapeutic agent is stabile extracellularly and labile intracellularly, for example, when present in the lysosomal compartment of the target cell. The tuning element provides a tunable stability to the phosphate-therapeutic agent linkage when the conjugate is within the lysosomal compartment of the target cell. The intracellular stability of the phosphate-therapeutic agent linkage or rate of intracellular release of the therapeutic agent from the conjugate may be adjusted or tuned by the particular tuning element adjacent to the phosphate group and/or by adjusting the number of the phosphate groups.

The link between the antibody and the anti-inflammatory therapeutic agent plays an important role in an antibody drug conjugate (ADC), as the type and structure of the linker may significantly affect the potency, selectivity, and the pharmacokinetics of the resulting conjugate (Widdeson et al, J. Med. Chem. 49: 4392-4408 (2006); Doronina et al., Bioconj. Chem. 17: 114-124 (2006); Hamann et al., Bioconj. Chem. 16: 346-353 (2005); King et al., J. Med. Chem. 45: 4336-4343 (2002); Alley et al., Bioconj. 19: 759-765 (2008); Blattler et al., Biochem. 24: 1517-1524 (1985). ADC delivery of a drug moiety to its intracellular target occurs via a multistep sequence of events: binding to the cell surface, endocytosis, trafficking (within an endosome) to a lysosome, proteolytic degradation of the conjugate, and diffusion of the released drug moiety across the lysosomal or endosomal membrane toward its intracellular target and its interaction with the target. Therefore, the linker should be sufficiently stable while in circulation to allow delivery of the intact ADC to the target cell but, on the other hand, sufficiently labile to allow release of the drug moiety from the ADC once inside the targeted cell. In general, four types of linkers have been used for preparation of ADCs that have currently entered the clinic: (a) acid-labile linkers, exploiting the acidic endosomal and lysosomal intracellular microenvironment (Hamann et al., op. cit.; Blattler et al., op. cit.); (b) linkers cleavable by lysosomal proteases (Dronina et al. op. cit.; King et al. op. cit.); (c) chemically stable thioether linkers that release a lysyl adduct after proteolytic degradation of the antibody inside the cell; (Lewis et al Cancer Res. 68: 9280-9290 (2008); Erickson et al., Cancer Res. 66: 4426-4433 (2006) and (d) disulfide containing linkers (Chari, Adv. Drug Delivery Rev. 31: 89-104 (1998); Widdeson et al., op. cit.), which are cleaved upon exposure to an intracellular thiol. While U.S. Pat. No. 5,094,848 discloses conjugates comprising a diphosphate or amidated diposphate group and a linker arm wherein the linker arm may preferably be an oligopeptide having preferably 2-10 amino acids, in particular embodiments the tuning element of the phosphate-based linkers disclosed herein may include a di-peptide.

The linker-therapeutic agent conjugates of the present invention wherein the therapeutic agent is covalently linked to a tuning element of the linker via a monophosphate, diphosphate, triphosphate, or tetraphosphate linkage have a differentiated and tunable stability of the phosphate linkage in blood vs. an intracellular environment (e.g. lysosomal compartment). Due to location of enzymes that recognize the phosphate linkage, conjugates that have a phosphate group linking a therapeutic agent to a tuning element of the linker are stable in circulation (plasma or blood) but reactive in intracellular compartments (e.g., lysosomes) making them suitable for intracellular delivery of therapeutic agent conjugates. The exemplary therapeutic agent-phosphate-based linker conjugates in the Examples show that the therapeutic agent-phosphate-based linker conjugates of the present invention are stable in blood, which is advantageous for extending the half-life and to prevent premature release of therapeutic agent from the conjugates.

Importantly, the inventors have discovered that by modifying the tuning element and/or V and/or W, and/or the number of phosphate groups, the ability to tune reactivity or cleavage of the phosphate linkage in a lysosomal environment so as to release the therapeutic agent from the conjugate. In general, the rate of release of the therapeutic agent is dependent on the proximal substitution of the tuning element. The ability to cleave the phosphate linkage between the payload and the tuning element efficiently in a lysosome is advantageous for the release of the therapeutic agent from the conjugate once it has been delivered to a cell and internalized through an endosomal pathway. Of note is that unlike other linkers known in the art, there is no need to for the phosphate-based linkers of the present invention to be self-immolative. In addition, the excellent solubility of the therapeutic agent-phosphate-based linker facilitates conjugation to a ligand or cell-targeting moiety and minimizes aggregation of the conjugates. In addition, the phosphate contributes to retention of the therapeutic agent to the conjugate within cell until phosphate linkage is fully cleaved and limits permeability of conjugates containing the payload from entering non-target cells.

The phosphate-based linkers provide greater solubility relative to disulfide linkers, cathepsin B-cleavable linkers, esters and acid-sensitive linkers such as hydrazones. They enable the release of the payload in its parent or unadultered form unlike some of the alternative linkers, and may offer an improved blood/lysosome stability profile. Specifically, these phosphate-based linkers will provide superior blood stability relative to esters and disuflides. Phosphate-based linkers, following lysosomal cleavage will release an alcohol or amine-containing payload whereas the other linker formats may require self-immolative tethers to accomplish this or leave residual linker on the therapeutic agent after lysosomal cleavage. The phosphate-based linker may have greater blood stability relative to the self-immolative cathepsin B linkers in the art, particularly when attached via the oxygen atom of a hydroxyl group of an alcohol-containing therapeutic agent. The enzymatic hydrolysis of the phosphate linkage may be more rapid than the acid-hydrolysis of hyrdazones. The phosphate-based linkers disclosed herein minimize the propensity for conjugates comprising particular therapeutic agent to aggregate. Thus, the phosphate-based linkers disclosed herein are particularly useful for conjugating therapeutic agents that are prone to forming aggregates to a cell-specific targeting ligand to provide a conjugate with a reduced or no detectable propensity for aggregation.

Phosphate Group

The phosphate group comprising the phosphate-based linkers disclosed herein may comprise 1, 2, 3, or 4 phosphate atoms. In particular embodiments, the phosphate group may be a phosphate ester

pyrophosphate ester

triphosphate ester

or tetraphosphate ester

In further embodiments, the phosphate group may be a phosphoramidate

pyrophosphoramidate

triphosphophoramidate

or tetraphosphoramidate

In further still embodiments, the phosphate group may be a phosphonate

a diposphonate

a phosphorthioate

or a diphosphorthioate

The wavy lines shown indicate the covalent attachment sites to the payload at the distal end (left) and the tuning element on the proximal end (right).

Anti-Inflammatory Therapeutic Agent

Anti-inflammatory therapeutic agents comprise glucocorticoid receptor agonists, which include but are not limited to glucocorticoids such as Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, and mometasone.

Linker Arm

The linker arm of the phosphate-based linkers disclosed herein comprises a tuning element at the distal end covalently linked to a phosphate group and a functional reactive group at the proximal end capable of covalent linkage to a cell-targeting ligand. Optionally, the linker arm may further include a spacer element interposed between the tuning element and the reactive functional group. Examples of tuning elements include but are not limited to

R1 and R2 each independently any amino acid

The wavy lines indicate the covalent attachment sites to the phosphate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.

Further examples of tuning elements include but are not limited to

The wavy lines indicate the covalent attachment sites to the phosphate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.

In general, the spacer element is to allow for distance control away from the cell-targeting ligand. In some embodiments, this distance may have an impact on the stability/cleavability of the linker. Examples of spacer elements include straight polyethylglycol (PEG) chains (of a defined length) and straight carbon chains with or without solubilizing groups attached thereto.

Targeting Antibody

The linker arm and anti-inflammatory therapeutic agent may be linked to an antibody that selectively delivers the therapeutic agent to a cell, organ, or region of the body that expresses the human CD25 protein, human CD74 protein, human CD74 protein, or human CD163 protein. Antibodies may be either polyclonal or monoclonal, but most preferably are monoclonal and may be human, humanized, or human chimeric antibodies. The antibody may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The term “CD” refers to “cluster of differentiation”.

The antibody may be attached to the linker arm by any available reactive group that can react with the reactive functional group on the proximal end of the linker arm. For example, the antibody may be attached through an amine, carboxyl, sulfhydryl, or hydroxyl group. Such a group may reside at N-terminus or at a site internal to the protein chain, for example, the side chain of an amino acid. The antibody may be further derivatized at one or more sites to allow for the attachment of appropriate reactive groups onto the peptide or protein. See, Chrisey et al. Nucleic Acids Res. 24:3031-3039 (1996). In addition, the antibody may be synthesized to contain one or more non-natural amino acids, the side chain thereof which may then serve as a site for attachment of the linker arm comprising the payload-phosphate-based linker. Antibodies comprising non-natural amino acids for conjugation and methods for making such antibodies have been disclosed in U.S. Pat. No. 7,632,924, which is incorporated herein by reference. As exemplified herein the antibody may comprise a substitution of an amino acid residue in the heavy chain or light chain with the non-natural amino acid para-azidophenylalanine (pAzF). The azido group on the side chain of the pAzF residue may be conjugated to a reactive functional group of the therapeutic agent-linker such as a strained cycloalkyne, for example, cyclooctyne.

In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSYLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYSSSPLT; SEQ ID NO: 3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYIIN; SEQ ID NO: 4), CDR2 (RIIPILGVENYAQKFQG; SEQ ID NO: 5), and CDR3 (KDWFDY; SEQ ID NO: 6). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSFLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYSSSPLT; SEQ ID NO: 3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYPIN; SEQ ID NO: 7), CDR2 (RIIPILGIADYAQRFQG; SEQ ID NO: 8), and CDR3 (RDWGDY; SEQ ID NO: 9). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSGSSSYLA; SEQ ID NO: 1), CDR2 (GASSRAT; SEQ ID NO: 2), and CDR3 (QQYGSSPIT; SEQ ID NO:10) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYAIN; SEQ ID NO:11), CDR2 (RIIPILDIADYAQKFQD; SEQ ID NO:12), and CDR3 (KDWFDP; SEQ ID NO:13). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSSFLA; SEQ ID NO:1), CDR2 (GASSRAT; SEQ ID NO:2), and CDR3 (QQYSSSPLT; SEQ ID NO:3) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (RYPIN; SEQ ID NO:14), CDR2 (RIIPILGIADYAQRFQG; SEQ ID NO:8), and CDR3 (RDWGDY; SEQ ID NO:9). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD25 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD25 antibody that competes with any one of the aforementioned antibodies for binding to the CD25. The aforementioned anti-CD70 antibodies comprising said CDR sequences have been disclosed in U.S. Pat. No. 7,438,907, which is incorporated herein by reference.

In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSYLA; SEQ ID NO:15), CDR2 (YDASNRAT; SEQ ID NO:16), and CDR3 (QQRTNWPLT; SEQ ID NO:17) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (SYIMH; SEQ ID NO:18), CDR2 (VISYDGRNKYYADSVK; SEQ ID NO:19), and CDR3 (DTDGYDFDY; SEQ ID NO:20). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSALA; SEQ ID NO:21), CDR2 (DASSLES; SEQ ID NO:22), and CDR3 (QQFNSYPFT; SEQ ID NO:23) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (YYAMH; SEQ ID NO:24), CDR2 (VISYDGSIKYYADSVK; SEQ ID NO:25), and CDR3 (EGPYSNYLDY; SEQ ID NO:26). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSWLA; SEQ ID NO:27), CDR2 (AASSLQS; SEQ ID NO:28), and CDR3 (QQYNSYPLT; SEQ ID NO:29) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (DYGMH; SEQ ID NO:30), CDR2 (VIWYDGSNKYYADSVK; SEQ ID NO:31), and CDR3 (DSIVMVRGDY; SEQ ID NO:32). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQGISSWLA; SEQ ID NO:33), CDR2 (AASSLQS; SEQ ID NO:34), and CDR3 (QQYNSYPLT; SEQ ID NO:35) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (DHGMH; SEQ ID NO:36), CDR2 (VIWYDGSNKYYADSVK; SEQ ID NO:37), and CDR3 (DSIMVRGDY; SEQ ID NO:38). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD70 antibody comprising the light chain CDR sequences CDR1 (RASQSVSSYLA; SEQ ID NO:15), CDR2 (DASNRAT; SEQ ID NO:39), and CDR3 (QQRSNWPLT; SEQ ID NO:40) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (SDYYYWS; SEQ ID NO:41), CDR2 (YIYYSGSTNYDPSLKS; SEQ ID NO:42), and CDR3 (GDGDYGGNCFDY; SEQ ID NO:43). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD70 antibody that competes with any one of the aforementioned antibodies for binding to the CD70. The aforementioned anti-CD70 antibodies comprising said CDR sequences have been disclosed in U.S. Pat. No. 8,124,738, which is incorporated herein by reference.

In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising the light chain complementarity-determining region (CDR) sequences CDR1 (RSSQSLVHRNGNTYLH; SEQ ID NO:44), CDR2 (TVSNRFS; SEQ ID NO:45), and CDR3 (SQSSHVPPT; SEQ ID NO:46) and the heavy chain CDR sequences CDR1 (NYGVN; SEQ ID NO:47), CDR2 (WINPNTGEPTFDDDFKG; SEQ ID NO:48), and CDR3 (SRGKNEAWFAY; SEQ ID NO:49). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) selected from SEQ ID NO:44, SEQ ID NO: 45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, and SEQ ID NO:49. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD74 antibody that competes with any one of the aforementioned antibodies for binding to the CD74. Antibodies comprising said CDR sequences have been disclosed in U.S. Pat. No. 7,772,373, which is incorporated herein by reference. In a particular aspect, the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73.

In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising the light chain complementarity-determining region (CDR) sequences CDR1 (QGISSW; SEQ ID NO:50), CDR2 (AAS), and CDR3 (QQYNSYPLT; SEQ ID NO:51) and the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ASGRYYGSGSYSSYFD; SEQ ID NO:54); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSIK; SEQ ID NO:55), and CDR3 (ARGREYTSQNIVILLD; SEQ ID NO:56); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (ISYDGSNK; SEQ ID NO:53), and CDR3 (ARGREITSQNIVILLD; SEQ ID NO:57); or the heavy chain CDR sequences CDR1 (GFTFSSYA; SEQ ID NO:52), CDR2 (IWYDGSNK; SEQ ID NO:58), and CDR3 (ARGGTLVRGAMYGTDV; SEQ ID NO:59). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD74 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from AAS, SEQ ID NO:50, SEQ ID NO: 51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD74 antibody that competes with any one of the aforementioned antibodies for binding to the CD74. Antibodies comprising said CDR sequences have been disclosed in U.S. Patent Application Publication No. 20140030273, which is incorporated herein by reference. In a particular aspect, the anti-CD74 antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78.

In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD163 antibody comprising the light chain CDR sequences CDR1 (ASQSVSSDV; SEQ ID NO:60), CDR2 (YAS), and CDR3 (QDYTSPRT; SEQ ID NO:61) and the heavy chain complementarity-determining region (CDR) sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63); or the light chain CDR sequences CDR1 (ASQSVSHDV; SEQ ID NO:54), CDR2 (YTS), and CDR3 (QDYSSPRT; SEQ ID NO:65) and the heavy chain CDR sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63). In particular aspects, the antibody is a chimeric, humanized, or fully human anti-CD163 antibody comprising at least one, two, three, four, five, or six CDR(s) having an amino acid sequence selected from YYAS, YSG, YTS, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, and SEQ ID NO:65. In particular aspects the antibody is a chimeric, humanized, or fully human anti-CD163 antibody that competes with any one of the aforementioned antibodies for binding to the CD163. Antibodies comprising said CDR sequences have been disclosed in U.S. Patent Application Publication No. 20120258107 and 20120276193, which are incorporated herein by reference.

In particular embodiments, the antibody has reduced effector function or lacks effector function compared to a wild-type or native IgG1 antibody. Reducing or eliminating effector function may be achieved by providing an antibody with an IgG4 framework or constant domain. In one embodiment, the IgG4 constant domain may differ from the native human IgG4 constant domain (Swiss-Prot Accession No. P01861.1) at a position corresponding to position 228 in the EU system and position 241 as determined in the KABAT numbering scheme (See, e.g., Elvin A. Kabat ((1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.)), where the native Ser108 is replaced with Pro, in order to prevent a potential inter-chain disulfide bond between Cys106 and Cys109 (corresponding to positions Cys226 and Cys229 in the EU system and positions Cys239 and Cys242 in the KABAT system) that could interfere with proper intra-chain disulfide bond formation (See Angal et al. Mol. Imunol. 30:105 (1993)). In other instances, a modified IgG1 constant domain which has been modified to increase half-life or reduce effector function can be used.

In particular aspects, the antibody that has reduced or lacks effector function is an aglycosylated antibody that lacks the N-glycan at position 297 of the heavy chain (as determined using the KABAT Numbering scheme). Aglycosylated antibodies may be produced in a prokaryote expression system, for example, E. coli. The antibody may be encoded by a nucleic acid molecule that introduces an amino acid substitution in any of positions 297-299 of the heavy chain such that the antibody is substantially aglycosylated when the nucleic acid molecule is expressed in a mammalian cell. In IgG1, the glycosylation site is Asn297 within the amino acid sequence QYNS (SEQ ID NO:66). In other immunoglobulin isotypes, the glycosylation site corresponds to Asn297 of IgG1. For example, in IgG2 and IgG4, the glycosylation site is the asparagine within the amino acid sequence QFNS (SEQ ID NO:67). Accordingly, a mutation of Asn297 of IgG1 removes the glycosylation site in an Fc portion derived from IgG1. In one embodiment, Asn297 is replaced with Gln. In other embodiments, the tyrosine within the amino acid sequence QYNS (SEQ ID NO:66) is further mutated to eliminate a potential non-self T-cell epitope resulting from asparagine mutation. As used herein, a T-cell epitope is a polypeptide sequence in a protein that interacts with or binds an MHC class II molecule. For example, the amino acid sequence QYNS (SEQ ID NO:66) within an IgG1 heavy chain can be replaced with a QAQS (SEQ ID NO:68) amino acid sequence. Similarly, in IgG2 or IgG4, a mutation of asparagine within the amino acid sequence QFNS (SEQ ID NO:67) removes the glycosylation site in an Fc portion derived from IgG2 or IgG4 heavy chain. In one embodiment, the asparagine is replaced with a glutamine. In other embodiments, the phenylalanine within the amino acid sequence QFNS (SEQ ID NO:67) is further mutated to eliminate a potential non-self T-cell epitope resulting from asparagine mutation. For example, the amino acid sequence QFNS (SEQ ID NO:67) within an IgG2 or IgG4 heavy chain can be replaced with a QAQS (SEQ ID NO:68) amino acid sequence.

In particular aspects, the antibody comprises a substitution of one or more of the amino acids at position 318, 320, 322, 234, 235, 236, 237, or 297 of the antibody wherein the antibody with the substitution has a reduced effector function compared to an antibody comprising the native or wild-type amino acid at the position. The effector function may be binding affinity for Clq and/or binding affinity for the Fc receptor. These amino acid substitutions and their effect on reducing effector function have been disclosed in U.S. Pat. No. 5,648,260, which is incorporated herein by reference.

In particular aspects, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In one embodiment, the Fc region is modified to decrease the ability of the antibody to mediate effector function and/or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328.

Pharmaceutical Formulations and Administration

The conjugates disclosed herein are useful for the manufacture of medicaments for the treatment of diseases or disorders such as an inflammatory disease or cancer. The conjugates disclosed herein may be formulated into pharmaceutical formulations for use in treating diseases or disorders such as an inflammatory disease or cancer.

The present invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier. The compounds described herein including pharmaceutically acceptable carriers such as addition salts or hydrates thereof, can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections.

In particular embodiments, the conjugates of the invention comprising an antibody or antibody fragment as the targeting moiety are administered parenterally, more preferably intravenously. As used herein, the terms “administering” or “administration” are intended to encompass all means for directly and indirectly delivering a compound to its intended site of action. The compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof, may be administered singly, in combination with other compounds of the invention, and/or in cocktails combined with other therapeutic agents. The choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated.

The active compound(s) of the invention are administered per se or in the form of a pharmaceutical composition wherein the active compound(s) is in admixture with one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutical compositions for use in accordance with the present invention are typically formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.

Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxyniethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g.,

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injection is a preferred method of administration for the compositions of the current invention. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (e.g., subcutaneously or intramuscularly), intramuscular injection or a transdermal patch. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

The following examples are intended to promote a further understanding of the present invention.

Example 1

The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (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) dihydrogen pyrophosphate (1-4) was as follows.

Step A: 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 dihydrogen phosphate (1-1)

To a stirred solution of dexamethasone (0.40 g, 1.02 mmol) in THF (2.0 mL) at −40° C. was added diphosphoryl chloride (0.31 mL, 2.24 mmol) and the resulting mixture was stirred at −40° C. for 1 hr. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase washed with brine, dried over sodium sulfate and concentrated to give 1-1 as a solid (497 mg, 103%). LRMS (ES) (M+H)+: observed=473.3, calculated=473.4.

Step B: (9H-fluoren-9-yl)methyl (2-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)ethyl)carbamate (1-2)

The title compound was prepared from N-(9-fluorenylmethoxycarbonyl)ethanolamine according to the protocol outlined in Example 1-1 to afford 1-2. LRMS (ES) (M+H)+: observed=414.3, calculated=414.4

Step C: (9H-fluoren-9-yl)methyl (2-(((((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)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)ethyl)carbamate (1-3)

To a stirred solution of 1-2 (0.15 g, 0.41 mmol) in DMF (1.2 mL) was added triethylamine (0.06 mL, 0.41 mmol) and CDI (0.17 g, 1.03 mmol). The resulting solution was stirred at room temperature for 30 minutes. To this mixture was added 1-1 (0.19 g, 0.41 mmol) and ZnCl2 (0.45 g, 3.31 mmol) and the mixture was allowed to stir at room temperature overnight. The reaction was diluted with 1 N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-35% MeCN/water w/ 0.1% NH4OH modifier over 20 min) gave 1-3 as a solid (134 mg, 40%). LRMS (ES) (M+H)+: observed=818.6, calculated=818.7.

Step D: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (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) dihydrogen pyrophosphate (1-4)

To a stirred solution of 1-3 (0.19 g, 0.23 mmol) in DCM (3 mL) was added piperidine (0.15 mL, 1.51 mmol) and the resulting mixture was stirred at room temperature for 3 hrs. The solution was concentrated to dryness and redissolved in DCM (2 mL). In a separate vial a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.045 g, 0.25 mmol) in dichloromethane (1 mL) was added HOAT (0.034 g, 0.25 mmol), EDC (0.056 g, 0.30 mmol) and triethylamine (0.1 mL, 0.68 mmol). The resulting solution was stirred at room temperature for 40 minutes. The two solutions were combined and stirred at room temperature. Additional 2-(cyclooct-2-yn-1-yloxy)acetic acid activated with HOAT/EDC was added as necessary to complete reaction. Upon completion, the mixture was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-30% MeCN/water w/ 0.1% NH4OH modifier over 20 min) gave 1-4 as a solid (59 mg, 34%). LRMS (ES) (M+H)+: observed=760.6, calculated=760.7. 1H NMR (400 MHz, DMSO-d6): δ 8.64 (br s, 1H), 7.29 (d, J=9.15 Hz, 1H), 6.21 (dd, J=10.05, 1.93 Hz, 1H), 6.00 (s, 1H), 4.57 (d, J=8.3 Hz, 2H), 4.31 (t, J=5.4 Hz, 1H), 4.12 (d, J=11.22 Hz, 1H), 3.92 (dd, J=14.43, 8.59 Hz, 1H), 3.80-3.76 (complex, 3H), 3.30-3.16 (complex, 2H), 3.02-2.91 (complex, 2H), 2.63 (m, 1H), 2.40-2.19 (complex, 3H), 2.17-2.03 (complex, 4H), 1.96-1.82 (m, 3H), 1.80-1.72 (complex, 3H), 1.66-1.52 (complex, 4H), 1.50 (s, 3H), 1.40-1.31 (complex, 2H), 1.07-1.02 (m, 1H), 0.88 (s, 3H), 0.77 (d, J=7.17 Hz, 3H)

Example 2

The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (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) trihydrogen triphosphate (2-7) was as follows.

Step A: 1,3-dioxoisoindolin-2-yl 2-(cyclooct-2-yn-1-yloxy)acetate (2-1)

To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.20 g, 1.10 mmol) in DCM (4.0 mL) was added N-hydroxyphthalimide (0.36 g, 2.20 mmol) and EDC (0.42 g, 2.20 mmol). The resulting mixture was stirred at room temperature for 45 minutes. The reaction was directly injected onto a silica gel column and flash column separation using a 0-50% ethyl acetate/hexane gradient gave 2-1 as a solid (335 mg, 93%)

Step B: (9H-fluoren-9-yl)methyl dihydrogen phosphate (2-2)

The title compound was prepared from (9H-fluoren-9-yl)methanol according to the protocol outlined in Example 1-1 to afford 2-2. LRMS (ES) (M+H)+: observed=277.1, calculated=276.2

Step C: ((9H-fluoren-9-yl)methyl) (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) dihydrogen pyrophosphate (2-3)

The title compound was prepared from 2-2 and 1-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 2-3. LRMS (ES) (M+H)+: observed=731.2, calculated=730.6

Step D: 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 trihydrogen diphosphate (2-4)

To a stirred solution of 2-3 (0.29 g, 0.39 mmol) in DCM (2 mL) was added piperidine (0.23 mL, 2.36 mmol) and the resulting mixture was stirred at room temperature for 80 minutes. The solution was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 3-25% MeCN/water w/ 0.1% NH4OH modifier over 20 min) gave 2-4 as a solid (123 mg, 56%). LRMS (ES) (M+H)+: observed=553.2, calculated=552.4

Step E: (9H-fluoren-9-yl)methyl (2-(((((((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)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)ethyl)carbamate (2-5)

The title compound was prepared from 2-4 and 1-2 according to the protocol outlined in Example 1 to produce 1-3 to afford 2-5. LRMS (ES) (M+H)+: observed=898.3, calculated=897.7

Step F: 2-amionethyl (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) trihydrogen triphosphate (2-6)

The title compound was prepared from 2-5 according to the protocol outlined in Example 2 to produce 2-4 to afford 2-6. LRMS (ES) (M+H)+: observed=676.2, calculated=675.4

Step G: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (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) trihydrogen triphosphate (2-7)

To a stirred solution of 2-6 (0.027 g, 0.04 mmol) in DMF (0.8 mL) was added triethylamine (0.02 mL, 0.16 mmol) and 2-1 and the resulting mixture was stirred at room temperature for 30 minutes. The solution was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-40% MeCN/water w/ 0.1% NH4OH modifier over 20 min) gave 2-7 as a solid (8 mg, 24%). 1H NMR (499 MHz, DMSO): 0.78 (d, J=7.1 Hz, 3H); 0.88 (s, 3H); 1.10-0.99 (complex, 1H); 1.44-1.28 (complex, 2H); 1.50 (s, 3H); 1.67-1.52 (complex, 4H); 1.80-1.67 (complex, 3H); 1.93-1.83 (m, 3H); 2.17-2.01 (complex, 3H); 2.42-2.17 (complex, 3H); 2.67-2.57 (complex, 1H); 2.81 (d, J=79.7 Hz, 1H); 3.02-2.91 (complex, 1H); 3.17 (s, 1H); 3.26-3.21 (complex, 2H); 3.84-3.74 (complex, 2H); 3.91 (d, J=14.5 Hz, 1H); 4.15 (d, J=11.4 Hz, 1H); 4.31 (t, J=5.1 Hz, 1H); 4.57 (dd, J=18.0, 8.1 Hz, 1H); 4.71 (dd, J=17.9, 7.1 Hz, 1H); 5.99 (s, 1H); 6.20 (d, J=10.1 Hz, 1H); 7.30 (d, J=10.6 Hz, 1H); 8.48 (s, 1H). LRMS (ES) (M+H)+: observed=840.4, calculated=839.6

Example 3

The synthesis of dexamethasone linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (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) dihydrogen pyrophosphate (3-4) was as follows.

Step A: (9H-fluoren-9-yl)methyl (4-hydroxyphenyl)carbamate (3-1)

To a stirred solution of 4-aminophenol (0.30 g, 2.75 mmol) in DCM (9 mL) was added (9H-fluoren-9-yl)methyl carbonochloridate (0.71 g, 2.75 mmol) and the resulting mixture was stirred at room temperature for 2 hours. The mixture was partitioned between ethyl acetate and 1 N HCl solution. To the organic phase was added methanol until the solution cleared. The organic phase was dried over sodium sulfate and concentrated onto silica gel and flash column separation using a 100% ethyl acetate gave 3-1 as a solid (634 mg, 70%). LRMS (ES) (M+H)+: observed=332.3, calculated=331.3

Step B: (9H-fluoren-9-yl)methyl (4-(phosphonooxy)phenyl)carbamate (3-2)

To a stirred solution of 3-1 (0.31 g, 0.95 mmol) in THF (1.9 mL) at −40° C. was added diphosphoryl chloride (0.31 mL, 2.24 mmol) and triethylamine (1.32 mL, 9.51 mmol) and the resulting mixture was stirred at −40° C. for 3 hr. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase washed with brine, dried over sodium sulfate and concentrated to give 3-2 as a solid (342 mg, 87%). LRMS (ES) (M+H)+: observed=412.3, calculated=411.3

Step C: (9H-fluoren-9-yl)methyl (4-(((((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)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)phenyl)carbamate (3-3)

The title compound was prepared from 3-2 and 1-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 3-3. LRMS (ES) (M+H)+: observed=866.5, calculated=865.7

Step D: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (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) dihydrogen pyrophosphate (3-4)

The title compound was prepared from 3-3 according to the protocol outlined in Example 1 to produce 1-4 to afford 3-4. LRMS (ES) (M+H)+: observed=808.4, calculated=807.7

Example 4

The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (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) hydrogen phosphate (4-3) was as follows.

Step A: tert-butyl (2-(((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)(hydroxy)phosphoryl)oxy)ethyl)carbamate (4-1)

To a stirred solution of dexamethasone (0.10 g, 0.26 mmol) in THF (0.5 mL) at −40° C. was added diphosphoryl chloride (0.12 g, 0.48 mmol) and the resulting mixture was stirred at −40° C. for 1 hr. To this was added tert-butyl N-(2-hydroxyethyl)carbamate (0.12 g, 0.76 mmol) and triethylamine (0.14 mL, 1.0 mmol). The resulting mixture was stirred at −40° C. for 4 hr. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase was concentrated onto silica gel. Flash column separation using a 0-10% isopropanol/dichloromethane gradient gave 4-1 as a solid (115 mg, 73%). LRMS (ES) (M+H)+: observed=616.5, calculated=616.6.

Step B: 2-aminoethyl (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) hydrogen phosphate, HCl (4-2)

To a stirred solution of 4-1 (0.11 g, 0.18 mmol) in ethyl acetate (1 mL) at 0° C. was bubbled in HCl gas until saturated. The resulting solution was stirred at 0° C. for 1 hr and concentrated to give 4-2 as a solid (99 mg, 100%). LRMS (ES) (M+H)+: observed=516.4, calculated=516.5.

Step C: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (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) hydrogen phosphate (4-3)

To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.036 g, 0.20 mmol) in dichloromethane (1 mL) was added HOAT (0.027 g, 0.20 mmol), EDC (0.041 g, 0.22 mmol) and triethylamine (0.05 mL, 0.36 mmol). The resulting solution was stirred at room temperature for 40 minutes. This solution was added to 4-2 (0.10 g, 0.18 mmol) in DCM (1 mL). Additional 2-(cyclooct-2-yn-1-yloxy)acetic acid activated with HOAT/EDC was added as necessary to complete reaction. Upon completion, the mixture was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) gave 4-3 as a solid (45 mg, 37%). LRMS (ES) (M+H)+: observed=680.6, calculated=680.7. 1H NMR (400 MHz, DMSO-d6): δ 8.05 (br t, J=5.6 Hz, 1H), 7.30 (d, J=10.15 Hz, 1H), 6.21 (d, J=10.12 Hz, 1H), 6.00 (s, 1H), 5.55 (s, 1H), 5.37 (s, 1H), 4.70 (dd, J=17.2, 6.7 Hz, 1H), 4.29 (br t, J=6.4 Hz, 1H), 4.18-4.10 (complex, 2H), 3.87 (d, J=14.8 Hz, 1H), 3.74 (d, J=14.8 Hz, 1H), 3.69-3.64 (complex, 2H), 3.27-3.18 (complex, 2H), 2.91 (m, 1H), 2.61 (m, 1H), 2.39-2.05 (complex, 7H), 1.97-1.83 (complex, 2H), 1.80-1.69 (complex, 3H), 1.64-1.52 (complex, 3H), 1.48 (s, 3H), 1.47-1.30 (complex, 3H), 1.05 (m, 1H), 0.85 (s, 3H), 0.76 (d, J=7.13 Hz, 3H).

Example 5

The synthesis of dexamethasone linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (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) hydrogen phosphate (5-3) was as follows.

Step A: tert-butyl (4-(((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)(hydroxy)phosphoryl)oxy)phenyl)carbamate (5-1)

To a stirred solution of dexamethasone (0.20 g, 0.51 mmol) in THF (1.0 mL) at −40° C. was added diphosphoryl chloride (0.24 g, 0.97 mmol) and the resulting mixture was stirred at −40° C. for 1 hr 15 min. To this was added N-BOC-4-aminophenol (0.32 g, 1.53 mmol) and triethylamine (0.56 mL, 4.0 mmol). The resulting mixture was stirred at −40° C. for 30 minutes. The reaction was quenched with water, and treated with saturated sodium bicarbonate solution until pH ˜8. The solution was made acidic using 1N HCl solution and extracted several times with ethyl acetate. The combined organic phase was concentrated onto silica gel. Flash column separation using a 0-70% isopropanol/dichloromethane gradient gave 5-1 as a solid (370 mg, 88%). LRMS (ES) (M+H)+: observed=664.5, calculated=664.6.

Step B: 4-aminophenyl (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) hydrogen phosphate, HCl (5-2)

The title compound was prepared from 5-1 according to the protocol outlined in Example 4 to produce 4-2 to afford 2-2. LRMS (ES) (M+H)+: observed=564.4, calculated=564.5

Step C: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (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) hydrogen phosphate (5-3)

The title compound was prepared from 5-2 according to the protocol outlined in Example 4 to produce 4-3 to afford 5-3. LRMS (ES) (M+H)+: observed=728.6, calculated=728.7 1H NMR (400 MHz, DMSO-d6): δ 9.53 (s, 1H), 7.43 (d, J=8.54 Hz, 2H), 7.30 (d, J=10.14 Hz, 1H), 7.04 (d, J=8.54 Hz, 2H), 6.22 (dd, J=10.08, 1.92 Hz, 1H), 6.00 (s, 1H), 5.41 (s, 1H), 5.37 (d, J=4.22 Hz, 1H), 4.78 (dd, J=17.50, 6.20 Hz, 1H), 4.37 (t, J=5.59 Hz, 1H), 4.27 (dd, J=17.47, 9.02, 1H), 4.15-4.12 (m, 1H), 4.06 (d, J=14.53 Hz, 1H), 3.94 (d, J=14.59 Hz, 1H), 3.05 (q, J=7.26 Hz, 1H), 2.92 (m, 1H), 2.61 (m, 1H), 2.40-2.06 (complex, 7H), 1.98 (m, 1H), 1.87 (m, 1H), 1.82-1.73 (complex, 3H), 1.67-1.52 (complex, 3H), 1.48 (s, 3H), 1.43 (d, J=13.54 Hz, 2H), 1.35 (m, 1H), 1.05 (m, 1H), 0.84 (s, 3H), 0.76 (d, J=7.14 Hz, 3H).

Example 6

The synthesis of dexamethasone linker 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 hydrogen (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphoramidate (6-2) was as follows.

Step A: N-(2-aminoethyl)-2-(cyclooct-2-yn-1-yloxy)acetamide (6-1)

To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.10 g, 0.55 mmol) in dichloromethane (2 mL) was added HOAT (0.075 g, 0.55 mmol) and EDC (0.126 g, 0.66 mmol). The resulting solution was stirred at room temperature for 20 minutes. This solution was added to 1,2-ethylenediamine (0.49 g, 8.23 mmol) in DCM (1 mL) dropwise. The mixture was concentrated and purified. (Phenomenex Gemini NX C18, 5 um particle size, 21.2 mm i.d. by 5 cm length, 10-50% CH3CN/water w/ 0.1% NH4OH modifier over 10 min at 40 mL/min) (50 mg, 40%).

Step B: 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 hydrogen (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphoramidate (6-2)

To a stirred solution of 6-1 (0.05 g, 0.22 mmol) and 1-1 (0.035 g, 0.074 mmol) in a solution of t-butanol (1.2 mL) and water (0.25 mL) was added DCC (0.06 g, 0.30 mmol) and the resulting mixture was heated to 100 C for 4 hr. The reaction mixture was allowed to cool and concentrated. The residue was dissolved in a 1:1:1 MeOH:water:MeCN solution and syringe filtered. The mixture was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-45% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 6-2 as a solid (15 mg, 30%). LRMS (ES) (M+H)+: observed=679.5, calculated=678.7.

Example 7

The synthesis of dexamethasone linker 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl (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) dihydrogen pyrophosphate (7-1) was as follows.

To a stirred solution of 1-3 (0.19 g, 0.23 mmol) in DCM (3 mL) was added piperidine (0.15 mL, 1.51 mmol) and the resulting mixture was stirred at room temperature for 3 hrs. The solution was concentrated to dryness. The crude mixture was taken into a 2:1:1 methanol:acetonitrile:water mixture and filtered. The filtrate was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-35% MeCN/water w/ 0.1% NH4OH modifier over 20 min). A portion of the resulting purified amine (0.07 g, 0.11 mmol) was dissolved in DMF (0.8 mL). To this solution was added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.09 mg, 0.34 mmol) and DMAP (0.014 g, 0.11 mmol) and the resulting solution was stirred 20 minutes. The crude reaction mixture was directly purified using reverse phase preparative chromatography (Sunfire Prep C18 OBD 5 um 30×150 mm; 10-35% CH3CN/water w/ 0.1% TFA modifier over 20 min) gave 7-1 as a solid (13 mg, 15%). LRMS (ES) (M+H)+: observed=747.2, calculated=746.6.

Example 8

The synthesis of dexamethasone linker ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((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)(hydroxy)phosphoryl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)carbamate (8-5) was as follows.

Step A: (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) (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) phosphite (8-1)

To a stirred mixture of (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) diisopropylphosphoramidite (1.20 g, 1.40 mmol) and dexamethasone (0.50 g, 1.27 mmol) in acetonitrile (12 mL) was added 5-(ethylthio)-1H-tetrazole (0.33 g, 2.55 mmol). The resulting mixture was stirred for 20 minutes. To the homogenous solution that resulted was added 5M tert-butyl hydroperoxide (0.51 mL, 2.55 mmol). The reaction was stirred 1 hr at room temperature and concentrated onto silica gel. Flash column separation using a 0-100% ethyl acetate/hexane gradient gave 8-1 as a solid (1.67 g, 100%) LRMS (ES) (M+H)+: observed=1168.3, calculated=1168.3.

Step B: (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl (2-cyanoethyl) (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) phosphate (8-2)

To a stirred solution of 8-1 (1.48 g, 1.26 mmol) in DCM (30 mL) was added TFA (0.3 mL, 3.89 mmol) at room temperature. The reaction was stirred for 30 minutes, washed with saturated bicarbonate solution and the organic phase was concentrated onto silica gel. Flash column separation using a 0-10% isopropanol/DCM gradient gave 8-2 as a solid (0.79 g, 72%) LRMS (ES) (M+H)+: observed=866.5, calculated=865.9.

Step C: ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((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)(hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methyl (4-nitrophenyl) carbonate (8-3)

To a stirred solution of 8-2 (0.30 g, 0.35 mmol) in DCM (5 mL) was added triethylamine (0.15 mL, 1.04 mmol) and 4-nitrophenyl carbonochloridate (0.15 g, 0.76 mmol) and the resulting solution was stirred at room temperature. Additional 4-nitrophenyl carbonochloridate was added until reaction was complete by LCMS. The reaction was directly loaded onto a silica gel column and flash column separation using a 0-50% isopropanol/DCM gradient gave 8-3 as a solid (0.32 g, 93%) LRMS (ES) (M+H)+: observed=978.4, calculated=977.9.

Step D: ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((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)(hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methyl (2-aminoethyl)carbamate (8-4)

To a stirred solution of ethylenediamine (0.28 mL, 4.17 mmol) in DMF (1 mL) was added a solution of 8-3 (0.20 g, 0.20 mmol) in DMF (1 mL) dropwise. The reaction was stirred at room temperature for 10 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 8-4 as a solid (115 mg, 61%). LRMS (ES) (M+H)+: observed=899.5, calculated=898.9.

Step E: ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((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)(hydroxy)phosphoryl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)carbamate (8-5)

The title compound was prepared from 8-4 according to the protocol outlined in Example 4 to produce 4-3 to afford 8-5. LRMS (ES) (M+H)+: observed=949.4, calculated=948.9

Example 9

The synthesis of Budesonide linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (9-4) was as follows.

Step A: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl dihydrogen phosphate (9-1)

The title compound was prepared from budesonide according to the protocol outlined in Example 1 to produce 1-1 to afford 9-1. LRMS (ES) (M+H)+: observed=511.2, calculated=510.5

Step B: (9H-fluoren-9-yl)methyl (2-((hydroxy((hydroxy(2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,7′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)phosphoryl)oxy)ethyl)carbamate (9-2)

The title compound was prepared from 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 9-2. LRMS (ES) (M+H)+: observed=856.3, calculated=855.8

Step C: 2-aminoethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (9-3)

The title compound was prepared from 9-2 according to the protocol outlined in Example 2 to produce 2-6 to afford 9-3. LRMS (ES) (M+H)+: observed=634.3, calculated=633.5

Step D: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (9-4)

The title compound was prepared from 9-3 according to the protocol outlined in Example 2-7 to afford 9-4. Mixture of isomers 1H NMR (499 MHz, DMSO): 0.86-0.83 (complex, 6H); 0.89 (s, 6H); 0.99-0.90 (complex, 2H); 1.09 (t, J=7.0 Hz, 2H); 1.15 (t, J=7.2 Hz, 6H); 1.35-1.25 (complex, 6H); 1.39 (s, 6H); 2.32-1.46 (complex, 32H); 3.02 (q, J=7.0 Hz, 4H); 3.21 (br s, 2H); 3.81-3.73 (complex, 6H); 3.94-3.89 (complex, 2H); 4.33-4.25 (complex, 4H); 4.39 (d, J=18.4 Hz, 2H); 4.56 (t, J=4.3 Hz, 1H); 4.79-4.63 (complex, 3H); 5.01 (d, J=7.2 Hz, 1H); 5.17 (dd, J=4.9, 4.6 Hz, 1H); 5.91 (s, 2H); 6.15 (d, J=10.1 Hz, 2H); 7.31 (d, J=10.3 Hz, 2H); 8.84 (br s, 2H). LRMS (ES) (M+H)+: observed=798.4, calculated=797.7

Example 10

The synthesis of Budesonide linker 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (10-1) was as follows.

The title compound was prepared from 9-2 according to the protocol outlined in Example 7 to produce 7-1 to afford 10-1. LRMS (ES) (M+H)+: observed=785.4, calculated=784.6

Example 11

The synthesis of Budesonide linker 1-(cyclooct-2-yn-1-yloxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (11-5) was as follows.

Step A: (9H-fluoren-9-yl)methyl (2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)ethyl)carbamate (11-1)

To a stirred solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (1.00 g, 5.17 mmol) in DCM (15 mL) was added 9-fluorenylmethyl chloroformate (1.34 g, 5.17 mmol) and triethylamine (1.08 mL, 7.76 mmol). The resulting solution was stirred at room temperature for 10 minutes. The reaction was concentrated onto silica gel and flash column separation using a 0-10% isopropanol/dichloromethane gradient gave 11-1 as an oil (1.43 g, 66%) LRMS (ES) (M+H)+: observed=416.1, calculated=415.4.

Step B: (9H-fluoren-9-yl)methyl (2-(2-(2-(2-(phosphonooxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (11-2)

The title compound was prepared from 11-1 according to the protocol outlined in Example 1 to produce 1-1 to afford 11-2. LRMS (ES) (M+H)+: observed=496.3, calculated=495.4

Step C: (9H-fluoren-9-yl)methyl (2-(2-(2-(2-((hydroxy((hydroxy(2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,7′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)phosphoryl)oxy)ethoxy)ethoxy)ethoxy)ethyl)carbamate (11-3)

The title compound was prepared from 11-2 and 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 11-3. LRMS (ES) (M+H)+: observed=988.6, calculated=987.9

Step D: 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,7′:4,5]indeno[,2-d][,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (11-4)

The title compound was prepared from 11-3 according to the protocol outlined in Example 2 to produce 2-6 to afford 11-4. LRMS (ES) (M+H)+: observed=766.5, calculated=765.7

Step E: 1-(cyclooct-2-yn-1-yloxy)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,7′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (11-5)

The title compound was prepared from 11-4 according to the protocol outlined in Example 2 to produce 2-7 to afford 11-5. LRMS (ES) (M+H)+: observed=930.6, calculated=929.9

Example 12

The synthesis of Budesonide linker 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-3) was as follows.

Step A: (9H-fluoren-9-yl)methyl ((2S)-1-(((2S)-1-((4-((((2-cyanoethoxy)(2((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (12-1)

To a stirred solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.20 g, 0.33 mmol) in DMF (4.7 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (0.11 g, 0.37 mmol). To this mixture was added 0.45M tetrazole in acetonitrile (0.81 mL, 0.37 mmol) dropwise and the resulting mixture was stirred for 20 minutes at room temperature. To this was added Budesonide (0.22 g, 0.50 mmol) and 5-(ethylthio)-1H-tetrazole (0.09 g, 0.67 mmol) and allowed to stir to 30 minutes at room temperature. To this was added 6 M tertbutyl hydroperoxide in decane (0.12 mL, 0.73 mmol) and allowed to stir at room temperature for 1 hour. The crude reaction was loaded directly onto silica gel and flash column separation using a 0-100% ethyl acetate/hexane gradient followed by a 0-50% isopropanol/DCM gradient gave 12-1 as a solid. (0.10 g, 27%) LRMS (ES) (M+H)+: observed=1147.7, calculated=1147.2.

Step B: 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-2)

To a stirred solution of 12-1 (0.10 g, 0.09 mmol) in DCM (1.8 mL) was added DBU (0.05 mL, 0.35 mmol) and the resulting solution was stirred 20 minutes at room temperature. The reaction was concentrated and dissolved in a 2:1:1 methanol:water:acetonitrile mixture and syringe filtered. The filtrate was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 12-2 as a solid (42 mg, 53%). LRMS (ES) (M+H)+: observed=872.6, calculated=871.9.

Step C: 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-3)

The title compound was prepared from 12-2 according to the protocol outlined in Example 4 to produce 4-3 to afford 12-3. LRMS (ES) (M+H)+: observed=1036.8, calculated=1036.1

Example 13

The synthesis of Budesonide linker 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-1) was as follows.

Step A: synthesis 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-1)

To a stirred solution of budesonide (2.00 g, 4.65 mmol) in pyridine (20.0 mL) at room temperature was added acetic anhydride (2.0 mL, 21.20 mmol) and the resulting mixture was stirred for 2.5 hours. The reaction was chilled in an ice bath and quenched with saturated sodium bicarbonate solution (20.0 mL). The solution was extracted several times with ethyl acetate. The combined organic phase washed with brine, dried over sodium sulfate and concentrated to give 13-1 as a solid (2.30 g, 105%). LRMS (ES) (M+H)+: observed=473.4, calculated=472.5.

Step B: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-((hydroxyhydrophosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-2)

To a stirred solution of 13-1 (0.50 g, 1.06 mmol) in THF (10.0 mL) at −78° C. was added phosphorus trichloride (0.18 mL, 2.12 mmol) dissolved in THF (2.0 mL) followed by triethylamine (0.74 mL, 5.29 mmol) dissolved in THF (2.0 mL). The resulting mixture was stirred at −78° C. for 10 minutes and allowed to warm to room temperature for 45 minutes. The reaction was chilled in an ice bath and quenched with water (0.50 mL). The solution was allowed to warm to room temperature and saturated sodium bicarbonate solution was added until pH 9 and stirred for 10 minutes. The mixture was acidified with 1N HCl and was extracted several times with ethyl acetate. The combined organic phase was dried over sodium sulfate and concentrated to give 13-2 as a solid (0.55 g, 97%). LRMS (ES) (M+H)+: observed=537.3, calculated=536.5.

Step C: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-((hydroxy(1H-imidazol-1-yl) phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-3)

To a stirred solution of 13-2 (0.55 g, 1.03 mmol) and imidazole (0.35 g, 5.13 mmol) in pyridine (8.0 mL) at room temperature was added TMS-Cl (1.31 mL, 10.25 mmol) and the resulting solution was stirred for 10 minutes. To this mixture was added iodine (0.52 g, 2.05 mmol) dissolved in pyridine (2 mL) and stirred room temperature for 50 minutes. The reaction was then cooled in and ice bath and quenched with water (0.5 mL). The reaction was concentrated, dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 13-3 as a solid (282 mg, 45%). LRMS (ES) (M+H)+: observed=603.4, calculated=602.6.

Step D: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-(((((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-4)

To a stirred solution of 13-3 (0.20 g, 0.33 mmol) and 1-2 (0.12 g, 0.33 mmol) in DMF (1.4 mL) was added ZnCl2 (0.36 g, 2.66 mmol) and the mixture was allowed to stir at room temperature overnight. The reaction was diluted with 1 N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated, dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 13-4 as a solid (166 mg, 55%). LRMS (ES) (M+H)+: observed=898.4, calculated=897.8.

Step E: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-(((((2-aminoethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-5)

The title compound was prepared from 13-4 according to the protocol outlined in Example 2 to prepare 2-4 to afford 13-5. LRMS (ES) (M+H)+: observed=676.4, calculated=675.6.

Step F: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-6)

To a stirred solution of 13-5 (0.037 g, 0.055 mmol) and 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.032 g, 0.175 mmol) in DMF (0.8 mL) was added HATU (0.066 g, 0.175 mmol) and triethylamine (0.03 mL, 0.22 mmol). The reaction was stirred at room temperature for 20 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 13-6 as a solid (36 mg, 78%). LRMS (ES) (M+H)+: observed=840.5, calculated=839.8.

Step G: 2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl acetate (13-7)

To a stirred solution of 13-6 (0.035 mg, 0.042 mmol) in methanol (0.50 mL) was added 70% perchloric acid (7.2 μL, 0.083 mmol) and the resulting solution was stirred room temperature overnight. The reaction was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 μM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 13-7 as a solid (16 mg, 47%). LRMS (ES) (M+H)+: observed=798.4, calculated=797.7.

Example 14

The synthesis of Fluticasone linker (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (14-5) was as follows.

Step A: (6S,8S,9R,10S,1S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-((hydroxyhydrophosphoryl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (14-1)

The title compound was prepared from fluticasone propionate according to the protocol outlined in Example 13 to prepare 13-2 to afford 14-1. LRMS (ES) (M+H)+: observed=565.3, calculated=564.5.

Step B: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)-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 propionate (14-2)

The title compound was prepared from 14-1 according to the protocol outlined in Example 13 to prepare 13-3 to afford 14-2. LRMS (ES) (M+H)+: observed=631.3, calculated=630.6.

Step C: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (14-3)

The title compound was prepared from 14-2 according to the protocol outlined in Example 13 to prepare 13-4 to afford 14-3. LRMS (ES) (M+H)+: observed=943.4 (M+H+NH3), calculated=925.8.

Step D: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-aminoethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (14-4)

The title compound was prepared from 14-3 according to the protocol outlined in Example 2 to prepare 2-4 to afford 14-4. LRMS (ES) (M+H)+: observed=704.3, calculated=703.6.

Step F: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (14-5)

The title compound was prepared from 14-4 according to the protocol outlined in Example 13 to prepare 13-6 to afford 14-5. LRMS (ES) (M+H)+: observed=868.4, calculated=867.8.

Example 15

The synthesis of Fluticasone linker (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy) phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (15-5) was as follows.

Step A: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-11-((methylthio)methoxy)-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-1)

To a stirred solution of fluticasone propionate (0.50 g, 1.00 mmol) in MeCN (5.0 mL) at 0° C. was added dimethyl sulfide (0.59 mL, 8.00 mmol) followed by benzoyl peroxide (0.97 g, 4.00 mmol) added in four portions over 20 minutes. The resulting mixture was stirred at 0° C. for 1 hour. The reaction was concentrated, taken up in ethyl acetate and washed with saturated sodium bicarbonate. The combined organic phase was concentrated. The crude was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 40-80% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give to give 15-1 as a solid (0.07 g, 12.7%). LRMS (ES) (M+H)+: observed=561.3, calculated=560.6.

Step B: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-11-((phosphonooxy)methoxy)-6, 7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-2)

Phosphoric acid (0.09 g, 0.89 mmol) was heated under nitrogen at 120° C. for 30 minutes. This was allowed to cool and to it was added molecular seives and 15-1 (0.07 g, 0.13 mmol). This mixture was dissolved in THF (1.3 mL) and NIS (0.04 g, 0.19 mmol) was added. The resulting solution was allowed to stir overnight at room temperature. The mixture was filtered and concentrated. The crude was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 10-50% MeCN/water w/ 0.1% NH40H modifier over 20 min) to give to give 15-2 as a solid (0.05 g, 63%). LRMS (ES) (M+H)+: observed=611.3, calculated=610.5.

Step C: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (15-3)

The title compound was prepared from 15-2 according to the protocol outlined in Example 1 to prepare 1-3 to afford 15-3. LRMS (ES) (M+H)+: observed=956.5, calculated=955.8.

Step D: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-aminoethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (15-4)

The title compound was prepared from 15-3 according to the protocol outlined in Example 12 to prepare 12-2 to afford 15-4. LRMS (ES) (M+H)+: observed=734.5, calculated=733.6.

Step E: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-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 propionate (15-5)

The title compound was prepared from 15-4 according to the protocol outlined in Example 13 to prepare 13-6 to afford 15-5. LRMS (ES) (M+H)+: observed=898.4, calculated=897.8.

Example 16

The synthesis of Budesonide linker 1-(cyclooct-2-yn-1-yloxy)-2-oxo-3-aza-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tricosaoxahenheptacont-74-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-5) was as follows.

Step A: (9H-fluoren-9-yl)methyl (71-hydroxy-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontyl)carbamate (16-1)

The title compound was prepared from 71-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontan-1-ol and (9H-fluoren-9-yl)methyl (2,5-dioxopyrrolidin-1-yl) carbonate according to the protocol outlined in Example 11 to produce 11-1 to afford 16-1. LRMS (ES) (M+H)+: observed=1314.1, calculated=1296.5

Step B: (9H-fluoren-9-yl)methyl (71-(phosphonooxy)-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontyl)carbamate (16-2)

The title compound was prepared from 16-1 according to the protocol outlined in Example 1 to produce 1-1 to afford 16-2. LRMS (ES) (M+H)+: observed=1394.0, calculated=1376.5

Step C: (9H-fluoren-9-yl)methyl (71-((hydroxy((hydroxy(2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)phosphoryl)oxy)-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontyl)carbamate (16-3)

The title compound was prepared from 16-2 and 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 16-3. LRMS (ES) (M+H)+: observed=1886.7, calculated=1869.0

Step D: 72-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontanyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-4)

The title compound was prepared from 16-3 according to the protocol outlined in Example 12 to produce 12-2 to afford 16-4. LRMS (ES) (M+H)+: observed=1664.0, calculated=1646.7

Step E: 1-(cyclooct-2-yn-1-yloxy)-2-oxo-3-aza-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tricosaoxahenheptacont-74-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-5)

The title compound was prepared from 16-4 according to the protocol outlined in Example 13 to produce 13-6 to afford 16-5. LRMS (ES) (M+H)+: observed=1828.7, calculated=1810.9

Example 17

The synthesis of Budesonide linker 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (17-2) was as follows.

Step A: 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (17-1)

To a stirred solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.05 g, 0.083 mmol) in DMF (1.2 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (29 uL, 0.09 mmol). To this mixture was added 0.45M tetrazole in acetonitrile (0.20 mL, 0.09 mmol) dropwise and the resulting mixture was stirred for 20 minutes at room temperature. To this was added 9-1 (0.042 g, 0.083 mmol) and 5-(ethylthio)-1H-tetrazole (0.02 g, 0.16 mmol) and allowed to stir to 30 minutes at room temperature. To this was added 6 M tertbutyl hydroperoxide in decane (0.03 mL, 0.18 mmol) and allowed to stir at room temperature for 30 minutes. To this was added DBU (0.12 mL, 0.83 mmol) and the resulting solution was stirred overnight at room temperature. The crude reaction was purified by direct injection using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30×100 mm; 5-40% MeCN/water w/ 0.1% NH4OH modifier over 20 min) to give 17-1 as a solid (18.5 mg, 23%). LRMS (ES) (M+H)+: observed=952.6, calculated=951.9.

Step B: 4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (17-2)

The title compound was prepared from 17-1 according to the protocol outlined in Example 13 to produce 13-6 to afford 17-2. LRMS (ES) (M+H)+: observed=1116.6, calculated=1116.1.

Example 18

The solubility of exemplary drug-linker conjugates in aqueous solutions was evaluated. Linkers utilized in drug conjugates may have aqueous solubility to enable conjugation in aqueous media amenable to protein solubilization. Furthermore, linkers with aqueous solubility are considered hydrophilic, and may confer to the drug conjugate a reduced propensity to aggregate relative to standard hydrophobic linkers in the literature. The following exemplary drug-linkers were tested in an aqueous solution comprising 20% acetonitrile (MeCN/H2O) for solubility at a concentration of 10 mg/mL. As shown in Table 1, the exemplary linker-drug conjugates displayed high solubility, which may be a result of the contribution of the polarity and charge of the phosphate containing linker to the drug-linker conjugate.

TABLE 1 Measured Solubility in Drug Linker 20% MeCN/H2O 1-4 >10 mg/mL 2-7 >10 mg/mL 3-4 >10 mg/mL 4-3 >10 mg/mL 5-3 >10 mg/mL

Example 19 In Vitro Stability Studies of Exemplary Drug-Linkers in Blood and Lysosomal Lysates.

Exemplary dexamethasone-linkers 4-3, 5-3, 1-4, 3-4, and 2-7 were incubated in relevant biomatrices to measure their stability and propensity to release free drug (Tables 2 to 4).

The exemplary dexamethasone-linkers were studied for their stability (Table 1) and propensity to release dexamethasone (Table 2) in human blood. As shown in the tables, all the dexamethasone linkers were stable in blood with little detectable degradation or release of free dexamethasone.

TABLE 1 Time Course of Calculated Conc. for each compound in human whole blood (nM) Dexa- Ma- meth- trix Time asone 4-3 5-3 1.4 3-4 2-7 Matrix 2939 2821 n/a 3059 n/a 1059 spiking 0 m 1687 1539 1299 1617 1382 1040 5 m 1251 1325 10 m 15 m 1276 1398 20 m 2409 1861 1390 30 m 1111 1250 1 hr 2224 1619 937 1421 1141 1515 2 hr 1480 1703 3 hr 1502 1677 1899 6 hr 2792 2040 1400 123

TABLE 2 Time Course of Calculated Conc. for dexamethasone in human whole blood (nM) Dexa- Ma- meth- trix Time asone 4-3 15-3 1.4 3-4 2-7 Matrix 2939 4 0 2 0 0 spiking 0 m 1687 0 0 0 0 0 5 m 0 0 10 m 15 m 0 0 20 m 2409 0 0 30 m 0 0 1 hr 2224 0 0 0 0 0 2 hr 0 0 3 hr 1502 1 0 6 hr 2792 3 0 0

General Experimental Procedure Human Blood Incubation

Human blood was collected the morning of the experiment from at least 3 individuals using K2EDTA as the anticoagulant. An equal volume from each individual was combined for use in the experiment. The experiment started no more than 2 hours after the blood collection. All drug-linker conjugates were solubilized in DMSO to form each 10 mM stock solution. Dosing solution for each linker was prepared by serial dilution of each stock solution using 1:3 acetonitrile: water. All solutions were kept on ice during the experiment.

Human blood was pre-warmed in a 37° C. water bath in an appropriate volume to collect samples over a time course from 0 through 6 hours. Incubating blood was mixed well just prior to sampling to give a homogenous mixture. Aliquots of blood were removed at appropriate time points, added to cold stopping solution, which was methanol containing an appropriate internal standard, and mixed rigorously. The samples were centrifuged at 4000 RPM for 10 minutes after which equal volumes of the supernatant fractions were diluted with cold deionized water. The samples were then ready for analysis. A time 0 sample was prepared by spiking blood, which had been pretreated with the same stopping reagent used above with the drug-linker. This sample is referred to in the tables as the matrix spiking.

Representative dexamethasone-linkers 4-3, 5-3, 1-4, 3-4, and 2-7 were studied for their stability (Table 4) and propensity to release dexamethasone (Table 5) in purified rat liver lysosomal lysates. Tables 4 and 5 show that the different dexamethasone-linkers released dexamethasone at different rates depending on the structure of the tuning element. For example, 3-4 was no longer detectable after 10 minutes incubation in the rat lysosomal extract whereas for 4-3 and 5-3 the dexamethasone was more slowly released with 5-3 releasing dexamethasone faster than 4-3.

TABLE 3 Time Course of Calculated Conc. for each compound in rat lysosomal lysate (nM) Dexa- Ma- meth- trix Time asone 4-3 5-3 1.4 3-4 2-7 Matrix 2059.0 1590.7 2555.29 2487.3 3128.83 2228 spiking 0 m 1456.4 888.8 1394.08 1148.2 1252 1327 5 m 1851.2 1156.1 1295.86 158.2 501.28 56 10 m 1640.8 983.4 1278.4 19.0 149.18 512 15 m 1666.7 1032.6 1407.73 3.7 N/A 0 30 m 1602.6 980.1 1175.39 N/A N/A 37 1 hr 1576.8 882.4 970.65 N/A N/A 0 2 hr 1681.0 880.1 743.45 N/A N/A 0 3 hr 1689.7 903.8 500.41 N/A N/A 0 6 hr 1689.8 858.9 221.03 N/A N/A 0

TABLE 4 Time Course of Calculated Conc. for dexamethasone in rat lysosomal lysate (nM) Ma- Dexameth- trix Time asone 4-3 5-3 1.4 3-4 2-7 Matrix 2059.0 N/A N/A N/A N/A 17 spiking 0 m 1456.4 0.9 N/A 4.7 15.74 44 5 m 1851.2 14.5 10.25 320.7 100.44 415 10 m 1640.8 14.2 21.56 559.4 209.07 682 15 m 1666.7 22.3 31.68 831.7 285.67 808 30 m 1602.6 28.9 78.6 1059.6 425.28 1039 1 hr 1576.8 41.6 162.45 1148.8 526.5 1159 2 hr 1681.0 72.5 290.66 854.5 534.84 1309 3 hr 1689.7 90.2 379.36 841.9 584.61 1207 6 hr 1689.8 141.9 550.43 826.6 561.88 1155

General Experimental Procedure Rat Lysosome Incubation:

Rat Liver lysosomes were available commercially with a pool of 6 animals. All linker compounds were solubilized in DMSO to form each 10 mM stock solution. Dosing solution for each linker was prepared by serial dilution of each stock solution using 1:3 acetonitrile: water. All solutions were kept on ice during the experiment.

Rat lysosomes were pre-warmed in a 37° C. water bath in an appropriate volume to collect samples over a time course from 0 minutes through 6 hours. Incubating lysosomes were mixed well just prior to sampling to give a homogenous mixture. Aliquots of lysosomes were removed at appropriate time points, added to cold stopping solution, which was methanol containing an appropriate internal standard, and mixed rigorously. The samples were centrifuged at 4000 RPM for 10 minutes after which equal volumes of supernatant were diluted with cold deionized water. The samples were ready for analysis. A time 0 sample was prepared by spiking the drug-linker to lysosomes which had been pretreated with the same stopping reagent as above. This sample is referred to as matrix spiking in the tables.

Liquid Chromatography—Tandem Mass Spectrometry Analysis

A Thermo LX-2 ultra-performance liquid chromatography system coupled with a Sciex API5000 triple quadrupole mass spectrometer was used for the analysis. The payload and drug-linkers were retained and separated by a C18 column and detected by the mass spectrometer. The standard curve for each analyte was prepared to obtain the quantitative results. Samples were kept in cold stack set at 5° C.

Example 20 Synthesis, Purification and Analysis of ADC Using Exemplary Drug-Linker 1-4

To establish the chemical reactivity of this linker design to form a drug conjugate, exemplary drug-linker 1-4 was conjugated to an anti-mouse CD25 antibody (IgG1) (mCD25) to produce antibody-drug conjugate ADC 12-1 or anti-human CD70 antibody (hCD70) to produce ADC 12-2. Specifically, the drug-linker was conjugated to the unnatural amino acid para-azido-phenylalanine (pAzF) replacing the alanine at position 1 of CH1 of the antibody using copper-free 3+2 cycloaddition chemistry. Synthesis of antibodies containing an unnatural amino acid has been described in U.S. Pat. No. 7,632,924, incorporated herein by reference, and copper-free 3+2 cycloaddition chemistry has been described in U.S. Pat. No. 7,807,619, incorporated herein by reference. Conjugation, purification, and analysis confirmed synthesis of the Anti-CD25 antibody-drug conjugate ADC 12-1 and the anti-CD70 ADC 12-2.

Experimental for Conjugation of Phosphate Linker 1-4 to antibodies. Antibodies were purified over protein A column (NovaSep) followed by SP 6505 column (Tosoh Biosciences). The heavy chain and light chain of the anti-CD25 antibody comprise SEQ ID NO:81 and SEQ ID NO:82, respectively, wherein X is pAzF. The heavy chain and light chain of the anti-CD70 antibody comprise SEQ ID NO:89 and SEQ ID NO:80, respectively, wherein X is pAzF.

Site specific conjugation using click (2+3) chemistry. Para-azidophenylalanine containing antibodies were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; 0-20% Dimethylamine, pH 6.0 and concentrated to 1-20 mg/mL. 10-15 molar equivalents of cyclooctyne drug-linker were added and reacted for 16-72 hours at 28-30° C. The antibody conjugates were purified over a SP 650S column (Tosoh Biosciences) to remove excess reagents. The conjugates were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; pH 6.0, 0.22 m filtered, and stored at 4° C.

Conjugation analysis. Conjugation efficiency and DAR values were determined by reversed phase HPLC. The ADC was run over a Zorbax 300SB-C3 column, 4.6×150 mm (Agilent) at 80° C. and eluted with a linear gradient from 30% B to 90% B (A: water, 0.1% TFA; B: acetonitrile, 0.1% TFA). An Agilent 1200 series HPLC system and Chemstation software were used to resolve and quantify percentage of antibody conjugated with drug-linker.

Example 21 Serum Stability of ADC 12-1

Drug conjugates may be designed with a stable linker to ensure that the attached payload adopts the pharmacokinetic properties of the carrier. In the example of antibody drug conjugates, premature release of the payload will reduce the total payload delivered to a target cell. To establish the potential for circulatory stability of this linker design in the context of a drug conjugate, ADC 12-1 was incubated in mouse serum and monitored for degradation or loss of payload (dexamethasone). As shown in FIG. 2, no measurable loss of dexamethasone was observed over three weeks incubation in serum.

In Vitro Stability Study Design in DBA1 Mice Serum

For the in vitro stability study, the ADC 12-1 was spiked in DBA1 mice serum at 0.1 mg/mL. Samples were sealed under nitrogen, placed at 37° C. in a cell culture incubator and stored at −80° C. until analysis. Time points from 0 min to 21 days of incubation were evaluated.

In Vitro Stability—Free Payload Analysis

Samples were evaluated for free dexamethasone by LC-MS/MS. For the in vitro stability study, 40 μL of serum for each time point underwent protein precipitation with 400 μL acetonitrile containing dexamethasone-d4 (internal standard). Tubes were centrifuged at 14000 RPM (4° C.) for 10 minutes and the supernatant fraction removed and dried in a speed vac. Samples were reconstituted with methanol/water (50/50) and injected in an Acquity/TSQ Vantage triple quad mass spectrometer equipped with an Xbridge C18 column (Waters, Milford). Mobile phases consisted of buffers A (water:acetonitrile:formic acid, 95:5:0.1%) and B (acetonitrile:water:formic acid, 80:20:0.1%) and a linear gradient was performed with buffer B increasing from 30 to 65% in 3 minutes. Flow rate was set at 0.3 mL/minutes. Transitions for acetate adduct of dexamethasone and dexamethasone d-4 were 437>361 and 441>363, respectively. Compounds were detected using negative electrospray ionization. Free dexamethasone was not be detected in the in vitro stability serum samples over the period evaluated.

In Vitro Stability—Immunoprecipitation Coupled to Intact Mass Analysis

ADC 12-1 was pulled down from mice serum using immunoprecipitation (IP) with streptavidin magnetic beads (Dynabeads M-280) coupled to biotinylated CD25 (antigen). Beads were washed 3 times with 100 μL of TBS 1×. Two microliters of 1 mg/mL biotinylated CD25 was added to 30 μL of each sample (serum from each time point) and incubated for 10 minutes at room temperature (RT). Samples were added to the pre-washed beads and incubated for 30 minutes at RT under gentle shaking. The flow through was discarded and beads were washed twice with 0.02% Rapigest in TBS 1×. Elution was performed with 30 μL of TFA 0.1% and 5 μL of each sample was analyzed in an UPLC/Synapt G2-S (Waters, Milford) equipped with a POROS column (ABSciex) using reverse phase gradient.

Spectra deconvolution was performed using MaxEntl software (Waters, Milford) and results were compared against the intact mass obtained for the ADC from a stock solution (FIG. 3). FIG. 3 shows a deconvoluted intact mass spectrum for the ADC 12-1 stock solution. G0F, G1F and G2F in the figure refer to the carbohydrate isoforms on the antibody portion of the antibody-drug conjugate.

The deconvoluted spectra of the stock solution (FIG. 3) revealed that the antibody-drug conjugate 12-1 intact mass in its predominant DAR 2 form contained two GOF sugar motifs. Additional peaks at about 162 Da apart showed two other glycoforms containing GOF/G1F (peak at 148701 Da) and 2×G1F (peak at 148863 Da). The glycan profile is typical of that for an IgG.

FIG. 4A-4D shows the deconvoluted spectra of time points 1 hour, 8 hours, 14 days and 21 days from the in vitro stability study. The data analysis showed that no significant mass change occurred for the ADC over the incubation period evaluated in the study (Table 6: intact mass results for the in-vitro stability study).

TABLE 6 In vitro Stability MW of the ADC 12-1 Incubation (2xG0F form) Time 148539 1 hour 148539 8 hours 148539 2 days 148539 3 days 148540 7 days 148540 14 days 148540 21 days

In Vivo Stability of ADC 12-1

Effective linker designs will not only provide stable tethering to a carrier in drug conjugates, but should also have minimal to no effect on the pharmacokinetic properties of the carrier itself. To establish the potential of this linker design for in vivo circulatory stability and to understand its impact on the pharmacokinetics of the conjugated carrier in the context of a drug conjugate, ADC 12-1 was dosed to DB1 mice and was monitored for degradation of the mAb, intactness of the antibody-drug conjugate, and loss of payload (dexamethasone). Importantly, the study showed that the inherent pharmacokinetics of naked (non-conjugated) anti-mouse CD25 was not adversely affected by the conjugation of two molecules of drug-linker 1-4 to the antibody. Furthermore, the study showed that no loss of drug linker and no measurable dexamethasone were observed over the course of the 5 day study. The data shows that drug-linker 1-4 was stable in circulation and retained payload on the carrier in this example of an antibody-drug conjugate.

In Vivo Pharmacokinetic (PK)/Stability Study General Experimental for In Vivo PK Study of ADC 12-1

An in vivo study in DBA1 mice was performed in order to evaluate stability and pharmacokinetics of ADC 12-1. Naked antibody was administered intravenously in a single bolus to all groups. Group 1 was given a dose of 2mpk; Group 2 and Group 3 were dosed 2 and 4 mpk of ADC 12-1, respectively. Plasma samples were taken from all three groups at 1, 2, 6, 16 hrs and 1, 2, 3, 5 days after dosing. At each of these time points, three animals per group were sacrificed to obtain plasma sample. Samples from group 1 were analyzed for total naked antibody contents, samples from groups 2 and 3 were submitted for total antibody, intact antibody and free-payload analysis. FIG. 5 shows the In vivo stability of ADC 12-1 following IV dosing to DBA1 mice.

In Vivo Pharmacokinetic (PK)/Stability Study—Free Payload Analysis

PK study samples were evaluated for free dexamethasone using the method described above. For increased sensitivity, 100 μL of serum from one mice of each time point was submitted to protein precipitation with acetonitrile. Free dexamethasone was not detected in any PK samples.

In Vivo Pharmacokinetic (PK)/Stability Study—Intact Antibody Drug Conjugate Mass Analysis

Samples from the ADC 12-1 PK study were also evaluated for stability using immunoprecipitation coupled to intact mass analysis. 50 μL of each sample was processed as described above. The results showed no molecular weight change for the antibody-drug conjugate over the study time range (from 1 hour to 5 days) (FIG. 6A-6B, Table 7).

TABLE 7 In vivo PK/Stability Intact Mass Results MW of the 12-1 PK Time Sample (2xG0F motif) Group Point B1 148548 G2 1 hour C1 148547 G2 1 hour E1 148546 G2 2 hours F1 148548 G2 2 hours A2 148546 G2 6 hours C2 148546 G2 16 hours D2 148547 G2 16 hours F2 148545 G2 1 day G2 148548 G2 1 day A3 148546 G2 2 days D3 148548 G2 3 days G3 148550 G2 5 days H3 148542 G2 5 days B7 148542 G3 1 hour C7 148548 G3 1 hour E7 148547 G3 2 hours F7 148545 G3 2 hours A8 148547 G3 6 hours C8 148546 G3 16 hours D8 148546 G3 16 hours F8 148546 G3 1 day G8 148546 G3 1 day A9 148544 G3 2 days B9 148547 G3 2 days D9 148549 G3 3 days E9 148550 G3 3 days G9 148547 G3 5 days H9 148545 G3 5 days

In Vivo Pharmacokinetic (PK)/Stability Study Naked/Total mAb Analysis

Plasma samples from in vivo PK study were analyzed for naked Antibody/total Antibody ADC 12-1 concentrations using Meso Scale Discovery (MSD) based electro-chemiluminescence method. The capture reagent is recombinant mouse IL-2R alpha (CD25) for both assays. The detection reagent may be goat anti-rat IgG for naked antibody/total Antibody and anti-dex mAb (e.g., Rabbit polyclonal anti-dexamethasone (Abcam Cat # ab35000)) may be used to detect ADC 12-1, respectively. Briefly, 96 well MSD plates were coated with the capture reagent and then washed. Plates were blocked for 1 hour and washed again. Samples were then added and incubated for 2 hours. Following incubation, plates were washed, incubated with the detection antibody for 1 hour, and washed again. The reading buffer was added and the plates were read using MSD plate reader.

Example 22

In this example, anti-CD70 antibody 2H5 was conjugated to exemplary drug-linker 1-4 to produce ADC 12-2 (FIG. 1) as described for making ADC 12-1 in Example 20. In vitro activity and targeted delivery of ADC 12-2, naked antibody, and anti-hexon conjugate control were assessed by transfecting into 786-0 (renal cell) and then measuring glucocorticoid-induced leucine zipper (GILZ) mRNA, a widely expressed dexamethasone-induced mRNA transcript. As shown in FIG. 7, ADC 12-2 displayed potent in vitro activity (0.7 ug/ml IP value) in 786-0 cells that were confirmed to express CD70. This activity reflects dexamethasone conjugation and targeted delivery as the nonconjugated IgG variant and anti-hexon controls did not induce and observable GILZ in this cell line.

786-O cells were plated at 30K cells/well overnight at 37° C. in RPMI Media as suggested by ATCC (+10% HI FBS). Cells were stimulated with ADCs for 2, 6, or 24 hours at 37° C. Cells were lysed using RLT and RNA is isolated using RNeasy 96 Kits. PCR was used to measure GAPDH, PER1, or TSC22D3

Quantitation of Glucocorticoid-induced leucine zipper (GILZ) mRNA expression was determined as follows. Cellular quantitation of GILZ mRNA was conducted using the following method. Cells suspension were prepared in HBSS+2% FBS (assay buffer) and plated at 5×104 cells per well. Dosing solutions for free drug, ADCs and parental antibodies were prepared by serial dilution of each stock solution using 1:3 in in HBSS+2% FBS supplemented with 1% final concentration of (50 mM Histidine, 100 mM NaCl, 5% Trehalose, pH 6.0), and incubated with cells final concentrations ranging from 20 to 0.002 μg/ml and 100 to 0.01 ng dexamethasone/ml for 18 hours. Cell lysis, cDNA synthesis, and real-time PCR were performed according to manufacturer's instructions using TaqMan Gene Expression Cells-to-CT™ Kit (Invitrogen, Carlsbad, Calif.). Specific primers against human GILZ and GAPDH were purchased from the Life Technologies (Invitrogen, Carlsbad, Calif.). Real-time PCR reactions were performed on the Applied Biosystems 7900 HT Fast Real-Time PCR System. Thermal cycling conditions consisted of an initial UDG incubation hold (50° C., 2 min) denaturing and enzyme activation step (95° C., 2 min) followed by 40 cycles of denaturing (95° C., 15 s), annealing and extending (60° C., 1 min). The mRNA levels were normalized to GAPDH (internal control) using the formula A threshold cycle (CT)=CT target−CT reference. The differential expression signal were expressed as delta Ct (ΔCt) by subtracting the Ct values of the un-stimulated samples (containing only assay buffer or DMSO vehicle) from those of the stimulated samples and expressed as relative fold of change using the formula: 2ΔΔCT.

Example 23

The example shows that conjugates comprising the phosphate-based links have little or no propensity to form aggregates.

Aggregation Assay

An SE-HPLC method was used to conduct the aggregation/% monomer analysis. An isocratic gradient using 0.2 M potassium phosphate, 0.25 M potassium chloride pH 6.0 was used as the mobile phase at a flowrate of 0.5 mL/min. The column used was a Sepax Zenix-C SEC-300, 3 μm, 300 A, 7.8×300 mm (Cat #233300-7830). Detection of signal was monitored at 214 nm (280 for FIO). For a representative run, the analyte load was 10 μg.

The results are shown in Table 8.

TABLE 8 % High Molecular Sample Weight Name Drug-Linker (aggregate) % Monomer ADC 12-1 Phos-21Dex365 (1-4) 1.4 98.6 ADC 12-2 Phos-21Dex365 (1-4) 0.9 99.1

Example 24

In this example, anti-CD74 antibody LL1 (or negative control IgG1 isotype) was conjugated to exemplary drug-linkers 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 9-4 to produce ADC 12-4, 12-5, 12-6, 12-7, 12-9, 12-10, 12-11, 12-12 (Table 9) as described for making ADC 12-1 in Example 20. The anti-CD74 antibody LL1 comprises a heavy chain (HC) having the amino acid sequence shown in SEQ ID NO:69 and a light chain (LC) having the amino acid sequence shown in SEQ ID NO:73. The amino acid at position 121 is para-azido phenylalanine (pAzF), which was the site for conjugating the exemplary drug-linkers using copper-free 3+2 cycloaddition chemistry to produce the ADCs. The construction of ADC 12-4 is illustrated in FIG. 1.

TABLE 9 Antibody Linker ADC Anti-hCD74 (LL1) Linker 1-4 ADC 12-4 Anti-hCD74 (LL1) Linker 2-7 ADC 12-5 Anti-hCD74 (LL1) Linker 3-4 ADC 12-6 Anti-hCD74 (LL1) Linker 4-3 ADC 12-7 Anti-hCD74 (LL1) Linker 6-2 ADC 12-9 Anti-hCD74 (LL1) Linker 9-4 ADC 12-10 IgG1 (neg control) Linker 1-4 ADC 12-11 IgG1 (neg control) Linker 9-4 ADC 12-12 Anti-hCD74 (clone11) Linker 9-4 ADC 12-13 Anti-hCD74 (clone11) Linker 15-5 ADC 12-14 Anti-hCD74 (clone11) Linker 16-5 ADC 12-15

In vitro activity and targeted delivery of ADC 12-4, 12-5, 12-6, 12-7, 12-9, 12-10, the isotype-matched negative controls 12-11, 12-12, and naked anti-hCD74 (LL1) control were assessed in HUT-78 an SUDHL-6 cells (CD74 positive T and B lymphoma cell lines respectively) as well as 786-0 cells (a CD74 negative renal carcinoma cell line). The cells were treated with individual ADC, anti-hCD74 antibody, or budesonide (Bud) for 18 hours and then glucocorticoid-induced leucine zipper (GILZ) mRNA, a glucocorticoid-regulated gene, was measured by RT-PCR. As shown in FIGS. 8 and 9, the tested ADCs displayed a range of potency in up-regulating GILZ mRNA in both HUT-78 and SUDHL-6 cells, with ADC 12-10 most active with an EC50 value of about 0.2 pig/mL in both cell lines (see Table 10). In contrast, the isotype-matched negative control ADCs did not induce measurable GILZ mRNA in both cell lines. Furthermore, all the ADCs did not induce measurable GILZ mRNA except at the highest concentration (30 μg/mL) in 786-0 cells (FIG. 10). (The activity at the highest concentration in 786-0 cells may be due to impurity of the sample or possible low CD74 expression in cells.). The overall activity profile of the ADC is consistent with targeted delivery of a payload (e.g. dexamethasone or budesonide) that is dependent on the binding and internalization of the antibody with its antigen on the cell surface, and on the efficiency of linker cleavage inside the cells.

TABLE 10 The Max effects and the EC50 values of ADC and budesonide on human cell lines 786-0 cells HUT-78 cells SUDHL-6 cells (CD74(−)) ADC or EC50 EC50 EC50 Antibody Emax (μg/mL) Emax (μg/mL) Emax (μg/mL) ADC 12-4 3.95 1.08 3.44 0.99 1.71 >30 (LL1) ADC 12-5 4.13 1.32 3.4 0.4 1.34 >30 (LL1) ADC 12-6 4.61 1.87 3.33 0.47 2.26 >30 (LL1) ADC 12-7 1.93 >30 1.52 14.3 1.73 >30 (LL1) ADC 12-9 2.55 >30 6.48 >30 4.02 >30 (LL1) ADC 12-10 6.47 0.2 5.89 0.21 3.52 >30 (LL1) ADC 12-11 0.99 N/A 1.13 >30 1.14 >30 (IgG1) ADC 12-12 0.99 N/A 1.28 >30 2.66 >30 (IgG1) ADC12-13 4.94 0.2 6.77 0.63 9.19 >30 (Clone 11) CD74 Ab 1.34 N/A 1.02 N/A 1.02 N/A (LL1) ADC 12-14 8.36 0.055 14.2 0.023 18.78 4.45 (Clone11) ADC 12-15 8.34 0.26 7.53 0.67 5.26 >30 (Clone11) Budesonide 9.03 0.65 (nM) 14.33 2.21 (nM) 25.68 0.58 (nM) Fluticasone 10.27 0.45 (nM) 15.78 0.52 (nM) 25.38 0.46 (nM) Propionate N/A: EC50 value can't be generated.

Materials and Methods

The HUT-78 (ATCC TIB-161), SUDHL-6 (ATCC CRL-2959) and 786-0 (ATCC CRL-1932) cells were purchased from ATCC and maintained in culture medium as suggested by ATCC, in which Iscove's MDM/20% HI FBS for HUT-78 cells and RPMI-1640/10% FBS for SUDHL-6 and 786-0 cells.

The quantitation of Glucocorticoid-induced leucine zipper (GILZ) mRNA expression was determined by real time-PCR. In brief, actively growing cells were harvested and then resuspended in the assay buffer (HBSS plus 2% FBS) at concentration of 1.1×106/ml. 5×104 cells/well in 45 μl volume were plated to Greiner 384 well v-bottom reagent plates (Ref #781280). Dosing solutions for free drug, ADCs and parental antibodies were prepared in the v-bottom Greiner reagent plates at 10-fold over the final concentration by serial dilution of each stock solution using 1:3 in in HBSS+2% FBS supplemented with 1% final concentration of (50 mM Histidine, 100 mM NaCl, 5% Trehalose, pH 6.0), and 5 μl of the 10-fold solutions were added to each well to reach final concentrations ranging from 30 to 0.0005 μg/ml for ADC/parental antibody and 100 to 0.002 nM for dexamethasone or budesonide (11 concentrations). After 18 hour incubation, the cells were lysed, and the lysates were used for cDNA synthesis and real-time PCR, according to manufacturer's instructions in TaqMan Gene Expression Cells-to-CT™ Kit (Invitrogen, Carlsbad, Calif.). Specific primers against human GILZ and GAPDH were purchased from the Life Technologies (Invitrogen, Carlsbad, Calif.). Real-time PCR reactions were performed on the Applied Biosystems 7900 HT Fast Real-Time PCR System. Thermal cycling conditions consisted of an initial UDG incubation hold (50° C., 2 min) denaturing and enzyme activation step (95° C., 2 min) followed by 40 cycles of denaturing (95° C., 15 s), annealing and extending (60° C., 1 min). The mRNA levels were normalized to GAPDH (internal control) using the formula A threshold cycle (CT)=CT target−CT reference. The differential expression signal were expressed as delta Ct (ΔCt) by subtracting the Ct values of the un-stimulated samples (containing only assay buffer or DMSO vehicle) from those of the stimulated samples and expressed as relative fold of change using the formula: 2ΔΔCT. The graphs were generated in GraphPad Prism and the EC50 values were calculated with non-linear regression curve fit of the data in GraphPad Prism.

Example 25

The anti-CD74 antibody LL1 (or negative control isotype) was conjugated to exemplary drug-linkers 15-5, 16-5, or 17-2 as described for making ADC 12-1 to make ADC 12-13, ADC 12-14, and ADC 12-15, respectively. The anti-CD74 antibody LL1 comprises a heavy chain (HC) having the amino acid sequence shown in SEQ ID NO:69 and a light chain (LC) having the amino acid sequence shown in SEQ ID NO:73. The amino acid at position 121 is para-azido phenylalanine (pAzF) and may serve as the site for conjugating the exemplary drug-linkers using copper-free 3+2 cycloaddition chemistry to produce the ADCs.

TABLE of Sequences SEQ ID NO: Description Amino Acid Sequence   1 Anti-CD25 LC CDR1 RASQSVSSSYLA   2 Anti-CD25 LC CDR2 GASSRAT   3 Anti-CD25 LC CDR3 QQYSSSPLT   4 Anti-CD25 HC CDR1 RYIIN   5 Anti-CD25 HC CDR2 RIIPILGVENYAQKFQG   6 Anti-CD25 HC CDR3 KDWFDY   7 Anti-CD25 HC CDR1 RYPIN   8 Anti-CD25 HC CDR2 RIIPILGIADYAQRFQG   9 Anti-CD25 HC CDR3 RDWGDY  10 Anti-CD25 LC CDR3 QQYGSSPIT  11 Anti-CD25 HC CDR1 RYAIN  12 Anti-CD25 HC CDR2 RIIPILDIADYAQKFQD  13 Anti-CD25 HC CDR3 KDWFDP  14 Anti-CD25 HC CDR1 RYPIN  15 Anti-CD70 LC CDR1 RASQSVSSYLA  16 Anti-CD70 LC CDR2 YDASNRAT  17 Anti-CD70 LC CDR3 QQRTNWPLT  18 Anti-CD70 HC CDR1 SYIMH  19 Anti-CD70 HC CDR2 VISYDGRNKYYADSVK  20 Anti-CD70 HC CDR3 DTDGYDFDY  21 Anti-CD70 LC CDR1 RASQGISSALA  22 Anti-CD70 LC CDR2 DASSLES  23 Anti-CD70 LC CDR3 QQFNSYPFT  24 Anti-CD70 HC CDR1 YYAMH  25 Anti-CD70 HC CDR2 VISYDGSIKYYADSVK  26 Anti-CD70 HC CDR3 EGPYSNYLDY  27 Anti-CD70 LC CDR1 RASQGISSWLA  28 Anti-CD70 LC CDR2 AASSLQS  29 Anti-CD70 LC CDR3 QQYNSYPLT  30 Anti-CD70 HC CDR1 DYGMH  31 Anti-CD70 HC CDR2 VIWYDGSNKYYADSVK  32 Anti-CD70 HC CDR3 DSIVMVRGDY  33 Anti-CD70 LC CDR1 RASQGISSWLA  34 Anti-CD70 LC CDR2 AASSLQS  35 Anti-CD70 LC CDR3 QQYNSYPLT  36 Anti-CD70 HC CDR1 DHGMH  37 Anti-CD70 HC CDR2 VIWYDGSNKYYADSVK  38 Anti-CD70 HC CDR3 DSIMVRGDY  39 Anti-CD70 LC CDR2 DASNRAT  40 Anti-CD70 LC CDR3 QQRSNWPLT  41 Anti-CD70 HC CDR1 SDYYYWS  42 Anti-CD70 HC CDR2 YIYYSGSTNYDPSLKS  43 Anti-CD70 HC CDR3 GDGDYGGNCFDY  44 Anti-CD74 LC CDR1 RSSQSLVHRNGNTYLH  45 Anti-CD74 LC CDR2 TVSNRFS  46 Anti-CD74 LC CDR3 SQSSHVPPT  47 Anti-CD74 HC CDR1 NYGVN  48 Anti-CD74 HC CDR2 WINPNTGEPTFDDDFKG  49 Anti-CD74 HC CDR3 SRGKNEAWFAY  50 Anti-CD74 LC CDR1 QGISSW  51 Anti-CD74 LC CDR3 QQYNSYPLT  52 Anti-CD74 HC CDR1 GFTFSSYA  53 Anti-CD74 HC CDR2 ISYDGSNK  54 Anti-CD74 HC CDR3 ASGRYYGSGSYSSYFD  55 Anti-CD74 HC CDR2 ISYDGSIK  56 Anti-CD74 HC CDR3 ARGREYTSQNIVILLD  57 Anti-CD74 HC CDR3 ARGREITSQNIVILLD  58 Anti-CD74 HC CDR2 IWYDGSNK  59 Anti-CD74 HC CDR3 ARGGTLVRGAMYGTDV  60 Anti-CD163 LC CDR1 ASQSVSSDV  61 Anti-CD163 LC CDR3 QDYTSPRT  62 Anti-CD163 HC CDR1 GYSITSDY  63 Anti-CD163 HC CDR3 CVSGTYYFDYWG  64 Anti-CD163 LC CDR1 ASQSVSHDV  65 Anti-CD163 LC CDR3 QDYSSPRT  66 Glycosylation site QYNS atN297 of IgG1  67 Glycosylation site QFNS atN297 of IgG4  68 Mutated glycosylation QAQS site of IgG1 or IgG4  69 Anti-CD74 IgG1; QVQLQQSGSELKKPGASVKVSCKASGYTFT X at position 121 is NYGVNWIKQAPGQGLQWMGWINPNTGEP para-azido- TFDDDFKGRFAFSLDTSVSTAYLQISSLKAD phenylalanine (pAzF) DTAVYFCSRSRGKNEAWFAYWGQGSLVTV (CDRs bold type; Fc SSXSTKGPSVFPLAPSSKSTSGGTAALGCLV underlined) KDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK  70 Anti-CD74 IgG4; QVQLQQSGSELKKPGASVKVSCKASGYTFT X at position 121 is NYGVNWIKQAPGQGLQWMGWINPNTGEP para-azido- TFDDDFKGRFAFSLDTSVSTAYLQISSLKAD phenylalanine (pAzF) DTAVYFCSRSRGKNEAWFAYWGQGSLVTV (CDRs bold type; Fc SSXSTKGPSVFPLAPCSRSTSESTAALGCLVK underlined) DYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK  71 Anti-CD74 IgG1 QVQLQQSGSELKKPGASVKVSCKASGYTFT (CDRs bold type; Fc NYGVNWIKQAPGQGLQWMGWINPNTGEP underlined) TFDDDFKGRFAFSLDTSVSTAYLQISSLKAD DTAVYFCSRSRGKNEAWFAYWGQGSLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK  72 Anti-CD74 IgG4 QVQLQQSGSELKKPGASVKVSCKASGYTFT (CDRs bold type; Fc NYGVNWIKQAPGQGLQWMGWINPNTGEP underlined) TFDDDFKGRFAFSLDTSVSTAYLQISSLKAD DTAVYFCSRSRGKNEAWFAYWGQGSLVTV SSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK  73 Anti-CD74 LC DIQLTQSPLSLPVTLGQPASISCRSSQSLVHR (CDRs bold type) NGNTYLHWFQQRPGQSPRLLIYTVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDVGVYFC SQSSHVPPTFGAGTRLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC  74 Anti-CD74 IgG1; QVQLVESGGGVVQPGRSLRLSCAASGFTFS X at position 126 is SYAMHWVRQAPGKGLEWVAVISYDGSIKY para-azido- YADSVKGRFTISRDNSKNTLYLQMNSLRVE phenylalanine (pAzF) DTAVFYCARGREEITSQNIVILLDYWGQGT (CDRs bold type; Fc LVTVTSXSTKGPSVFPLAPSSKSTSGGTAAL underlined) GCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG K  75 Anti-CD74 IgG4; QVQLVESGGGVVQPGRSLRLSCAASGFTFS X at position 126 is SYAMHWVRQAPGKGLEWVAVISYDGSIKY para-azido- YADSVKGRFTISRDNSKNTLYLQMNSLRVE phenylalanine (pAzF) DTAVFYCARGREEITSQNIVILLDYWGQGT (CDRs bold type; Fc LVTVTSXSTKGPSVFPLAPCSRSTSESTAALG underlined) CLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK  76 Anti-CD74 IgG1 QVQLVESGGGVVQPGRSLRLSCAASGFTFS (CDRs bold type; Fc SYAMHWVRQAPGKGLEWVAVISYDGSIKY underlined) YADSVKGRFTISRDNSKNTLYLQMNSLRVE DTAVFYCARGREEITSQNIVILLDYWGQGT LVTVTSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG K  77 Anti-CD74 IgG4 QVQLVESGGGVVQPGRSLRLSCAASGFTFS (CDRs bold type; Fc SYAMHWVRQAPGKGLEWVAVISYDGSIKY underlined) YADSVKGRFTISRDNSKNTLYLQMNSLRVE DTAVFYCARGREEITSQNIVILLDYWGQGT LVTVTSASTKGPSVFPLAPCSRSTSESTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDH KPSNTKVDKRVESKYGPPCPPCPAPEFLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK  78 Anti-CD74 LC DIQMTQSPSSLSASVGDRVTITCRASQGISS (CDRs bold type) WLAWYQQKPEKAPKSLIYAASSLQSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCQQYNS YPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC  79 Anti-CD70 2H5 IgG1 QVQLVESGGGVVQPGRSLRLSCAASGFTFSS X at position 119 is YIMHWVRQAPGKGLEWVAVISYDGRNKY para-azido- YADSVKGRFTISRDNSKNTLYLQMNSLRAE phenylalanine (pAF) DTAVYYCARDTDGYDFDYWGQGTLVTVSS (CDRs bold type; Fc XSTKGPSVFPLAPSSKSTSGGTAALGCLVKD underlined) YFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSCDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG  80 Anti-CD70 Kappa light EIVLTQSPATLSLSPGERATLSCRASQSVSSY chain LAWYQQKPGQAPRLLIYDASNRATGIPARF (CDRs bold type) SGSGSGTDFTLTISSLEPEDFAVYYCQQRTN WPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC  81 Anti-murine CD25 QVKLLQSGAALVKPGASVKMSCKASGYSFP muIgG1 D265A DSWVTWVKQSHGKSLEWIGDIFPNSGATNF X at position 115 is NEKFKGKATLTVDKSTSTAYMELSRLTSEDS para-azido- AIYYCTRLDYGYWGQGVMVTVSSXKTTPPS phenylalanine (pAF) VYPLAPGSAAQTNSMVTLGCLVKGYFPEPV TVTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITL TPKVTCVVVAISKDDPEVQFSWFVDDVEVH TAQTQPREEQFNSTFRSVSELPIMHQDWLNG KEFKCRVNSAAFPAPIEKTISKTKGRPKAPQ VYTIPPPKEQMAKDKVSLTCMITDFFPEDITV EWQWNGQPAENYKNTQPIMDTDGSYFVYS KLNVQKSNWEAGNTFTCSVLHEGLHNHHTE KSLSHSPGK  82 Anti-murine CD25 DVVLTQTPPTLSATIGQSVSISCRSSQSLLHS muKappa NGNTYLNWLLQRPGQPPQLLIYLASRLESGV PNRFSGSGSGTDFTLKISGVEAEDLGVYYCV QSSHFPNTFGVGTKLELKRADAAPTVSIFPPS SEQLTSGGASVVCFLNNFYPKDINVKWKIDG SERQNGVLNSWTDQDSKDSTYSMSSTLTLT KDEYERHNSYTCEATHKTSTSPIVKSFNRNE C

While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

Claims

1: A composition comprising a compound having formula (I) an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group;

wherein V is selected from O and S;
W is selected from O, N, and CH2;
X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom;
Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group;
T is an NR, CR2, O, or S;
D is an anti-inflammatory agent;
antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD74 protein or human CD163 protein;
Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody;
each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;
n is 1, 2, 3, or 4;
m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
and a pharmaceutically acceptable carrier.

2: The compound of claim 1, wherein the anti-inflammatory agent comprises a glucocorticoid receptor agonist.

3: The compound of claim 1, wherein the anti-inflammatory agent comprises Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, or mometasone.

4: The compound of claim 1, wherein the antibody comprises a substitution of an amino acid in the heavy chain or light chain of the antibody with para-azidophenylalanine (pAzF) and the reactive functional group comprises a strained cycloalkyne.

5-6. (canceled)

7: The composition of claim 1, wherein the antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73.

8-12. (canceled)

13: The composition of claim 1, wherein the antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78.

14-16. (canceled)

17: A method for treating an inflammatory disease or disorder by providing to a subject having the disease or disorder a composition comprising a compound having formula (I) an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group;

wherein V is selected from O and S;
W is selected from O, N, and CH2;
X is a tuning element selected from a covalent bond; a carbon atom; a heteroatom;
Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, C(O)O—, OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O)2—, —N(R)SO2—, SO2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group;
T is an NR, CR2, O, or S;
D is an anti-inflammatory agent;
antibody is a chimeric, humanized, or human antibody having a light chain and a heavy chain wherein the antibody binds the human CD74 protein or human CD163 protein;
Z is a linkage formed between (i) a reactive functional group selected from the group consisting of N-hydroxysuccinimide, para-nitrophenyl carbonate, para-nitrophenyl carbamate, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadience, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S, and (ii) the side chain of an amino acid in the heavy chain or light chain of the antibody;
each occurrence of R is independently hydrogen, a suitable protecting group, an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;
n is 1, 2, 3, or 4;
m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
and a pharmaceutically acceptable carrier to treat the inflammatory disease or disorder.

18: The method of claim 17, wherein the anti-inflammatory agent comprises a glucocorticoid receptor agonist.

19: The method of claim 17, wherein the anti-inflammatory agent comprises Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, or mometasone.

20: The method of claim 17, wherein the antibody comprises a substitution of an amino acid in the heavy chain or light chain of the antibody with pAzF and the reactive functional group comprises a strained cycloalkyne.

21: The method of claim 17, wherein the inflammatory disease or disorder comprises Alzheimer's disease, ankylosing spondylitis arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis), asthma, atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematous (SLE), nephritis, Parkinson's disease, or ulcerative colitis.

22-23. (canceled)

24: The method of claim 17, wherein the antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO: 69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73.

25-29. (canceled)

30: The method of claim 17, wherein the antibody comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78.

31-50. (canceled)

51: An antibody drug conjugate comprising

(a) an antibody that binds a CD74 protein, wherein the antibody comprises a para-azidophenylalanine (pAzF) conjugated to a molecule selected from the group of molecules consisting of 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-1, 10-1, 11-5, 21-1, 13-1, 14-5, 15-1, 16-5, and 17-2;
(b) an antibody that binds a CD163 protein, wherein the antibody comprises a para-azidophenylalanine (pAzF) conjugated to a molecule selected from the group of molecules consisting of 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-1, 10-1, 11-5, 21-1, 13-1, 14-5, 15-5, 16-5, and 17-2: or
(c) antibody drug conjugate selected from the group consisting of ADC 12-1, ADC 12-2, ADC 12-3, ADC 12-4, ADC 12-5, ADC 12-6, ADC 12-7, ADC 12-8, ADC 12-9, ADC 12-10, ADC 12-13, ADC 12-14, and ADC 12-15.

52-53. (canceled)

54: The antibody drug conjugate of claim 51, wherein the antibody that binds CD74 protein comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:69, 70, 71, and 72 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:73.

55-59. (canceled)

60: The antibody drug conjugate of claim 51, wherein the antibody that binds CD74 protein comprises a heavy chain (HC) comprising an amino acid sequence selected from SEQ ID NO:74, 75, 76, and 77 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:78.

61. (canceled)

62: The antibody drug conjugate of claim 51, wherein the antibody that binds CD163 protein comprises light chain CDR sequences CDR1 (ASQSVSSDV; SEQ ID NO:60), CDR2 (YAS), and CDR3 (QDYTSPRT; SEQ ID NO:61) and heavy chain complementarity-determining region (CDR) sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63).

63: The antibody drug conjugate of claim 51, wherein the antibody that binds CD163 protein comprises light chain CDR sequences CDR1 (ASQSVSHDV; SEQ ID NO:54), CDR2 (YTS), and CDR3 (QDYSSPRT; SEQ ID NO:65) and heavy chain CDR sequences CDR1 (GYSITSDY; SEQ ID NO:62), CDR2 (YSG), and CDR3 (CVSGTYYFDYWG; SEQ ID NO:63).

64-65. (canceled)

Patent History
Publication number: 20230099074
Type: Application
Filed: Oct 26, 2022
Publication Date: Mar 30, 2023
Applicants: Merck Sharp & Dohme LLC (Rahway, NJ), Ambrx, Inc. (La Jolla, CA)
Inventors: Philip E. Brandish (Needham, MA), Robert M. Garbaccio (Lansdale, PA), Jeffrey Kern (Gilbertsville, PA), Linda Liang (Mountain View, CA), Sanjiv Shah (Wakefield, MA), Dennis Zaller (Boston, MA), Andrew Beck (San Diego, CA), Dennis Gately (San Diego, CA), Nick Knudsen (Escondido, CA), Anthony Manibusan (San Diego, CA), Jianing Wang (San Diego, CA), Ying Sun (San Diego, CA)
Application Number: 18/049,711
Classifications
International Classification: A61K 47/68 (20060101); C07K 16/28 (20060101);