Phosphonate linkers and their use to facilitate cellular retention of compounds

Abstract

Phosphonate linkers and their use for delivering compounds with passive cell permeability into a cell wherein the phosphonate group facilitates cellular retention of the compound are described.

Claims

1. A payload-ligand conjugate compound comprising a cell-specific targeting ligand conjugated to a payload wherein said conjugate comprises the formula ##STR00075## wherein V is selected from O and S; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; X is 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 C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; D is a payload; 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) an S, NR, or O group on the cell-specific targeting ligand (L); 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; L is a cell-specific targeting ligand; and n is 1, 2, 3, or 4.

2. The compound of claim 1, wherein the payload is a therapeutic agent, a detectable label, radionuclide, or protecting group.

3. The compound of claim 2, wherein the therapeutic agent is a cytotoxic agent, an anti-inflammatory agent, peptide, or nucleic acid or nucleic acid analog.

4. The compound of claim 3, wherein the anti-inflammatory agent is a glucocorticoid receptor agonist.

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

6. The compound of claim 1, wherein the targeting ligand is an antibody or monoclonal antibody, ligand for a receptor, lectin, saccharide, poly(ethylene glycol), polysaccharide, or polyamino acid.

7. The compound of claim 1, wherein the targeting ligand is an anti-Her2 antibody, anti-CD4 antibody, anti-CD20 antibody, anti-EGFR antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD25 antibody, anti-CD52 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD40L antibody, anti-CD70 antibody, anti-CD74 antibody, anti-CD80 antibody, anti-CD163 antibody, anti-Mucl8 antibody, anti-integrin antibody, anti-PSMA antibody, anti-CEA antibody, anti-CD1 Ia antibody, anti-CTLA4 antibody, or anti-BLys antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention provides phosphonate-based linkers comprising a monophosphonate, diphosphonate, triphosphonate, or tetraphosphonate group having the general formula

(2) ##STR00012##
Wherein R1 is a first linker arm comprising a tuning element, an optional spacer element, and a reactive functional group capable of reacting with a group on a ligand or targeting moiety in which the tuning element is linked to the O group of the phosphonate group; and, R2 is a second linker arm comprising a payload linked to the P atom of the phosphonate group via a linker, V is selected from O or S, and wherein n=1, 2, 3, or 4. Interspersed between the tuning element and the reactive functional group of the linker arm may be an optional spacer element.

(3) In general, the phosphonate-based linker comprises a compound that has the following formula (I)

(4) ##STR00013##
Wherein V is selected from O and S; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; Z is a reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, 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; D is a payload; 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; and n is 1, 2, 3, or 4.

(5) In a further embodiment, the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload. Such a compound comprises formula (II)

(6) ##STR00014##
Wherein V is selected from O and S; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; D is a payload; 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) an S, NR, or O group on L; 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; L is a cell-specific targeting ligand; and n is 1, 2, 3, or 4.

(7) In particular embodiments, the payload-ligand conjugate compound comprising a cell-specific targeting ligand conjugated to a drug moiety comprises the formula (III)

(8) ##STR00015##
where the wavy lines indicate the covalent attachment sites to the cell-specific targeting ligand and the payload and wherein V is selected from O and S; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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) an S, NR, or O group on the cell-specific targeting ligand; 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; and n is 1, 2, 3, or 4.

(9) In particular embodiments, the phosphonate-based linker comprises a compound that has the following formula (IV)

(10) ##STR00016##
Wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; Z is a reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, 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; D is a payload; 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; and n is 1, 2, 3, or 4.

(11) In a further embodiment, the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload. Such a compound comprises formula (V)

(12) ##STR00017##
Wherein; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; D is a payload; 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) an S, NR, or O group on L; 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; L is a cell-specific targeting ligand; and n is 1, 2, 3, or 4.

(13) In particular embodiments, the payload-ligand conjugate compound comprising a cell-specific targeting ligand conjugated to a drug moiety comprises the formula (VI)

(14) ##STR00018##
where the wavy lines indicate the covalent attachment sites to the cell-specific targeting ligand and the payload; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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) an S, NR, or O group on the cell-specific targeting ligand; 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; and n is 1, 2, 3, or 4.

(15) In particular embodiments, the phosphonate-based linker comprises a compound that has the following formula (VII)

(16) ##STR00019##
Wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; Z is a reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, 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; D is a payload; 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; and n is 1, 2, 3, or 4.

(17) In a further embodiment, the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload. Such a compound comprises formula (VIII)

(18) ##STR00020##
Wherein; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; D is a payload; 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) an S, NR, or O group on L; 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; L is a cell-specific targeting ligand; and n is 1, 2, 3, or 4.

(19) In particular embodiments, the payload-ligand conjugate compound comprising a cell-specific targeting ligand conjugated to a drug moiety comprises the formula (IX)

(20) ##STR00021##
where the wavy lines indicate the covalent attachment sites to the cell-specific targeting ligand and the payload; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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) an S, NR, or O group on the cell-specific targeting ligand; 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; and n is 1, 2, 3, or 4.

(21) In particular embodiments, the phosphonate-based linker is a compound that has the following formula (X)

(22) ##STR00022##
Wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; Z is a reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, 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; D is a payload; 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; and n is 1, 2, 3, or 4.

(23) In a further embodiment, the reactive functional group at the proximal end of the payload-phosphonate-based linker may be covalently linked to a ligand or targeting moiety to provide a conjugate wherein in particular embodiments, the ligand is capable of targeting the conjugate to a particular cellular target when administered to a subject in need of the payload. Such a compound comprises formula (XI)

(24) ##STR00023##
Wherein; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; D is a payload; 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) an S, NR, or O group on L; 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; L is a cell-specific targeting ligand; and n is 1, 2, 3, or 4.

(25) In particular embodiments, the payload-ligand conjugate compound comprising a cell-specific targeting ligand conjugated to a drug moiety comprises the formula (XII)

(26) ##STR00024##
where the wavy lines indicate the covalent attachment sites to the cell-specific targeting ligand and the payload; W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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) an S, NR, or O group on the cell-specific targeting ligand; 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; and n is 1, 2, 3, or 4.

(27) In particular embodiments, the phosphonate-based linker comprises a compound that has a formula selected from

(28) ##STR00025##
wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; Z is a reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, 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; D is a payload; 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; and each V is independently O or S.

(29) In particular embodiments, the phosphonate-based linker comprises a compound that has a formula selected from

(30) ##STR00026##
wherein W is selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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 optional but when present is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C.sub.1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 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) an S, NR, or O group on L; 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; L is a cell-specific targeting ligand; and each V is independently O or S.

(31) In particular aspects of the above wherein Z is a reactive group, Z may have the structure

(32) ##STR00027##
wherein the wavy line marks the covalent bond between Z and Y, or X when Y is a covalent bond.

(33) In particular aspects, the linkage Z when conjugated to a cell-specific targeting ligand L, Z may have the structure

(34) ##STR00028##
wherein the wavy lines mark the covalent bond between Z and Y, or X when Y is a covalent bond on the left and Z and L on the right.

(35) The O—X linkage of the phosphonate group is stabile extracellularly and labile intracellularly, for example, when present in the lysosomal compartment of the target cell the O—X linkage is cleaved. However, the phosphonate-W-payload linkage is stable both intracellularly and extracellularly. When the phosphonate group is cleaved intracellularly, the payload-W is released with the W attached to a monophosphonate group, which renders the payload charged or polar and facilitates retention of the payload within the cell. This is illustrated schematically below wherein n=1, 2, 3, or 4 and the other consituents are as above.

(36) ##STR00029##
It should be noted that even in the case where the conjugate comprises a diphosphonate, triphosphonate, or tetraphosphonate, upon intracellular cleavage, the payload-W is released with the W linked to a monphosphonate.

(37) The tuning element provides a tunable stability to the phosphonate linkage when the conjugate is within the lysosomal compartment of the target cell. The intracellular stability of the phosphonate group or rate of intracellular release of the payload from the conjugate may be adjusted or tuned by the particular tuning element adjacent to the phosphonate group and/or by adjusting the number of the phosphonate groups. The conjugates disclosed herein are particularly useful in embodiments in which the ligand is an antibody or antibody fragment and the payload is a therapeutic agent, for example, a cytotoxin or a glucocorticoid receptor agonist, which herein is referred to as an “antibody drug conjugate” or “ADC”.

(38) The link between the antibody and the drug moiety 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.

(39) The payload-linker conjugates of the present invention wherein the payload is covalently linked to a tuning element of the linker via a monophosphonate, diphosphonate, triphosphonate, or tetraphosphonate linkage have a differentiated and tunable stability of the phosphonate linkage in blood vs. an intracellular environment (e.g. lysosomal compartment). Due to location of enzymes that recognize the phosphonate linkage, conjugates that have a phosphonate group linking a payload 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 payload conjugates where it desired to render the poayload charged to facilitate intracellular retention. The exemplary payload-phosphonate-based linker conjugates in the Examples show that the payload-phosphonate-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 payload from the conjugates but when cleaved intracellularly provides a payload comprising the phosphonate group, which facilitates intracellular retention.

(40) Importantly, the inventors have discovered that by modifying the tuning element and/or V and/or W, and/or the number of phosphonate groups, the ability to tune reactivity or cleavage of the phosphonate linkage in a lysosomal environment so as to release the payload-phosphonate from the conjugate. In general, the rate of release of the payload conjugated to a phosphonate group is dependent on the proximal substitution of the tuning element. The ability to cleave the linkage between the phosphonate and the tuning element efficiently in a lysosome is advantageous for the release of the payload from the conjugate once it has been delivered to a cell and internalized through an endosomal pathway. In addition, the excellent solubility of the payload-phosphonate-based linker facilitates conjugation to a ligand or cell-targeting moiety and minimizes aggregation of the conjugates. In addition, the phosphonate contributes to retention of the payload to the conjugate within cell and limits permeability of conjugates containing the payload from entering non-target cells.

(41) The phosphonate-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 conjugated to a phosphonate unlike some of the alternative linkers, and may offer an improved blood/lysosome stability profile.

(42) Specifically, these phosphonate-based linkers will provide superior blood stability relative to esters and disuflides. Phosphonate-based linkers, following lysosomal cleavage will release a phosphonate-containing payload. The enzymatic hydrolysis of the phosphonate may be more rapid than the acid-hydrolysis of hyrdazones. The phosphonate-based linkers disclosed herein minimize the propensity for conjugates comprising particular payloads to aggregate. For example, antibody-drug conjugates comprising duocarmycin are known to have a propensity to aggregate. However, antibodies conjugated to duocarmycin via a phosphonate-based linker disclosed herein did not produce detectable aggregates. Thus, the phosphonate-based linkers disclosed herein are particularly useful for conjugating payloads 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.

(43) Thus, the phosphonate-based linkers disclosed herein provide an ideal design for antibody-drug conjugates and the like.

(44) Phosphonate Group

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

(46) ##STR00030##
a diposphonate

(47) ##STR00031##
a triphosphonate

(48) ##STR00032##
tetraphosphonate

(49) ##STR00033##
a phosphorthionate

(50) ##STR00034##
or a diphosphorthionate.

(51) ##STR00035##

(52) The wavy lines shown indicate the bond between the P and a linker W connected to a payload (left) and the bond between the O and the tuning element on the proximal end (right).

(53) Payload

(54) Payloads, depicted as “D” herein, are provided in the current invention as part of a payload-ligand conjugate where the payload is linked to a ligand via a phosphonate-based linker comprising reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, azide, hydrazine, 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. The payload must possess a desired biological activity and contain a reactive functional group capable of forming a covalent linkage to the linker that attached to the phosphonate group. The desired biological activity includes the diagnosis, cure, mitigation, treatment, or prevention of disease in an animal such as a human. Thus, so long as it has the needed reactive functional group, the term “payload” refers to chemicals recognized as drugs in the official United States Pharmacopeia, official Homeopathic Pharmacopeia of the United States, or official National Formulary, or any supplement thereof. Exemplary drugs are set forth in the Physician's Desk Reference (PDR) and in the Orange Book maintained by the U.S. Food and Drug Administration (FDA). New drugs are being continually being discovered and developed, and the present invention provides that these new drugs may also be incorporated into the payload-ligand complex of the current invention. In particular embodiments, the functional groups on the drug include primary or secondary amines, hydroxyls, sulfhydryls, carboxyls, aldehydes, and ketones. The drug must have at least one, but may have 2, 3, 4, 5, 6 or more reactive functional groups. The payload may also be a biomolecule such as a peptide, polypeptide, or protein; a nucleic acid molecule or analog thereof, a carbohydrate, polysaccharide, a saccharide, or any other therapeutic agent that has a biological effect.

(55) The payload-ligand conjugate is effective for the usual purposes for which the corresponding drugs are effective, but have superior efficacy because of the ability, inherent in the ligand, to transport the drug to the desired cell where it is of particular benefit. Exemplary drugs include proteins, peptides, and small molecule drugs containing a functional group for linkage to the phosphonate moiety of the linker. More specifically, these drugs include, for example, the enzyme inhibitors such as dihydrofolate reductase inhibitors, and thymidylate synthase inhibitors, DNA intercalators, glutocorticoid receptor agonists, nuclear recemptor agonists, antinflammatory agents, DNA cleavers, topoisomerase inhibitors, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the pteridine family of drags, diynenes, the podophyllotoxins, differentiation inducers, and taxols.

(56) In one embodiment, the drugs of the current invention include cytotoxic drugs useful in cancer therapy and other small molecules, proteins or polypeptides with desired biological activity, such as a toxin. The drug may be selected to be activated at a tumor cells by conjugation to a tumor-specific ligand. These tumor specific drug-ligand conjugates have tumor specificity arising from the specificity of the ligand. Examples of this are drug-ligand conjugates that are highly selective substrates for tumor specific enzymes, where these enzymes are present in the proximity of the tumor in sufficient amounts to generate cytotoxic levels of free drug in the vicinity of the tumor.

(57) Cytotoxic drugs useful in the current invention include, for example, duocarmycins and CC-1065, and analogues thereof, including CBI (1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one)-based analogues, MCBI (7-methoxy-1,2,9,9a-tetra-hydrocyclopropa[c]benz[e]indol-4-one)-based analogues and CCBI (7-cyano-1,2,9,9a-tetra-hydrocyclo-propa[c]benz[e]-indol-4-one)-based analogues of the duocarmycins and CC-1065, doxorubicin and doxorubicin conjugates such as morpholino-doxorubicin and cyanomorpholino-doxorubicin, dolastatins such as dolestatin-10, combretastatin, calicheamicin, maytansine, maytansine analogs, DM-I, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), 5-benzoylvaleric acid-AE ester (AEVB), tubulysins, disorazole, epothilones, Paclitaxel, docetaxel, SN-38, Topotecan, rhizoxin, echinomycin, colchicine, vinblastin, vindesine, estramustine, cemadotin, eleutherobin, methotrexate, methopterin, dichloro methotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin and daunorubicin conjugates, mitomycin C, mitomycin A, carminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vincristine, taxol, taxotere retinoic acid, butyric acid, N-acetyl spermidine, camptothecin, and their analogues.

(58) Anti-inflammatory agents, such as glucocorticoid receptor agonists include glucocorticoids such as Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, fluticasone propionate, fluticasone furoate, or mometasone.

(59) Linker Arms

(60) The phosphonate-based linkers disclosed herein comprise a first arm comprising a tuning element having a distal end and a proximal end wherein the distal end is covalently linked to an oxygen atom of the phosphonate group and the proximal end is covalently linked to a functional reactive group capable of covalent linkage to a cell-targeting ligand and a second arm comprising a linker having a distal end and a proximal end wherein the distal end is covalently linked to a payload or drug and a proximal end covelntly linked to a phophorus atom of the phosphonate group. Optionally, the linker arm may further include a spacer element interposed between the tuning element and the reactive functional group.

(61) Examples of tuning elements comprising the first arm include but are not limited to

(62) ##STR00036##

(63) R.sub.1 and R.sub.2 each independently any amino acid

(64) ##STR00037##
The wavy lines indicate the covalent attachment sites to an oxygen atom of the phosphonate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.

(65) Further examples of tuning elements include but are not limited to

(66) ##STR00038##
The wavy lines indicate the covalent attachment sites to an oxygen atom of the phosphonate group at the distal end (left) and the functional reactive group on the proximal end (right), or optionally, a spacer element.

(67) 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. The spacer element may be selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group. In particular aspects, the spacer element may be a straight polyethylglycol (PEG) chain (of a defined length, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polyethylene groups) and straight carbon chains of C1-30 hysdrocarbons with or without solubilizing groups attached thereto.

(68) In general, the linker comprising the second arm is to allow for distance control away from the phosphonate group. In some embodiments, this distance may have an impact on the efficacy of the payload or drug. The linker may be selected from a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units 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).sub.2—, —N(R)SO.sub.2—, SO.sub.2N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group. In particular aspects, the linker may be a straight polyethylglycol (PEG) chain (of a defined length, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polyethylene groups) and straight carbon chains of C1-30 hysdrocarbons with or without solubilizing groups attached thereto.

(69) 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), straight carbon chains with or without solubilizing groups attached thereto, a dipeptide, a tripeptide, a tetrapeptide, an enzyme cleavage site, for eample, a cathepsin cleavage site haing the structure

(70) ##STR00039##
wherein the wavy line on the left marks the covalent bond to an atom in the tuning element and the wavy line on the right marks the covalent bond to an atom of a reactive group Z.
Targeting Ligand

(71) The phosphonate linker and payload may be linked to a targeting ligand that selectively delivers a pay load to a cell, organ, or region of the body. Exemplary targeting ligands such as antibodies (e.g., chimeric, humanized and human), ligands for receptors, lectins, saccharides, and the like are recognized in the art and are useful without limitation in practicing the present invention. Other targeting ligands include a class of compounds that do not include specific molecular recognition motifs include macromolecules such as poly(ethylene glycol), polysaccharide, polyamino acids and the like, which add molecular mass to the cytotoxin. The additional molecular mass affects the pharmacokinetics of the payload, e.g., serum half-life.

(72) In an exemplary embodiment, the invention provides a payload, linker or payload-linker conjugate with a targeting ligand that is a biomolecule, e.g, an antibody, receptor, peptide, lectin, saccharide, nucleic acid or a combination thereof. Biomolecules useful in practicing the present invention may be derived from any source. The biomolecules may be isolated from natural sources or may be produced by synthetic methods. Proteins may be natural proteins or mutated proteins. Mutations may be effected by chemical mutagenesis, site-directed mutagenesis or other means of inducing mutations known to those of skill in the art. Proteins useful in practicing the instant invention include, for example, enzymes, antigens, antibodies and receptors. Antibodies may be either polyclonal or monoclonal, but most preferably are monoclonal and may be human, humanized, or human chimeric antibodies. Peptides and nucleic acids may be isolated from natural sources or can be wholly or partially synthetic in origin.

(73) In a particular embodiment, the targeting ligand is an antibody, or antibody fragment, that is selected based on its specificity for an antigen expressed on a target cell, or at a target site, of interest. A wide variety of tumor-specific or other disease-specific antigens have been identified and antibodies to those antigens have been used or proposed for use in the treatment of such tumors or other diseases. The antibodies that are known in the art may be used in the conjugates of the invention, in particular for the treatment of the disease with which the target antigen is associated. Non-limiting examples of target antigens (and their associated diseases) to which a conjugate of the invention may be targeted include: Her2 (breast cancer), CD4 (lymphomas, autoimmune diseases, including rheumatoid arthritis), CD20 (lymphomas), EGFR (solid tumors), CD22 (lymphomas, including non-Hodgkin's lymphoma), CD23 (asthma), CD25, CD52 (chronic lymphocytic leukemia), CD30 (lymphomas, including non-Hodgkin's lymphoma), CD33 (acute myelogenous leukemia), CD40L (immune thromobcytopenic purpura), CD70, CD74, CD80 (psoriasis), CD163, Mucl8 (melanoma), integrins (solid tumors), PSMA (prostate cancer, benign prostatic hyperplasia), CEA (colorectal cancer), CDl Ia (psoriasis), CTLA4 (T cell lymphomas) and BLys (autoimmune diseases, including systemic lupus erythematosus).

(74) Targeting ligands 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, peptides and proteins may be attached through an amine, carboxyl, sulfhydryl, or hydroxyl group. Such a group may reside at a peptide terminus or at a site internal to the peptide chain. Nucleic acids may be attached through a reactive group on a base (e.g., exocyclic amine) or an available hydroxyl group on a sugar moiety (e.g., 3′- or 5′-hydroxyl). The peptide or protein 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 protein or peptide may be synthesized to contain one or more nonnatural amino acids which may then serve as a site for attachment of the linker arm comprising the payload-phosphonate-based linker. Antibodies comprising nonnatural amino acids for conjugation and methods for making such antibodies have been disclosed in U.S. Pat. No. 7,632,924.

(75) In particular aspects, an anti-inflammatory therapeutic agent may be linked by the phophonate linker disclosed herein 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”.

(76) In one embodiment, the IgG4 constant domain can 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 in the KABAT system, wherein the native serine at position 108 (Ser108) of the HC constant domain is replaced with proline (Pro), in order to prevent a potential inter-chain disulfide bond between the cysteine at position 106 (Cys106) and the cysteine at position 109 (Cys109), which correspond to 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); see also (Schuurman et. al., Mol. Immunol. 38: 1-8, (2001); SEQ ID NOs:14 and 41). In other instances, a modified IgG1 constant domain which has been modified to reduce effector function can be used, for example, the IgG1 isotype may include substitutions of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 to greatly reduce ADCC and CDC (Armour et al., Eur J Immunol. 29(8):2613-24 (1999); Shields et al., J Biol Chem. 276(9):6591-604(2001)). In another embodiment, the IgG HC is modified genetically to lack N-glycosylation of the asparagine (Asn) residue at around position 297. The consensus sequence for N-glycosylation is Asn-Xaa-Ser/Thr (wherein Xaa is any amino acid except Pro); in IgG1 the N-glycosylation consensus sequence is Asn-Ser-Thr. The modification may be achieved by replacing the codon for the Asn at position 297 in the nucleic acid molecule encoding the HC with a codon for another amino acid, for example Gln. Alternatively, the codon for Ser may be replaced with the codon for Pro or the codon for Thr may be replaced with any codon except the codon for Ser. Such modified IgG1 molecules have little or no detectable effector function. Alternatively, all three codons are modified.

(77) The antibody may be attached to the first linker arm by any available reactive group that can react with the reactive functional group on the proximal end of the first 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.

(78) 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.

(79) 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.

(80) 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.

(81) 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.

(82) 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.

(83) 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 Cys 242 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.

(84) 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.

(85) 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 C1q 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.

(86) 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.

(87) Pharmaceutical Formulations and Administration

(88) 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.

(89) 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.

(90) 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.

(91) 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.

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

(93) 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.

(94) 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.

(95) 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.

(96) 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.

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

(98) 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.

(99) 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.

(100) 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.

(101) 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.

(102) 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.

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

Example 1

(104) This example shows the synthesis of the C21 Analogs.

(105) ##STR00040##

(106) Preparation of C21 Analog (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl)phosphonic acid (3) was as follows.

(107) In a dry round bottom flask equipped with a stir bar under nitrogen, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyl-oxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H cyclopenta[a] phenanthrene-17-carbothioic S-acid (1, 200 mg, 0.427 mmol) and (10-bromodecyl)phosphonic acid (2, 141 mg, 0.470 mmol, 1.10 eq.) were dissolved in anhydrous DMF (2.1 mL, 0.2M). After 10 minutes of stirring, diisopropylethylamine (176 μL, 1.07 mmol, 2.50 eq.) was added and the reaction stirred overnight at ambient temperature. Upon completion as determined by LCMS, the reaction was concentrated, diluted in DMSO and directly injected on a reverse phase acidic prep HPLC (Sunfire C18 30×150) with 5 to 95 gradient of organic (0.1% TFA/acetonitrile)/aqueous (0.1% TFA/water). The isolated fractions containing product were evaporated using a Genevac and isolated (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio)decyl)phosphonic acid (3, 185 mg, 63% yield) as a white, foamy solid. LRMS (ES) (M+H).sup.+. observed=689.6, calculated=689.8. .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ.sub.H 7.26 (1H, d, J=10.2 Hz), 6.29 (1H, d, J=10.2 Hz), 6.11 (1H, s), 5.63 (1H, ddd, J=48.7, 11.1, 6.7 Hz), 4.20 (1H, d, J=9.3 Hz), 2.86 (2H, dddd, J=20.9, 18.9, 13.5, 7.4 Hz), 2.31 (2H, q, J=7.7 Hz), 2.24 (1H, br s), 2.05-2.11 (2H, m), 1.80-1.87 (2H, m), 1.47 (8H, d, J=12.3 Hz), 1.31 (5H, br s), 1.25 (9H, s), 0.98-1.02 (6H, m), 0.89 (3H, d, J=7.1 Hz).

(108) The following C21 analogs were made by a similar experimental procedure:

(109) TABLE-US-00001 Compound Mass number Structure Spectrometry Data 4 C.sub.28H.sub.39F.sub.2O.sub.8PS LRMS (ES) (M + H).sup.+: observed 605.2, calculated = 605.2. 5 C.sub.25H.sub.35F.sub.2O.sub.7PS LRMS (ES) (M + H).sup.+: observed 549.2, calculated = 549.2. 6 C.sub.30H.sub.43F.sub.2O.sub.10PS LRMS (ES) (M + H).sup.+: observed 665.4, calculated = 665.2. 7 C.sub.30H.sub.43F.sub.2O.sub.8PS LRMS (ES) (M + H)+: observed 633.4, calculated = 633.2. 8 C.sub.31H.sub.47F.sub.2O.sub.7PS LRMS (ES) (M + H).sup.+: observed 633.4, calculated = 633.3. 9 C.sub.27H.sub.39F.sub.2O.sub.9PS LRMS (ES) (M + H).sup.+: observed 609.3, calculated = 609.2. 10 C.sub.27H.sub.39F.sub.2O.sub.7PS LRMS (ES) (M + H).sup.+: observed 577.5, calculated = 577.2. 11 C.sub.23H.sub.31F.sub.2O.sub.7PS LRMS (ES) (M + H).sup.+: observed 521.3, calculated = 521.2. 12 C.sub.26H.sub.35F.sub.2O.sub.8PS LRMS (ES) (M + H).sup.+: observed 577.2, calculated = 577.2. 13 0 C.sub.24H.sub.33F.sub.2O.sub.7PS LRMS (ES) (M + H).sup.+: observed 535.2, calculated = 535.2. 14 C.sub.27H.sub.37F.sub.2O.sub.8PS LRMS (ES) (M + H).sup.+: observed 591.2, calculated = 591.2.

Example 2

(110) This example shows the synthesis of the C17 Analogs.

(111) ##STR00052##

(112) Preparation of C17 Analog (9-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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)oxy)-9-oxononyl)phosphonic acid (18) was follows.

Step A: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-17-(non-8-enoyloxy)-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic S-acid (16)

(113) In a dry round bottom flask equipped with a stir bar under nitrogen, (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-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]phenanthrene-17-carbothioic S-acid (15, 100 mg, 0.242 mmol) and triethylamine (84 μL, 0.606 mmol, 2.50 eq.) were dissolved in anhydrous DCM (12.0 mL, 0.02M). To this was added non-8-enoyl chloride (93 mg, 0.533 mmol, 2.20 eq.) and the reaction stirred 20 minutes at ambient temperature. To this was then added N,N-dimethylethylenediamine (64 mg, 0.727 mmol, 3.00 eq.) and stirred at ambient temperature for 1 hour. Upon completion as determined by LCMS, the reaction was washed with 1N HCl twice and concentrated. The crude (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-7-(non-8-enoyloxy)-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic S-acid 16 was carried on directly without further purification. LRMS (ES) (M+H).sup.+: observed=551.1, calculated=550.7.

Step B: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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 non-8-enoate (17)

(114) To a round bottom flask containing the crude 16 from step A under nitrogen, was added dioxane (8.0 mL, 0.03M), diisopropylethylamine (118 μL, 0.678 mmol, 2.80 eq.) and bromofluoromethane 2M in DMF (230 μL, 0.460 mmol, 1.90 eq.). The resulting reaction was stirred at ambient temperature for 30 minutes. To this was added additional diisopropylethylamine (480 μL, 2.71 mmol, 11.20 eq.) and bromofluoromethane 2M in DMF (920 μL, 1.84 mmol, 7.6 eq.) and the resulting reaction as stirred at ambient temperature for 3 days. The reaction was concentrated and the residue was dissolved in ethyl acetate and washed several times with 1H HCl solution. The crude material was flash column purified using a 0-50% ethyl aceate/hexane gradient to give (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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 non-8-enoate (17, 37 mg, 26.5% for 2 steps) LRMS (ES) (M+H).sup.+: observed=583.2, calculated=582.7.

Step C: Preparation of (9-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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)oxy)-9-oxononyl)phosphonic Acid (18)

(115) To a vial containing 17 (37 mg, 0.063 mmol) under nitrogen, was added 4,4,5,5-tetramethyl-1,3,2-dioxaphospholane 2-oxide (10.42 mg, 0.063 mmol, 1.00 eq.), tris(triphenylphosphine)rhodium(I) chloride (11.75 mg, 0.013 mmol, 0.20 eq.), and 1,4-bis(diphenylphosphino)butane (10.83 mg, 0.025 mmol, 0.40 eq.). Dioxane (0.25 mL, 0.25M) was added and the resulting mixture was heated at 100 C for 23 hours. The mixture was allowed to cool and concentrated to dryness. The residue was dissolved in a mixture of 4N HCl dioxane (100 μL) and water (100 μL) and BHT (4.16 mg, 0.019 mmol, 0.30 eq.) was added. The resulting mixture was heated to 50 C overnight. The reaction was concentrated, dissolved in methanol and directly injected on a reverse phase acidic prep HPLC (Sunfire C18 30×150) with 20 to 80 gradient of organic (0.1% TFA/acetonitrile)/aqueous (0.1% TFA/water to give (9-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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)oxy)-9-oxononyl)phosphonic acid (18, 20.3 mg, 48.5% yield). LRMS (ES) (M+H).sup.+: observed=665.2, calculated=664.7.

(116) The following C17 Analog was made by a similar experimental procedure:

(117) TABLE-US-00002 Compound Mass number Structure Spectrometry Data 19 C.sub.28H.sub.38F.sub.3O.sub.8PS LRMS (ES) (M + H).sup.+: observed 623.2, calculated = 622.6.

Example 3

(118) This example shows the synthesis of intermediate 25 for constructing A-Ring Analogs.

(119) ##STR00054## ##STR00055##

(120) Preparation of intermediate 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(4-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (23) was as follows.

Step A: Preparation of 4-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-13b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-8(3aH,4bH,13bH)-yl)phenyl Pivalate (22)

(121) In a dry 20 mL microwave vial equipped with a stir bar under nitrogen, (20, 500 mg, 0.870 mmol, synthesis described in WO 2009082342) and 4-hydrazinylphenyl pivalate HCl (21, 234 mg, 0.957 mmol, 1.10 eq.) were dissolved in ethanol (5.1 mL, 0.17M). To this was added potassium acetate (128 mg, 1.305 mmol, 1.50 eq.) and the vial was microwave irradiated to 90 C for 15 minutes. The crude reaction was concentrated and flash column purified using a 0-30% ethyl acetate/hexane gradient to gave 4-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-13b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-8(3aH,4bH,13bH)-yl)phenyl pivalate. (22, 497 mg, 76% yield). LRMS (ES) (M+H).sup.+. observed=747.6, calculated=747.0.

Step B: Preparation of 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(4-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1,′2′:5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (Intermediate 23)

(122) In a round bottom flask equipped with a stir bar 4-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-13b-(2-((tert-butyldimethylsilyl)oxy)acetyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[,2-f]indazol-8(3aH,4bH,13bH)-yl)phenyl pivalate. (22, 850 mg, 1.138 mmol) was dissolved in THE (7.1 mL, 0.16M). To this was added 1M aq lithium hydroxide (2276 μL, 2.276 mmol, 2.00 eq.) and stirred at ambient temperature overnight. The reaction was poured into saturated aq ammonium chloride solution and extracted several times with ethyl acetate. The combined organic layers were concentrated and flash column purified using a 0-50% ethyl aceate/hexane gradient to gave 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(4-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[,2-f]indazol-13b-yl)ethanone. (23, 560 mg, 74.2% yield). LRMS (ES) (M+H).sup.+. observed=663.5, calculated=662.9.

(123) Preparation of 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(3-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[,2-f]indazol-13b-yl)ethanone (25) was as follows.

(124) In a dry 20 mL microwave vial equipped with a stir bar under nitrogen, (20, 450 mg, 0.783 mmol, synthesis described in WO 2009082342) and 3-hydrazinylphenol, HCl (24, 143 mg, 0.892 mmol, 1.14 eq.) were dissolved in ethanol (4.6 mL, 0.17M). To this was added potassium acetate (115 mg, 1.174 mmol, 1.50 eq.) and the vial was microwave irradiated to 90 C for 15 minutes. The crude reaction was concentrated and flash column purified using a 0-50% ethyl aceate/hexane gradient to gave 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(3-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-3b-yl)ethanone. (25, 380 mg, 73.2% yield). LRMS (ES) (M+H).sup.+. observed=663.4, calculated=662.9.

Example 4

(125) This example shows the synthesis of A-Ring Analogs.

(126) ##STR00056##

(127) Preparation of A-Ring Analog (6-(3-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-13b-(2-hydroxyacetyl)-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[,2-f]indazol-8(3aH,4bH,13bH)-yl)phenoxy)hexyl)phosphonic acid (27) was follows.

Step A: Preparation of 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-8-(3-(hex-5-en-1-yloxy)phenyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (26)

(128) In a vial equipped with a stir bar under nitrogen, 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-12-hydroxy-8-(3-hydroxyphenyl)-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (25, 140 mg, 0.211 mmol) and 5-hexen-1-ol (21.15 mg, 0.211 mmol, 1.00 eq.) and triphenylphosphine (55.40 mg, 0.211 mmol, 1.00 eq.) were dissolved in dioxane (1.0 mL, 0.21M). To this was added Di-tert-butyl azodicarboxylate (58.4 mg, 0.253 mmol, 1.20 eq.) and the resulting mixture was stirred at ambient temperature until complete based upon LCMS. The crude reaction was concentrated and flash column purified using a 0-50% ethyl aceate/hexane gradient to gave 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-8-(3-(hex-5-en-1-yloxy)phenyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-13b-yl)ethanone. (26, 119 mg, 76% yield). LRMS (ES) (M+H).sup.+: observed=745.5, calculated=745.0.

Step B: Preparation of (6-(3-((3aR,4aS,4bS,1aR,11bS,12S,13aS,13bS)-12-hydroxy-13b-(2-hydroxyacetyl)-11a,13a-dimethyl-2-propyl-4,4a,5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[,2-f]indazol-8(3aH,4bH,13bH)-yl)phenoxy)hexyl)phosphonic Acid (27)

(129) In a similar manner to Step C of Example 2, 2-((tert-butyldimethylsilyl)oxy)-1-((3aR,4aS,4bS,11aR,11bS,12S,13aS,13bS)-8-(3-(hex-5-en-1-yloxy)phenyl)-12-hydroxy-11a,13a-dimethyl-2-propyl-3a,4,4a,4b,5,6,8,11,11a,11b,12,13,13a,13b-tetradecahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2-f]indazol-13b-yl)ethanone (26) and tetramethyl-1,3,2-dioxaphospholane 2-oxide were used to synthesize (6-(3-((3aR,4aS,4bS,11aR,11bS,12,13aS,13bS)-2-hydroxy-3b-(2-hydroxyacetyl)-11a,13a-dimethyl-2-propyl-4,4a, 5,6,11,11a,11b,12,13,13a-decahydro-[1,3]dioxolo[4″,5″:3′,4′]cyclopenta[1′,2′:5,6]naphtho[1,2f]indazol-8(3aH,4bH,13bH)-yl)phenoxy)hexyl)phosphonic acid (27). LRMS (ES) (M+H).sup.+: observed=713.3, calculated=712.8.

(130) The following A-Ring Analogs were made by a similar procedure:

(131) TABLE-US-00003 Compound Mass number Structure Spectrometry Data 28 C.sub.36H.sub.49N.sub.2O.sub.9P LRMS (ES) (M + H).sup.+: observed 685.4, calculated = 684.7. 29 C.sub.41H.sub.59N.sub.2O.sub.9P LRMS (ES) (M + H).sup.+: observed 755.4, calculated = 754.8. 30 C.sub.36H.sub.49N.sub.2O.sub.9P LRMS (ES) (M + H).sup.+: observed 685.3, calculated = 684.7. 31 0 C.sub.38H.sub.53N.sub.2O.sub.9P LRMS (ES) (M + H).sup.+: observed 713.4, calculated = 712.8. 32 C.sub.41H.sub.59N.sub.2O.sub.9P LRMS (ES) (M + H).sup.+: observed 755.4, calculated = 754.8.

Example 5

(132) This example shows the synthesis of pyrophosphonate drug linkers.

(133) ##STR00062##

Step B: Preparation of ((9H-fluoren-9-yl)methyl-carbamoyl)-2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio) decyl) phosphonic Anhydride (34)

(134) In a dry glass vial equipped with a stir bar under nitrogen, (9H-fluoren-9-yl)methyl (2-(phosphonooxy)ethyl)carbamate (33, 95 mg, 0.261 mmol, 1 eq.) and carbonyldiimidazole (50.8 mg, 0.314 mmol, 1.2 eq.) were dissolved in anhydrous DMF (0.50 mL) and treated with triethylamine (36.4 μL, 0.261 mmol, 1 eq.). The reaction was stirred 20 min and found to be complete as measured by LCMS. In a separate dry glass vial (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio)decyl)phosphonic acid (3, 180 mg, 0.261 mmol, 1 eq.) and zinc (II) chloride (214 mg, 1.57 mmol, 6 eq.) were combined and dissolved in anhydrous DMF (0.80 mL, 0.2M final concentration of reaction). To this latter solution was added the solution of activated 33 and the resulting mixture was stirred overnight at ambient temperature. The reaction was judged to be complete by LCMS (basic conditions) after 12 h and diluted with 1N HCl (5 mL). The mixture was extracted with DCM (5×5 mL) and the combined extracts were concentrated. The resulting crude was dissolved in 1 mL of methanol and injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini—NX C18 OBD 5 uM 30×100 mm; 30-90% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min). The isolated fractions containing product were evaporated using a lyophilizer and isolated ((9H-fluoren-9-yl)methyl-carbamoyl)-2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,115,135,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl) phosphonic anhydride (34, 88.0 mg, 32% yield) as a white solid. LRMS (ES) (M+H).sup.+. observed=1034.6, calculated=1034.0.

Step C: Preparation of (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio) decyl) phosphonic Anhydride (35)

(135) In a dry round bottom flask equipped with a stir bar under nitrogen, ((9H-fluoren-9-yl)methyl-carbamoyl)-2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl) phosphonic anhydride (34, 88.0 mg, 0.085 mmol) was dissolved in anhydrous dichloromethane (0.42 mL, 0.2M) and treated with DBU (65 mL, 0.425 mmol, 5 eq). The resulting solution was stirred at ambient temperature for 1 h and determined to be complete as judged by LCMS. The reaction was concentrated and taken up in methanol and injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini—NX C18 OBD 5 uM 30×100 mm; 10-70% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min, 235 nM wavelength). The isolated fractions containing product were evaporated using a lyophilizer and isolated (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio) decyl)phosphonic anhydride (35, 27.0 mg, 39% yield) as a white solid. LRMS (ES) (M+H).sup.+: observed=812.4, calculated=812.8.

Step D: Preparation (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a] phenanthrene-17-carbonyl)thio)decyl)phosphonic Anhydride (37)

(136) In a dry round bottom flask equipped with a stir bar under nitrogen, (2-aminoethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodeca-hydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl)phosphonic anhydride (35, 9.0 mg, 11 mmol, 1.0 eq) and 2-(cyclooct-2-yn-1-yloxy)acetic acid (36, 3.0 mg, 17 mmol, 1.5 eq) were dissolved in anhydrous DMF (0.37 mL, 0.03M). To the resulting solution was added HATU (6.3 mg, 17 mmol, 1.5 eq) and triethylamine (6.2 μL, 44 mmol, 4 eq.). The reaction became yellow immediately and stirred 20 min before completion as judged by basic LCMS. The reaction was injected onto a on a reverse phase basic prep HPLC (Phenomenex Gemini—NX C18 OBD 5 uM 30×100 mm; 10-70% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min, 235 nM wavelength). The isolated fractions containing product were evaporated using a lyophilizer and isolated (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl phosphoric) (10-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-oxo-17-(propionyloxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbonyl)thio)decyl) phos-phonic anhydride (37, 6.0 mg, 55% yield) as a white solid. LRMS (ES) (M+H).sup.+: observed=976.6, calculated=977.0. .sup.1H NMR (DMSO-d.sub.6, 500 MHz): δ.sub.H 8.33 (1H, s), 7.35 (1H, d, J=10.1 Hz), 6.27 (1H, dd, J=10.0, 2.0 Hz), 6.10 (1H, d, J=2.1 Hz), 5.63 (1H, ddd, J=48.7, 10.7, 6.4 Hz), 4.29 (1H, t, J=5.2 Hz), 4.20 (1H, d, J=9.0 Hz), 3.89 (1H, d, J=14.6 Hz), 3.73-3.76 (3H, m), 3.22-3.27 (3H, m), 2.86 (2H, dddd, J=19.8, 13.9, 12.7, 7.0 Hz), 2.31 (2H, ddd, J=8.4, 8.2, 6.5 Hz), 2.21-2.25 (3H, m), 2.15 (1H, t, J=6.8 Hz), 2.05-2.11 (4H, m), 1.70-1.93 (7H, m), 1.45-1.58 (10H, m), 1.24-1.31 (15H, m), 0.98-1.01 (6H, m), 0.86-0.90 (4H, m).

Example 6

(137) This example shows the synthesis of pyrophosphonate drug linkers.

(138) ##STR00063##

Step A: 1,3-dioxoisoindolin-2-yl 2-(cyclooct-2-yn-1-yloxy)acetate (38)

(139) To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (36, 0.10 g, 0.55 mmol) in DCM (2 mL) was added 2-hydroxyisoindoline-1,3-dione (0.18 g, 1.10 mmol) and EDC (0.21 g 1.10 mmol) and the resulting mixture was stirred at room temperature for 1.5 hrs. The solution was directly purified by flash column separation using a 0-50% ethyl acetate/hexane gradient gave the title compound (38, 163 mg, 91%).

Step B: (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphonic Acid (40)

(140) To a stirred mixture of 1,3-dioxoisoindolin-2-yl 2-(cyclooct-2-yn-1-yloxy)acetate (38, 79.0 mg, 0.29 mmol) and 2-aminoethyl dihydrogen phosphate (39, 59.9 mg, 0.42 mmol, 1.5 eq) in 1:1 DMF/H.sub.2O (1.0 mL) was added triethylamine (59.1 uL, 0.42 mmol, 1.5 eq) and the mixture became a solution. The resulting solution stirred 2 h before being injected directly onto reverse phase acidic prep HPLC (Sunfire C18 30×150) with 5 to 95 gradient of organic (0.1% TFA/acetonitrile)/aqueous (0.1% TFA/water). The isolated fractions containing product were evaporated using a Genevac and isolated (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphonic acid (40, 45.0 mg, 54% yield) as a white solid. LRMS (ES) (M+H).sup.+. observed=306.3, calculated=306.3.

Step C: (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl phosphoric) (6-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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)oxy)-6-oxohexyl)phosphonic Anhydride (41)

(141) In a dry glass vial equipped with a stir bar under nitrogen, (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphonic acid (40, 14.7 mg, 0.048 mmol, 1 eq.) and carbonyldiimidazole (9.4 mg, 0.058 mmol, 1.2 eq.) were dissolved in anhydrous DMF (0.19 mL) and treated with triethylamine (6.7 μL, 0.048 mmol, 1 eq.). The reaction was stirred 15 min and found to be complete as measured by LCMS. In a separate dry glass vial (6-(((6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio) carbonyl)-11-hydroxy-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)oxy)-6-oxohexyl)phosphonic acid (19, 30 mg, 0.048 mmol, 1 eq.) and zinc (II) chloride (39.4 mg, 0.289 mmol, 6 eq.) were combined and dissolved in anhydrous DMF (0.05 mL, 0.2M final concentration of reaction). To this latter solution was added the solution of activated 40 and the resulting mixture was stirred 2 days at ambient temperature. The reaction was judged to be complete by LCMS (basic conditions) and diluted with 1N HCl. The mixture was extracted with DCM several times and the combined extracts were concentrated. The resulting crude was dissolved in DMF and injected onto a reverse phase basic prep HPLC (Phenomenex Gemini—NX C18 OBD 5 uM 30×100 mm; 10-60% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min to give (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl phosphoric) (6-(((6S,8S,9R,105,11S,135,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy-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)oxy)-6-oxohexyl)phosphonic anhydride (41, 13.0 mg, 30% yield) as a white solid. LRMS (ES) (M+H).sup.+: observed=910.4, calculated=909.8.

(142) The following compounds were made by a similar procedure:

(143) TABLE-US-00004 Compound Mass number Structure Spectrometry Data 42 C.sub.40H.sub.57F.sub.2NO.sub.13P.sub.2S LRMS (ES) (M + H).sup.+: observed 892.4, calculated = 891.8. 43 C.sub.50H.sub.71N.sub.3O.sub.14P.sub.2 LRMS (ES) (M + H).sup.+: observed 1000.6, calculated = 1000.0. 44 C.sub.50H.sub.71N.sub.3O.sub.14P.sub.2 LRMS (ES) (M + H).sup.+: observed 1000.6, calculated = 1000.0.

Example 7

(144) This example shows the synthesis of cathepsin phosphonate drug linkers

(145) ##STR00067## ##STR00068##

Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-(((10-(((4-(2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)decyl)thio)carbonyl)-6,9-difluoro-11-hydroxy-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 (47)

Step A: Preparation of 2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (46)

(146) In a dry vial equipped with a stir bar, was dissolved 2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (45, 100 mg, 0.264 mmol) in anhydrous DMF (0.6 mL, 0.44 M). To this was added 2,5-dioxopyrrolidin-1-yl 2-(cyclooct-2-yn-1-yloxy)acetate (81 mg, 0.290 mmol, 1.10 eq.) and the reaction stirred 40 minutes at ambient temperature. Additional 2,5-dioxopyrrolidin-1-yl 2-(cyclooct-2-yn-1-yloxy)acetate was added as necessary to complete reaction by LCMS. This crude reaction was directly injected on a reverse phase basic prep HPLC (Phenomenex Gemini—NX C18 OBD 5 um 30×100 mm) with 10 to 60 gradient of organic (0.1% NH.sub.4OH/acetonitrile)/aqueous (0.1% NH.sub.4OH/water) to give 2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide. (46, 54 mg, 37.7% yield) LRMS (ES) (M+H).sup.+: observed=544.4, calculated=543.6.

Step B: Preparation of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-(((10-(((4-(2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)decyl)thio)carbonyl)-6,9-difluoro-11-hydroxy-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 (47)

(147) In a dry vial equipped with a stir bar, was added 3 (50 mg, 0.074 mmol), 46 (40 mg, 0.074 mmol, 1.00 eq.) and DCC (33 mg, 0.162 mmol, 2.20 eq.). To this was added anhydrous pyridine (1.22 mL, 0.06 M) and the resulting mixture was stirred for 3 days at ambient temperature. The pyridine was removed and the residue was dissolved in methanol and directly injected on a reverse phase acidic prep HPLC (Sunfire C18 30×150) with 20 to 90 gradient of organic (0.1% TFA/acetonitrile)/aqueous (0.1% TFA/water to give (6S,8S,9R,10S,11S,13S,14S,16R,17R)-17-(((10-(((4-(2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)decyl)thio)carbonyl)-6,9-difluoro-11-hydroxy-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 (47.30 mg, 33.6% yield). LRMS (ES) (M+H).sup.+: observed=1214.6, calculated=1214.4.

(148) The following compounds were made by a similar procedure:

(149) TABLE-US-00005 Compound Mass number Structure Spectrometry Data 48 C.sub.56H.sub.78F.sub.2N.sub.5O.sub.13PS LRMS (ES) (M + H).sup.+: observed 1129.5, calculated = 1130.3. 49 0 C.sub.58H.sub.82F.sub.2N.sub.5O.sub.13PS LRMS (ES) (M + H).sup.+: observed 1158.5, calculated = 1158.3. 50 C.sub.59H.sub.82F.sub.3N.sub.5O.sub.13PS LRMS (ES) (M + H)+: observed 1191.3, calculated = 1190.3.

Example 8

(150) This example shows the synthesis of various antibody drug conjugates (ADCs). Site specific conjugation using click (2+3) chemistry. Para-azido phenylalanine containing anti-CD74 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 μm filtered, and stored at 4° C.

(151) TABLE-US-00006 ADC number Structure αCD74-47 αCD74-50 αCD74-41

Example 9

(152) The potency of binding to gucocorticoid receptor by small molecule compounds was measured with the PolarScreen™ Glucocorticoid Receptor Competitor Assay Kit, Red (Life Technology, Catalog #A15898) according to the procedure in the kit. In brief, after compounds were diluted and transferred into assay plates, the fluormone GS red and GR full length protein were subsequently added into the assay plates, along with the positive and negative controls. The samples were mixed on a shaker for 1 minute and then the plates were incubated at room temperature for 2-4 hours with minimal light exposure. The plates were read with an Envision and the relative fluorescent signals were calculated. The data were plotted in GraphPad Prism and the EC50 values were calculated with non-linear regression curve fit of the data in GraphPad Prism.

(153) GILZ gene assays were conducted as follows. HUT78 cells were cultured in IMEM plus 20% heat inactivated FBS and cell density was maintained between 0.1 to 1.2 million/mL. 786-O cells were cultured in RPMI plus 10% heat inactivated FBS. Actively growing cells were harvested and resuspended in HBSS with 2% FBS at 1.1 million cells per mL then dispensed to 384-well V-bottom plates at 45 μL per well. Serially diluted ADC solution was added to the cell plate (5 μL per well) and mixed for 2 min. Cells were then cultured at 37° C., at 5% CO.sub.2 for a designated time before supernatant was removed. Cells were harvested in lysis buffer from the Cells-to-Ct kit (40 μL, Life Technologies, 4391851C) following the supplier's protocol and mixed for 10 min followed by addition of 5 μL per well of stop solution from the kit. cDNA was synthesized with a reverse transcription kit (Life Technologies, 4391852C) follow by qPCR using the TaqMan gene expression master mix (Life Technologies, 4369016) with GILZ gene assay (Life Technologies, Hs00608272_m1) and GAPDH assay (Hs2758991_g1) in a duplex format with 3-4 technical replicates.

(154) Determination of apparent permeability was as follows. MDCKII cells (kindly provided by the Netherlands Cancer Institute, under a licensing agreement) were seeded on to 96-well transwell culture plates (Millipore Corp, Billerica, Mass.) and used in experiments after five days in culture. Test compound (1 μM) was prepared in Hank's Balanced Salt Solution (HBSS), 10 mM (4-(2-hydroxyethyl)-1-piperrazineethanesulfonic acid) (HEPES, pH 7.4), with 10 μM cyclosporine A (to inhibit endogenous transport) and 1.2 μM dextran Texas red (to confirm monolayer integrity). Substrate solution (150 μL) was added to either the apical (A) or the basolateral (B) compartment of the culture plate, and buffer (150 μL; HBSS, 10 mM HEPES, pH 7.4) with 10 μM cyclosporine A was added to the compartment opposite to that containing the substrate. At t=3 hr, 50 μL samples were removed from both sides of monolayers dosed with test compound and placed in 96 well plates, 50 μL internal standard (1 μM labetolol) and 100 μL HBSS was added to the samples. Samples were analyzed by LC/MS/MS using an Applied Biosystems SCIEX API 5000 triple quadruple mass spectrometer (Concord, ON, Canada) with a TurboIonSpray ion source in the positive ion mode. A Thermo Scientific Transcend LX-2 system (Franklin, Mass.) was coupled to the API 5000 with a flow rate of 800 μL/min to direct sample into the mass spectrometer. The apparent permeability (P.sub.app) was calculated by the following formula for samples taken at t=3 hr:

(155) $\mathrm{Papp}=\frac{\mathrm{volume}\mathrm{of}\mathrm{receptor}\mathrm{chamber}\left(\mathrm{mL}\right)}{\left[\mathrm{area}\mathrm{of}\mathrm{membrane}\left({\mathrm{cm}}^2\right)\right]\times \left[\mathrm{initial}\mathrm{concentration}\left(\mathrm{.Math.M}\right)\right]}\times \frac{\Delta \mathrm{concentration}\left(\mathrm{.Math.M}\right)}{\Delta \mathrm{time}\left(s\right)}$
Where: Volume of Receiver Chamber is 0.15 mL; Area of membrane is 0.11 cm.sup.2; the initial concentration is the sum of the concentration measured in the donor plus concentration measured in receiver compartments at t=3 hr; Δ in concentration is concentration in the receiver compartment at 3 hr; and Δ in Time is the incubation time (3×60×60=10800 s). P.sub.app was expressed as 10-6 cm/s. The P.sub.app reported is the average of the A to B and B to A P.sub.app values control MDCKII cells at t=3 hr:

(156) $P_{app}=\frac{P_{app}\left(A.fwdarw.B\right)+P_{app}\left(B.fwdarw.A\right)}{2}$
The B-A/A-B ratio was calculated by dividing the P.sub.app from B to A by the P.sub.app from A to B at t=3 hr.

(157) The results of the aforementioned assays with compounds 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 27, 28, 29, 30, 31, and 32 compared to dexamehtsone, budesonide, anf fluticasone proprionate are shown in Table 1 below.

(158) TABLE-US-00007 TABLE 1 MDCK Permeability Compound GR Bind GILZ (cm × 10- Number IC50 (nM) GILZ EC50 Max 6/sec) Dexamethasone 2.1 3.7 100% 12.9 Budesonide 0.9 1 100% 31.6 Fluticasone 0.15 0.2 100% 21.5 Propionate 3 0.44 649 71% (at 1.04 10 uM) 4 0.71 >100 <10% (at 1.72 100 nM) 5 160.4 >10000 24% (at 1.84 100 nM) 6 0.32 >10000 40% (at n/a 10 uM) 7 0.45 6450 64% (at 2 10 uM) 8 0.25 970 66% (at 2.8 10 uM) 9 1.67 >10000 14.5% n/a (at 10 uM) 10 1.99 >10000 17.3% 1.9 (at 10 uM) 11 n/a 2865 81% (at n/a 10 uM) 12 162 6777 36% (at n/a 10 uM) 13 1225 >10,000 24% (at n/a 10 uM) 14 18.1 >10,000 15% (at n/a 10 uM) 18 0.1 3038 65% (at <0.9 10 uM) 19 0.14 1289 88% (at n/a 10 uM) 27 2 2759 80% (at 1.8 10 uM) 28 4.1 1267 101% (at 1.6 10 uM) 29 24.2 558 96% (at n/a 10 uM) 30 2.4 3008 61% (at 1.9 10 uM) 31 3.9 4175 47% (at 4.1 10 uM) 32 114.8 4154 86% (at n/a 10 uM)

Example 10

(159) The Analogs were conjugated to the antibodies as follows.

(160) To initiate conjugation, 10% v/v DMSO was added to the antibody solution, followed by a 15-fold molar excess of cyclooctyne-functionalized drug-linker. The solution was gently mixed and allowed to react at 28° C. for 48 hours. Removal of unreacted drug-linker and aggregates was performed via cation exchange as previously described. The final cation exchange pool was then concentrated and formulated into 50 mM histidine, 100 mM NaCl, 2.5% trehalose, pH 6.0 and 0.22 μm filtered.

(161) Analytical Data for ADCs

(162) UV: The concentration of conjugates was determined by absorbance at 280 nm with a background correction of 320 nm using Agilent U.V spectrometer (Model #8453).

(163) SEC: The ADCs were analyzed by SEC-HPLC (G3000SWXL Tosoh Column 7.8×300 mm (Serial #Y02322), Mobile Phase −200 mM KPO4, 250 mM KCl, pH 6.0+10% IPA; at flow rate 0.5 ml/min, isocratic gradient for 40 min, 10 μg load) on an Agilent 1100 HPLC. The data was processed using the software Agilent Chemstation and the percent monomer was reported.

(164) MS and DAR: The sample was denatured with 3M guanidine HCl, reduced with 150 mM DTT and analyzed by LC-MS (Agilent PLRP-S,4000° A, 8 um, 2.1×150 mm (Serial #0001023345-118) Mobile Phase A—0.05% TFA in Water and Mobile Phase B—0.05% TFA in Acetonitrile, at flow rate 0.3 ml/min, 80° C. for 23 min, 3 μg load) on Agilent Q-TOF LC/MS (Model #6510). Data was acquired and deconvoluted to monoisotopic and singly charged species using the software Agilent Mass Hunter Qualitative Analysis. The identity was confirmed from the mass of the light chain, the unconjugated and conjugated heavy chains. DAR was calculated from the DAD signal.

(165) Residual Drug Linker: Residual Drug Linker was extracted from Antibody Drug Conjugate by precipitating out the antibody with ACN:MeOH (50:50). The supernatant was analyzed by RP-HPLC (Waters X Bridge BEH C18 2.1×150 mm (PN 186003110), Mobile Phase A—4% NH.sub.4OH, 1.5% FA in water and Mobile Phase B—Acetonitrile; flow rate 0.3 ml/min, 60° C. for 28 min, 10 μl load) on Agilent Q TOF LC-MS (Model #6510). The data was processed using Agilent Mass Hunter Qualitative Analysis. The Extracted Ion chromatogram (EIC) was integrated and the molarity of the residual drug linker was extrapolated from linear regression analysis of the standard curve.

(166) TABLE-US-00008 Drug Linker Antibody No. ADC No. DAR % Monomer anti-hCD74 (IgG4) 47 1-474 1.6 90.6 50 1-477 1.7 98.5 41 1-496 1.5 94.6 anti-RSV (IgG4) 47 2-474 1.8 96.1 50 2-477 1.8 98.9 41 2-496 1.5 94.3
B-Cell Analysis

(167) The carrier solution for all ADCs and naked antibodies was 50 mM histidine pH 6.0, 100 mM NaCl and 5% trehelose. To assure consistency across experiments, ADCs were thawed on ice, dispensed to small aliquots, frozen to −80° C., and those aliquots were then used for experiments with unused material discarded at the end of the day. For all in vitro studies in this report, none of the read outs were measurably different in the presence or absence of the carrier solution. Nevertheless, the final concentration of antibody carrier solution was maintained constant at 1% v/v by serially diluting antibody solution prior to addition to the assay mixture and adding carrier solution to control wells. The same practice was observed for DMSO as the solvent for small molecules, with the final concentration being 0.1% v/v.

(168) For the studies of ADCs bearing reduced permeability payloads frozen human CD19+ B cells purchased from Precision Biosciences (catalog #84400, donor #13108) were thawed and re-suspended in RPMI1640 plus 10% heat-inactivated FBS (cell culture medium) at 1 million cells/mL. ADC or fluticasone was serially diluted to 100× the intended final concentrations. B cells were bulk treated in a 96-well block and subsequently dispensed to 96 well plates (100 μL and 1 million cells per well) and incubated at 37° C., 5% CO.sub.2 for 18 or 40 hr. Control wells containing equal percentages of DMSO (0.1%) and ADC buffer (1%) as sample wells were included. Cells were transferred to a 96 well v-bottom plate and spun at 500×g for 5 min. Medium was removed and cells were lysed in 150 μL RLT buffer containing beta-mercaptoethanol. RNA was isolated (RNeasy 96 kit, Qiagen, Cat #74181) and cDNA was synthesized (iScript cDNA Synthesis Kit, BioRad, Cat #170-8891) according to the supplier's protocol. Quantitative PCR was performed on Applied Biosystem's 7900 HT Real-Time PCR System to measure expression of ZBTB16 (Life Technologies, Hs00957433_m1) and GAPDH (Life Technologies, Hs02758991_g1) in duplex format.

(169) The ΔΔCt method was used to calculate fold change of expression of ZBTB16 relative to control sample, using 3 replicates per treatment condition. For free cell impermeable compounds, B cells were seeded at 100,000 cells/well in 90 μL in a 96-well flat bottom plate. Three fold serial dilutions were performed in DMSO, subsequent intermediate 10× stocks were made in tissue-culture medium and then added to the cell plate (10 μL/well). Cells were incubated at 37° C., 5% CO.sub.2 for 18 hr, harvested and processed for PCR as described above. EC50 curves were calculated using Graph Pad Prism 6 software using nonlinear fit (agonist) vs. response—variable slope (four parameters) calculation.

(170) The results are shown in Table 2. Potency and maximum activity of reduced permeability glucocorticoids (free payload) and the corresponding anti-CD74-drug conjugates on up-regulation of ZBTB16 mRNA levels in primary human B cells in vitro. Corresponding anti-RSV conjugates with 41, 47 and 50 were inactive at all doses tested.

(171) TABLE-US-00009 TABLE 2 ZBTB16 mRNA Max relative to free Compound ZBTB16 mRNA EC50 fluticasone Fluticasone propionate 0.019 nM   100% 3 1100 nM    75% 18 3200 nM  >77% αCD74-41 (DAR = 1.5) 0.70 ug/mL    22% (7.1 nM in payload) αCD74-47 (DAR = 1.6) 0.91 ug/mL    23% (9.8 nM in payload) αCD74-50 (DAR = 1.7) 0.13 ug/mL    16% (1.5 nM in payload)

(172) TABLE-US-00010 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 at N297 of IgG1 67 Glycosylation site QFNS at N297 of IgG4 68 Mutated glycosylation QAQS site of IgG1 or IgG4

(173) 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.