Phosphate based linkers for intracellular delivery of drug conjugates

Abstract

Phosphate-based linkers with tunable stability for intracellular delivery of drug conjugates are described. The phosphate-based linkers comprise a monophosphate, diphosphate, triphosphate, or tetraphosphate group (phosphate group) and a linker arm comprising a tuning element and optionally a spacer. A payload is covalently linked to the phosphate group at the distal end of the linker arm and the functional group at the proximal end of the linker arm is covalently linked to a cell-specific targeting ligand such as an antibody. These phosphate-based linkers have a differentiated and tunable stability in blood vs. an intracellular environment (e.g. lysosomal compartment).

Claims

1. A compound comprising the formula ##STR00082## wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; 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; a carbon atom linked to a trimethylammonium group by a C1-C5 hydrocarbon chain; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is selected from a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-30 hydrocarbon chain wherein one or more methylene units of Y are optionally and independently replaced by O, S, N(R), C(O), C(O)O, OC(O), N(R)C(O), C(O)N(R), S(O), S(O).sub.2, N(R)SO.sub.2, SO.sub.2N(R), a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, O, or S; Z is a cyclooctyne; D is an anti-inflammatory agent; 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.

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

3. The compound of claim 1, wherein the anti-inflammatory agent is Cortisol, cortisone acetate, beclometasone, prednisone, prednisolone, methylprednisolone, betamethasone, trimcinolone, budesonide, dexamethasone, fluticasone, fluticasone propionate, fluticasone furoate, compound 15-5, or mometasone.

4. The compound of claim 1, wherein the compound has a structure selected from the group consisting of ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a scheme for synthesis of ADC 12-1 and ADC 12-2.

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

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

(4) FIG. 4 is a graph showing deconvoluted intact mass spectra for in-vitro stability samples of ADC 12-1. incubated at 37 C. for: A) 1 hour, B) 8 hours, C) 14 days and D) 21 days.

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

(6) FIG. 6 is a graph showing deconvoluted intact mass spectra for the in vivo stability samples of ADC 12-1 from FIG. 5: A) sample B1 at 1 hour, B) sample H3 at 5 days, C) sample B7 at 1 hour and D) sample H9 at 5 days.

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

(8) FIG. 8 shows the activity of Her2-Duo-405 compared to that of a non-targeting antibody conjugated to Duo-405 on Her2 expressing SKBR3 cells.

(9) FIG. 9 shows the activity of Her2-Duo-405 on Her2 negative MDA-MD-468 cells.

DETAILED DESCRIPTION OF THE INVENTION

(10) The present invention provides phosphate-based linkers comprising a monophosphate, diphosphate, triphosphate, or tetraphosphate group and a linker arm comprising a tuning element, an optional spacer element, and a reactive functional group. The phosphate-based linkers have a distal end and a proximal end. The distal end of the phosphate-based linker comprises a monophosphate, diphosphate, or triphosphate group (phosphate group) linked to the distal end of the tuning element comprising the linker arm. The proximal end of the linker arm comprises a reactive functional group capable of reacting with a group on a ligand or targeting moiety to covalently link the phosphate-based linker to the ligand or targeting moiety. Interspersed between the tuning element and the reactive functional group of the linker arm may be an optional spacer element.

(11) In general, the phosphate-based linker is a compound that has the following formula (I)

(12) ##STR00021##
Wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; 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; a carbon atom linked to a trimethylammonium group by a C1-C5 hydrocarbon chain; nucleoside, protease sensitive group, cathepsin B sensitive group, or glycosidase sensitive group; Y is optional but when present it 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 of Y are optionally and independently replaced by O, S, N(R), C(O), C(O)O, OC(O), N(R)C(O), C(O)N(R), S(O), S(O).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, methyl ketone, azide, hydrazine, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadiene, wherein halo is iodine (I), bromine (Br), fluorine (F), or chlorine (Cl) and hetero is N, O, or S; 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.

(13) In particular aspect, the reactive group Z has the structure

(14) ##STR00022##
wherein the wavy line marks the bond between Z and Y, or X when Y is a covalent bond, or W when Y and X are a covalent bond.

(15) In a further embodiment, the distal end of the phosphate group is covalently linked to a payload, which may be a therapeutic agent such as a drug moiety or peptide, a radionuclide, or a protecting element, to provide a payload-phosphate-based linker compound comprising formula (II)

(16) ##STR00023##
Wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; 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; a carbon atom linked to a trimethylammonium group by a C1-C5 hydrocarbon chain; 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 of Y are optionally and independently replaced by O, S, N(R), C(O), C(O)O, OC(O), N(R)C(O), C(O)N(R), S(O), S(O).sub.2, N(R)SO.sub.2, SO.sub.2N(R), a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR.sub.2, O, or S; Z is a reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, methyl ketone, azide, hydrazine, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadiene, 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 particular aspect, the reactive group Z has the structure

(18) ##STR00024##
wherein the wavy line marks the covalent bond between Z and Y, or X when Y is a covalent bond, or W when Y and X are a covalent bond.

(19) In a further embodiment, the reactive functional group at the proximal end of the payload-phosphate-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.

(20) Such a compound comprises formula (III)

(21) ##STR00025##
Wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; 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; a carbon atom linked to a trimethylammonium group by a C1-C5 hydrocarbon chain; 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 of Y are optionally and independently replaced by O, S, N(R), C(O), C(O)O, OC(O), N(R)C(O), C(O)N(R), S(O), S(O).sub.2, N(R)SO.sub.2, SO.sub.2N(R), a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR.sub.2, O, or S; 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 azadiene, 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.

(22) In particular aspects, the linkage Z has the structure

(23) ##STR00026##
wherein the wavy lines mark the covalent bond between Z and Y, or X when Y is a covalent bond, or W when Y and X are a covalent bond (on the left) and Z and L (on the right), respectively.

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

(25) ##STR00027##
wherein wavy line 1 indicates the covalent attachment site to the payloand and wavy line 2 indicates the covalent attachment site of the cell-specific targeting ligand and wherein V is selected from O and S; W is selected from O, N, and CH.sub.2; 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; a carbon atom linked to a trimethylammonium group by a C1-C5 hydrocarbon chain; 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 of Y are optionally and independently replaced by O, S, N(R), C(O), C(O)O, OC(O), N(R)C(O), C(O)N(R), S(O), S(O).sub.2, N(R)SO.sub.2, SO.sub.2N(R), a heterocyclic group, an aryl group, or a heteroaryl group; T is an NR, CR.sub.2, O, or S of the 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 azadiene, 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.

(26) In particular aspects, the linkage Z has the structure

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

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

(29) 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); Blaittler 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.; Blittler 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.

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

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

(32) The phosphate-based linkers provide greater solubility relative to disulfide linkers, cathepsin B-cleavable linkers, esters and acid-sensitive linkers such as hydrazones. They enable the release of the payload in its parent or unadultered form unlike some of the alternative linkers, and may offer an improved blood/lysosome stability profile. Specifically, these phosphate-based linkers will provide superior blood stability relative to esters and disuflides. Phosphate-based linkers, following lysosomal cleavage will release an alcohol or amine-containing payload whereas the other linker formats may require self-immolative tethers to accomplish this or leave residual linker on the payload after lysosomal cleavage. The phosphate-based linker may have greater blood stability relative to the self-immolative cathepsin B linkers in the art, particularly when attached via the oxygen atom of a hydroxyl group of an alcohol-containing payload. The enzymatic hydrolysis of the phosphate linkage may be more rapid than the acid-hydrolysis of hyrdazones. The phosphate-based linkers disclosed herein minimize the propensity for conjugates comprising particular 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 phosphate-based linker disclosed herein did not produce detectable aggregates. Thus, the phosphate-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.

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

(34) Phosphate Group

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

(36) may be a phosphate ester

(37) ##STR00029##
pyrophosphate ester

(38) ##STR00030##
triphosphate ester

(39) ##STR00031##
or tetraphosphate ester

(40) ##STR00032##

(41) In further embodiments, the phosphate group may be a phosphoramidate

(42) ##STR00033##
pyrophosphoramidate

(43) ##STR00034##
triphosphophoramidate

(44) ##STR00035##
or tetraphosoramidate

(45) ##STR00036##
In further still embodiments, the phosphate group may be a phosphonate

(46) ##STR00037##
a diposphonate

(47) ##STR00038##
a phosphorthioate

(48) ##STR00039##
or a diphosphorthioate

(49) ##STR00040##

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

(51) Payload

(52) 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 phosphate-based linker comprising reactive functional group selected from the group consisting of N-hydroxysuccinimidyl ester, para-nitrophenyl carbonate, para-nitrophenyl carbamate, methyl ketone, azide, hydrazine, pentafluorophenyl, haloacetamide, maleimide, hydroxylamine, strained cycloalkyne, and heterocycloalkyne, alkyne, diene, azadiene, and heterocyclic azadiene, 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 phosphate group of the phosphate-based linker. 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.

(53) 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 phosphate 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.

(54) 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.

(55) 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 acidAE 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, Nacetyl spermidine, camptothecin, and their analogues.

(56) 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, compound 15-5, or mometasone.

(57) Linker Arm

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

(59) ##STR00041##

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

(61) ##STR00042##
wherein n=0-5, and wherein n=1-50, or 1-30, or 1-20, or 1-10. The wavy lines mark the covalent bond to the O, N, or CH.sub.2 of a phosphate group at the distal end (left) and the covalent bond to an atom of the functional reactive group on the proximal end (right), or optionally, a spacer element.

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

(63) ##STR00043## ##STR00044##
The wavy lines mark the covalent bond to the O, N, or CH.sub.2 of a phosphate group at the distal end (left) and the covalent bond to an atom of the functional reactive group on the proximal end (right), or optionally, a spacer element.

(64) 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 example, a cathepsin cleavage site having the structure

(65) ##STR00045##
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.

(66) The present invention further provides a compound comprising a glucocorticoid receptor agonist conjugated to a phosphate-based linker, wherein the compound has the structure of compound 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-4, 10-1, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, 19-5, 20-5, or 21-5. The structures of these compounds are shown in Examples 1-21.

(67) The present invention further provides antibody drug conjugates in which one or more of compounds 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-4, 10-1, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, 19-5, 20-5, or 21-5 is conjugated to the antibody. In particular embodiment, the antibody comprises one or more a non-natural amino acid having a reactive site capable of binding to compound 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-4, 10-1, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, 19-5, 20-5, or 21-5. In particular embodiments, the antibody comprises one or more para-azido-phenylalanine residues or para-acetyl-phenylalanine residues. In particular embodiments, the antibody-drug conjugate comprises compound 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 8-5, 9-4, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, or 19-5 conjugated to the azido group on a para-azido-phenylalanine residue within the antibody amino acid sequence. In particular embodiments, the antibody drug conjugate comprises compound 7-1 or 10-1 conjugated to a free thiol group within the amino acid sequence of the antibody, for example, the thio group of a cysteine residue, on the antibody. In particular embodiments, the antibody-drug conjugate comprises compound 20-5 or 21-5 conjugated to the methyl ketone group on a para-acetyl-phenylalanine residue within the antibody amino acid sequence

(68) The present invention further provides compositions comprising one or more antibody drug conjugates wherein at least one antibody drug conjugate is conjugated to compound 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-4, 10-1, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, 19-5, 20-5, or 21-5. In particular embodiment, the antibody comprises one or more a non-natural amino acid having a reactive site capable of binding to compound 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 7-1, 8-5, 9-4, 10-1, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, 19-5, 20-5, or 21-5.

(69) In particular embodiments, the antibody comprises one or more para-azido-phenylalanine residues or para-acetyl-phenylalanine residues. In particular embodiments, the antibody-drug conjugate comprises compound 1-4, 2-7, 3-4, 4-3, 5-3, 6-2, 8-5, 9-4, 11-5, 12-3, 13-7, 14-5, 15-5, 16-5, 17-5, 18-3, or 19-5 conjugated to the azido group on a para-azido-phenylalanine residue within the antibody amino acid sequence. In particular embodiments, the antibody drug conjugate comprises compound 7-1 or 10-1 conjugated to a free thiol group within the amino acid sequence of the antibody, for example, the thio group of a cysteine residue, on the antibody. In particular embodiments, the antibody-drug conjugate comprises compound 20-5 or 21-5 conjugated to the methyl ketone group on a para-acetyl-phenylalanine residue within the antibody amino acid sequence.

(70) Targeting Ligand

(71) The linker arm and payload of the invention 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, antibodies, 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), CD1 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 non-natural amino acids 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 in its entirety. Examples of non-natural amino acids include but are not limited to para-azido-phenylalanine and para-acetyl-phenylalanine.

(75) Pharmaceutical Formulations and Administration

(76) 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.

(77) 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.

(78) 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.

(79) 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.

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

(81) 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.

(82) 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.

(83) 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.

(84) 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.

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

(86) 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.

(87) 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.

(88) 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.

(89) 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.

(90) 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.

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

Example 1

(92) The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (1-4) was as follows.

(93) ##STR00046## ##STR00047##

Step A: 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl dihydrogen phosphate (1-1)

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

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

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

Step C: (9H-fluoren-9-yl)methyl (2-(((((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)ethyl)carbamate (1-3)

(96) To a stirred solution of 1-2 (0.15 g, 0.41 mmol) in DMF (1.2 mL) was added triethylamine (0.06 mL, 0.41 mmol) and CDI (0.17 g, 1.03 mmol). The resulting solution was stirred at room temperature for 30 minutes. To this mixture was added 1-1 (0.19 g, 0.41 mmol) and ZnCl.sub.2 (0.45 g, 3.31 mmol) and the mixture was allowed to stir at room temperature overnight. The reaction was diluted with 1 N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 5-35% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 1-3 as a solid (134 mg, 40%). LRMS (ES) (M+H).sup.+: observed=818.6, calculated=818.7.

Step D: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (1-4)

(97) To a stirred solution of 1-3 (0.19 g, 0.23 mmol) in DCM (3 mL) was added piperidine (0.15 mL, 1.51 mmol) and the resulting mixture was stirred at room temperature for 3 hrs. The solution was concentrated to dryness and redissolved in DCM (2 mL). In a separate vial a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.045 g, 0.25 mmol) in dichloromethane (1 mL) was added HOAT (0.034 g, 0.25 mmol), EDC (0.056 g, 0.30 mmol) and triethylamine (0.1 mL, 0.68 mmol). The resulting solution was stirred at room temperature for 40 minutes. The two solutions were combined and stirred at room temperature. Additional 2-(cyclooct-2-yn-1-yloxy)acetic acid activated with HOAT/EDC was added as necessary to complete reaction. Upon completion, the mixture was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 5-30% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 1-4 as a solid (59 mg, 34%). LRMS (ES) (M+H).sup.+: observed=760.6, calculated=760.7. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.64 (br s, 1H), 7.29 (d, J=9.15 Hz, 1H), 6.21 (dd, J=10.05, 1.93 Hz, 1H), 6.00 (s, 1H), 4.57 (d, J=8.3 Hz, 2H), 4.31 (t, J=5.4 Hz, 1H), 4.12 (d, J=11.22 Hz, 1H), 3.92 (dd, J=14.43, 8.59 Hz, 1H), 3.80-3.76 (complex, 3H), 3.30-3.16 (complex, 2H), 3.02-2.91 (complex, 2H), 2.63 (m, 1H), 2.40-2.19 (complex, 3H), 2.17-2.03 (complex, 4H), 1.96-1.82 (m, 3H), 1.80-1.72 (complex, 3H), 1.66-1.52 (complex, 4H), 1.50 (s, 3H), 1.40-1.31 (complex, 2H), 1.07-1.02 (m, 1H), 0.88 (s, 3H), 0.77 (d, J=7.17 Hz, 3H)

Example 2

(98) The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) trihydrogen triphosphate (2-7) was as follows.

(99) ##STR00048## ##STR00049## ##STR00050##

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

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

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

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

Step C: ((9H-fluoren-9-yl)methyl) (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (2-3)

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

Step D: 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl trihydrogen diphosphate (2-4)

(103) To a stirred solution of 2-3 (0.29 g, 0.39 mmol) in DCM (2 mL) was added piperidine (0.23 mL, 2.36 mmol) and the resulting mixture was stirred at room temperature for 80 minutes. The solution was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 3-25% MeCN/water w/0.10% NH.sub.4OH modifier over 20 min) gave 2-4 as a solid (123 mg, 56%). LRMS (ES) (M+H).sup.+: observed=553.2, calculated=552.4.

Step E: (9H-fluoren-9-yl)methyl (2-(((((((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)ethyl)carbamate (2-5)

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

Step F: 2-aminoethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) trihydrogen triphosphate (2-6)

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

Step G: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) trihydrogen triphosphate (2-7)

(106) To a stirred solution of 2-6 (0.027 g, 0.04 mmol) in DMF (0.8 mL) was added triethylamine (0.02 mL, 0.16 mmol) and 2-1 and the resulting mixture was stirred at room temperature for 30 minutes. The solution was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 5-40% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 2-7 as a solid (8 mg, 24%). .sup.1H NMR (499 MHz, DMSO): 0.78 (d, J=7.1 Hz, 3H); 0.88 (s, 3H); 1.10-0.99 (complex, 1H); 1.44-1.28 (complex, 2H); 1.50 (s, 3H); 1.67-1.52 (complex, 4H); 1.80-1.67 (complex, 3H); 1.93-1.83 (m, 3H); 2.17-2.01 (complex, 3H); 2.42-2.17 (complex, 3H); 2.67-2.57 (complex, 1H); 2.81 (d, J=79.7 Hz, 1H); 3.02-2.91 (complex, 1H); 3.17 (s, 1H); 3.26-3.21 (complex, 2H); 3.84-3.74 (complex, 2H); 3.91 (d, J=14.5 Hz, 1H); 4.15 (d, J=11.4 Hz, 1H); 4.31 (t, J=5.1 Hz, 1H); 4.57 (dd, J=18.0, 8.1 Hz, 1H); 4.71 (dd, J=17.9, 7.1 Hz, 1H); 5.99 (s, 1H); 6.20 (d, J=10.1 Hz, 1H); 7.30 (d, J=10.6 Hz, 1H); 8.48 (s, 1H). LRMS (ES) (M+H).sup.+: observed=840.4, calculated=839.6.

Example 3

(107) The synthesis of dexamethasone linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (3-4) was as follows.

(108) ##STR00051## ##STR00052##

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

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

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

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

Step C: (9H-fluoren-9-yl)methyl (4-(((((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)phenyl)carbamate (3-3)

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

Step D: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (3-4)

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

Example 4

(113) The synthesis of dexamethasone linker 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (4-3) was as follows.

(114) ##STR00053##

Step A: tert-butyl (2-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)ethyl)carbamate (4-1)

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

Step B: 2-aminoethyl (2-((8S,9R,10,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate, HCl (4-2)

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

Step C: 2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (4-3)

(117) To a stirred solution of 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.036 g, 0.20 mmol) in dichloromethane (1 mL) was added HOAT (0.027 g, 0.20 mmol), EDC (0.041 g, 0.22 mmol) and triethylamine (0.05 mL, 0.36 mmol). The resulting solution was stirred at room temperature for 40 minutes. This solution was added to 4-2 (0.10 g, 0.18 mmol) in DCM (1 mL). Additional 2-(cyclooct-2-yn-1-yloxy)acetic acid activated with HOAT/EDC was added as necessary to complete reaction. Upon completion, the mixture was concentrated and reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) gave 4-3 as a solid (45 mg, 37%). LRMS (ES) (M+H).sup.+: observed=680.6, calculated=680.7. .sup.1H NMR (400 MHz, DMSO-d.sub.6): 8.05 (br t, J=5.6 Hz, 1H), 7.30 (d, J=10.15 Hz, 1H), 6.21 (d, J=10.12 Hz, 1H), 6.00 (s, 1H), 5.55 (s, 1H), 5.37 (s, 1H), 4.70 (dd, J=17.2, 6.7 Hz, 1H), 4.29 (br t, J=6.4 Hz, 1H), 4.18-4.10 (complex, 2H), 3.87 (d, J=14.8 Hz, 1H), 3.74 (d, J=14.8 Hz, 1H), 3.69-3.64 (complex, 2H), 3.27-3.18 (complex, 2H), 2.91 (m, 1H), 2.61 (m, 1H), 2.39-2.05 (complex, 7H), 1.97-1.83 (complex, 2H), 1.80-1.69 (complex, 3H), 1.64-1.52 (complex, 3H), 1.48 (s, 3H), 1.47-1.30 (complex, 3H), 1.05 (m, 1H), 0.85 (s, 3H), 0.76 (d, J=7.13 Hz, 3H).

Example 5

(118) The synthesis of dexamethasone linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (5-3) was as follows.

(119) ##STR00054##

Step A: tert-butyl (4-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)phenyl)carbamate (5-1)

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

Step B: 4-aminophenyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate, HCl (5-2)

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

Step C: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)phenyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) hydrogen phosphate (5-3)

(122) The title compound was prepared from 5-2 according to the protocol outlined in Example 4 to produce 4-3 to afford 5-3. LRMS (ES) (M+H).sup.+: observed=728.6, calculated=728.7 .sup.1H NMR (400 MHz, DMSO-d.sub.6): 9.53 (s, 1H), 7.43 (d, J=8.54 Hz, 2H), 7.30 (d, J=10.14 Hz, 1H), 7.04 (d, J=8.54 Hz, 2H), 6.22 (dd, J=10.08, 1.92 Hz, 1H), 6.00 (s, 1H), 5.41 (s, 1H), 5.37 (d, J=4.22 Hz, 1H), 4.78 (dd, J=17.50, 6.20 Hz, 1H), 4.37 (t, J=5.59 Hz, 1H), 4.27 (dd, J=17.47, 9.02, 1H), 4.15-4.12 (m, 1H), 4.06 (d, J=14.53 Hz, 1H), 3.94 (d, J=14.59 Hz, 1H), 3.05 (q, J=7.26 Hz, 1H), 2.92 (m, 1H), 2.61 (m, 1H), 2.40-2.06 (complex, 7H), 1.98 (m, 1H), 1.87 (m, 1H), 1.82-1.73 (complex, 3H), 1.67-1.52 (complex, 3H), 1.48 (s, 3H), 1.43 (d, J=13.54 Hz, 2H), 1.35 (m, 1H), 1.05 (m, 1H), 0.84 (s, 3H), 0.76 (d, J=7.14 Hz, 3H).

Example 6

(123) The synthesis of dexamethasone linker 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl hydrogen (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphoramidate (6-2) was as follows.

(124) ##STR00055##

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

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

Step B: 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl hydrogen (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)phosphoramidate (6-2)

(126) To a stirred solution of 6-1 (0.05 g, 0.22 mmol) and 1-1 (0.035 g, 0.074 mmol) in a solution of t-butanol (1.2 mL) and water (0.25 mL) was added DCC (0.06 g, 0.30 mmol) and the resulting mixture was heated to 100 C for 4 hr. The reaction mixture was allowed to cool and concentrated. The residue was dissolved in a 1:1:1 MeOH:water:MeCN solution and syringe filtered. The mixture was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 5-45% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 6-2 as a solid (15 mg, 30%). LRMS (ES) (M+H).sup.+: observed=679.5, calculated=678.7.

Example 7

(127) The synthesis of dexamethasone linker 2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)ethyl (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) dihydrogen pyrophosphate (7-1) was as follows.

(128) ##STR00056##

(129) To a stirred solution of 1-3 (0.19 g, 0.23 mmol) in DCM (3 mL) was added piperidine (0.15 mL, 1.51 mmol) and the resulting mixture was stirred at room temperature for 3 hrs. The solution was concentrated to dryness. The crude mixture was taken into a 2:1:1 methanol:acetonitrile:water mixture and filtered. The filtrate was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 5-35% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min). A portion of the resulting purified amine (0.07 g, 0.11 mmol) was dissolved in DMF (0.8 mL). To this solution was added 2,5-dioxopyrrolidin-1-yl 3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoate (0.09 mg, 0.34 mmol) and DMAP (0.014 g, 0.11 mmol) and the resulting solution was stirred 20 minutes. The crude reaction mixture was directly purified using reverse phase preparative chromatography (Sunfire Prep C18 OBD 5 um 30150 mm; 10-35% CH3CN/water w/0.1% TFA modifier over 20 min) gave 7-1 as a solid (13 mg, 15%). LRMS (ES) (M+H).sup.+: observed=747.2, calculated=746.6.

Example 8

(130) The synthesis of dexamethasone linker ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3-(((2-((8S,9R,10S,11S,13 S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)(hydroxy)phosphoryl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)carbamate (8-5) was as follows.

(131) ##STR00057## ##STR00058##

Step A: (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl (2-cyanoethyl) (2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl)phosphite (8-1)

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

Step B: (2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-2-(hydroxymethyl)tetrahydrofuran-3-yl (2-cyanoethyl) (2-((8S,9R,10S,11S,13S,14S,16R,1.7R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl)phosphate (8-2)

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

Step C: ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methyl (4-nitrophenyl) carbonate (8-3)

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

Step D: ((2R,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)(hydroxy)phosphoryl)oxy)tetrahydrofuran-2-yl)methyl (2-aminoethyl)carbamate (8-4)

(135) To a stirred solution of ethylenediamine (0.28 mL, 4.17 mmol) in DMF (1 mL) was added a solution of 8-3 (0.20 g, 0.20 mmol) in DMF (1 mL) dropwise. The reaction was stirred at room temperature for 10 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 8-4 as a solid (115 mg, 61%). LRMS (ES) (M+H).sup.+: observed=899.5, calculated=898.9.

Step E: ((2R,3S,4R,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1 (2H)-yl)-3-(((2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy) (hydroxy)phosphoryl)oxy)-4-hydroxytetrahydrofuran-2-yl)methyl (2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethyl)carbamate (8-5)

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

Example 9

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

(138) ##STR00059## ##STR00060##

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

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

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

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

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

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

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

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

Example 10

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

(144) ##STR00061##

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

Example 11

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

(147) ##STR00062## ##STR00063##

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

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

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

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

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

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

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

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

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

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

Example 12

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

(154) ##STR00064## ##STR00065##

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

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

Step B: 4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl (2((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2,14,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) hydrogen phosphate (12-2)

(156) To a stirred solution of 12-1 (0.10 g, 0.09 mmol) in DCM (1.8 mL) was added DBU (0.05 mL, 0.35 mmol) and the resulting solution was stirred 20 minutes at room temperature. The reaction was concentrated and dissolved in a 2:1:1 methanol:water:acetonitrile mixture and syringe filtered. The filtrate was purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 12-2 as a solid (42 mg, 53%). LRMS (ES) (M+H)+: observed=872.6, calculated=871.9.

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

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

Example 13

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

(159) ##STR00066## ##STR00067##

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

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

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

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

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

(162) To a stirred solution of 13-2 (0.55 g, 1.03 mmol) and imidazole (0.35 g, 5.13 mmol) in pyridine (8.0 mL) at room temperature was added TMS-Cl (1.31 mL, 10.25 mmol) and the resulting solution was stirred for 10 minutes. To this mixture was added iodine (0.52 g, 2.05 mmol) dissolved in pyridine (2 mL) and stirred room temperature for 50 minutes. The reaction was then cooled in and ice bath and quenched with water (0.5 mL). The reaction was concentrated, dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-3 as a solid (282 mg, 45%). LRMS (ES) (M+H).sup.+: observed=603.4, calculated=602.6.

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

(163) To a stirred solution of 13-3 (0.20 g, 0.33 mmol) and 1-2 (0.12 g, 0.33 mmol) in DMF (1.4 mL) was added ZnCl.sub.2 (0.36 g, 2.66 mmol) and the mixture was allowed to stir at room temperature overnight. The reaction was diluted with 1 N HCl and extracted several times with ethyl acetate. The combined organic layers were concentrated, dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-4 as a solid (166 mg, 55%). LRMS (ES) (M+H).sup.+: observed=898.4, calculated=897.8.

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

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

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

(165) To a stirred solution of 13-5 (0.037 g, 0.055 mmol) and 2-(cyclooct-2-yn-1-yloxy)acetic acid (0.032 g, 0.175 mmol) in DMF (0.8 mL) was added HATU (0.066 g, 0.175 mmol) and triethylamine (0.03 mL, 0.22 mmol). The reaction was stirred at room temperature for 20 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-6 as a solid (36 mg, 78%). LRMS (ES) (M+H).sup.+ observed=840.5, calculated=839.8.

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

(166) To a stirred solution of 13-6 (0.035 mg, 0.042 mmol) in methanol (0.50 mL) was added 70% perchloric acid (7.2 L, 0.083 mmol) and the resulting solution was stirred room temperature overnight. The reaction was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 13-7 as a solid (16 mg, 47%). LRMS (ES) (M+H).sup.+: observed=798.4, calculated=797.7.

Example 14

(167) The synthesis of Fluticasone proprionate linker (6S,8S,9R,10S,11S,13 S,14S,16R,17R)-11-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-5) was as follows.

(168) ##STR00068## ##STR00069##

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

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

Step B: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-((fluoromethyl)thio)carbonyl)-11-((hydroxy(1H-imidazol-1-yl)phosphoryl)oxy)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-2)

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

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

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

Step D: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-aminoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-4)

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

Step F: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-(((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (14-5)

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

Example 15

(174) The synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-5) was as follows.

(175) ##STR00070## ##STR00071##

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

(176) To a stirred solution of fluticasone propionate (0.50 g, 1.00 mmol) in MeCN (5.0 mL) at 0 C. was added dimethyl sulfide (0.59 mL, 8.00 mmol) followed by benzoyl peroxide (0.97 g, 4.00 mmol) added in four portions over 20 minutes. The resulting mixture was stirred at 0 C. for 1 hour. The reaction was concentrated, taken up in ethyl acetate and washed with saturated sodium bicarbonate. The combined organic phase was concentrated. The crude was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 40-80% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give to give 15-1 as a solid (0.07 g, 12.7%). LRMS (ES) (M+H).sup.+: observed=561.3, calculated=560.6.

Step B: (6S,8S,9R,10S,11 S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl) thio)carbonyl)-10,13,16-trimethyl-3-oxo-1-((phosphonooxy)methoxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-2)

(177) Phosphoric acid (0.09 g, 0.89 mmol) was heated under nitrogen at 120 C. for 30 minutes. This was allowed to cool and to it was added molecular sieves and 15-1 (0.07 g, 0.13 mmol). This mixture was dissolved in THF (1.3 mL) and NIS (0.04 g, 0.19 mmol) was added. The resulting solution was allowed to stir overnight at room temperature. The mixture was filtered and concentrated. The crude was purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give to give 15-2 as a solid (0.05 g, 63%). LRMS (ES) (M+H).sup.+: observed=611.3, calculated=610.5.

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

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

Step D: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-aminoethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (15-4)

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

Step E: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((2-(2-(cyclooct-2-yn-1-yloxy)acetamido)ethoxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)methoxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (15-5)

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

Example 16

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

(182) ##STR00072## ##STR00073## ##STR00074##

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

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

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

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

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

(185) The title compound was prepared from 16-2 and 9-1 according to the protocol outlined in Example 1 to produce 1-3 to afford 16-3. LRMS (ES) (M+H).sup.+: observed=1886.7, calculated=1869.0 Step D: 72-amino-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69-tricosaoxahenheptacontanyl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2,1:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl) dihydrogen pyrophosphate (16-4)

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

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

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

Example 17

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

(189) ##STR00075##

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

(190) To a stirred solution of (9H-fluoren-9-yl)methyl ((S)-1-(((S)-1-((4(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (0.05 g, 0.083 mmol) in DMF (1.2 mL) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (29 uL, 0.09 mmol). To this mixture was added 0.45M tetrazole in acetonitrile (0.20 mL, 0.09 mmol) dropwise and the resulting mixture was stirred for 20 minutes at room temperature. To this was added 9-1 (0.042 g, 0.083 mmol) and 5-(ethylthio)-1H-tetrazole (0.02 g, 0.16 mmol) and allowed to stir to 30 minutes at room temperature. To this was added 6 M tertbutyl hydroperoxide in decane (0.03 mL, 0.18 mmol) and allowed to stir at room temperature for 30 minutes. To this was added DBU (0.12 mL, 0.83 mmol) and the resulting solution was stirred overnight at room temperature. The crude reaction was purified by direct injection using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 uM 30100 mm; 5-40% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 17-1 as a solid (18.5 mg, 23%). LRMS (ES) (M+H).sup.+: observed=952.6, calculated=951.9.

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

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

Example 18

(192) The synthesis of Fluticasone linker (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)(hydroxy)phosphoryl)oxy)(hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (18-3) was as follows.

(193) ##STR00076## ##STR00077##

Step A: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-11-(phosphonooxy)-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (18-1)

(194) To a stirred solution of 14-1 (0.20 g, 0.35 mmol) in pyridine (3.0 mL) at room temperature was added TMS-Cl (0.45 mL, 3.54 mmol) and the resulting solution was stirred for 10 minutes. To this mixture was added iodine (0.10 g, 0.42 mmol) dissolved in pyridine (0.5 mL) and stirred room temperature for 10 minutes. The reaction was then cooled in an ice bath and quenched with water (0.5 mL) and allowed to stir overnight at room temperature. The reaction was partitioned between ethyl acetate and 1N HCl. The organic phase was concentrated and dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 5-40% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 18-1 as a solid (89 mg, 44%). LRMS (ES) (M+H).sup.+: observed=581.3, calculated=580.5.

Step B: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido) benzyl)oxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl propionate (18-2)

(195) The title compound was prepared from 18-1 according to the protocol outlined in Example 17 to produce 17-1 to afford 18-2. LRMS (ES) (M+H).sup.+: observed=1022.6, calculated=1021.9.

Step C: (6S,8S,9R,10S,11S,13S,14S,16R,17R)-11-((((((4-((2S)-2-((2S)-2-(2-(cyclooct-2-yn-1-yloxy)acetamido)-3-methylbutanamido)-5-ureidopentanamido) benzyl)oxy) (hydroxy)phosphoryl)oxy) (hydroxy)phosphoryl)oxy)-6,9-difluoro-17-(((fluoromethyl)thio)carbonyl)-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-ylpropionate (18-3)

(196) The title compound was prepared from 18-2 according to the protocol outlined in Example 13 to produce 13-6 to afford 18-3. LRMS (ES) (M+H).sup.+: observed=1186.5, calculated=1186.1.

Example 19

(197) The synthesis of Budesonide linker 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)-1-(trimethylammonio)butan-2-yl (2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b, 11a,12,12a, 12b-dodecahydro-1H-naphtho[2,1:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl)phosphate (19-5) was as follows.

(198) ##STR00078## ##STR00079##

Step A: tert-butyl (2-(oxiran-2-yl)ethyl)carbamate (19-1)

(199) To a stirred solution of tert-butyl but-3-enylcarbamate (0.95 mL, 5.16 mmol) in chloroform (20 mL) chilled in an ice bath was added mCBPA (1.38 g, 8.0 mmol) and the resulting solution was stirred for 30 minutes. The reaction was allowed to warm to room temperature and monitored by TLC. Additional 1.5 equivalents of mCPBA was added every 30 minutes until complete. The mixture was diluted with additional 50 mL chloroform and washed with 10% aq sodium sulfite solution 3 times, and washed once with brine. The organic phase as dried over sodium sulfate and concentrated. The crude mixture was purified with flash column separation using a 0-50% ethyl acetate/hexane gradient to give 19-1 (800 mg, 83%). LRMS (ES) (M+H).sup.+: observed=188.0, calculated=187.2.

Step B: tert-butyl (4-(dimethylamino)-3-hydroxybutyl)carbamate (19-2)

(200) Compound 19-1 (0.79 g, 4.21 mmol) was dissolved in a solution of dimethylamine 2M (8.0 mL, 16.0 mmol) and microwave irradiated to 60 C. for 30 minutes. The reaction was allowed to cool and concentrated. The mixture was dissolved in aqueous acetonitrile and purified using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 10-50% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 19-2 (528 mg, 54%). LRMS (ES) (M+H).sup.+: observed=234.2, calculated=232.3.

Step C: 4-((tert-butoxycarbonyl)amino)-2-hydroxy-N,N,N-trimethylbutan-1-aminium (19-3)

(201) To a stirred solution of 9-2 (0.48 g, 2.05 mmol) in acetonitrile (9.5 mL) was added methyl iodide (0.13 mL, 2.05 mmol) and the resulting solution was stirred at room temperature for 1 hour and concentrated to give 19-3 as a white hydroscopic solid (509 mg, 100%). LRMS (ES) (M+H).sup.+: observed=248.3, calculated=247.3.

Step D: 4-((tert-butoxycarbonyl)amino)-2-((hydroxy(2-((6aR,6bS,7S,8aS,8bS,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2,1:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethoxy)phosphoryl)oxy)-N,N,N-trimethylbutan-1-aminium (19-4)

(202) The title compound was prepared from 19-3 and Budesonide according to the protocol outlined in Example 17 to produce 17-1 to afford 19-4. LRMS (ES) (M+H).sup.+: observed=739.5, calculated=739.8.

Step E: 4-(2-(cyclooct-2-yn-1-yloxy)acetamido)-1-(trimethylammonio)butan-2-yl (2((6aR,6bS,7S,8aS,8bS,11 aR, 12aS,12bS)-7-hydroxy-6a, 8a-dimethyl-4-oxo-10-propyl-2,4,6a,6b,7,8,8a, 8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2,1:4,5]indeno[1,2-d][1,3]dioxol-8b-yl)-2-oxoethyl)phosphate (19-5)

(203) To a stirred solution of 19-4 (0.03 g, 0.04 mmol) in dichloromethane (0.6 mL) in an ice bath was added TFA (0.3 mL, 3.89 mmol) and the resulting solution was allowed to stir 1 hour and concentrated. The resulting crude material was dissolved in DMF (0.4 mL) and 2-(cyclooct-2-yn-1-yloxy)acetic acid (9.6 mg, 0.053 mmol) and HATU (0.022 g, 0.057 mmol) and triethylamine (0.04 mL, 0.28 mmol) was added. The reaction was stirred at room temperature for 20 minutes, then purified directly using reverse phase preparative chromatography (Phenomenex Gemini-NX C18 OBD 5 M 30100 mm; 10-60% MeCN/water w/0.1% NH.sub.4OH modifier over 20 min) to give 19-5 as a solid (22 mg, 67%). LRMS (ES) (M+H).sup.+: observed=803.6, calculated=802.9.

Example 20

(204) This example shows the synthesis of a duocarmycin analog comprising a diphosphate linker.

(205) ##STR00080##

Step A: (9H-fluoren-9-yl)methyl 2-((((S)-1-(chloromethyl)-3-(5, 6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)ethylcarbamate (20-2)

(206) (S)-(1-(chloromethyl)-5-hydroxy-1H-benzo[e]indol-3(2H)-yl)(5,6,7-trimethoxy-1H-indol-2-yl)methanone (20-1) (60.0 mg, 0.13 mmol) was dissolved in dichloromethane (5 mL) and treated with phosphoryl chloride (36 L, 0.39 mmol) and diisopropylethylamine (67 L, 0.39 mmol) at 0 C. for 5 min. The reaction mixture was cooled down to 30 C. and (9H-fluoren-9-yl)methyl 2-(phosphonooxy)ethylcarbamate (234 mg, 0.64 mmol) was then added followed by another portion of diisopropylethylamine (115 L, 0.64 mmol). The reaction was kept at 30 C. for 30 min then allowed to warm up to 0 C. for 40 min. Additional amount of 1.0 eq of (9H-fluoren-9-yl)methyl 2-(phosphonooxy)ethylcarbamate (46.8 mg, 0.13 mmol) and diisopropylethylamine (22 L, 0.13 mmol) were added and kept for another 10 min at 0 C. The reaction was quenched with sodium phosphate monobasic (1M) at 4 C. overnight and purified by RP-HPLC (Phenomenex Gemini-NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to give the compound 20-2 as white powder (55 mg) with little impurity which is used directly for the next step without further purification. MS m/z 892 (M+H).sup.+.

Step B: 2-((((S)-1-(chloromethyl)-3-(5, 6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)ethylamine (20-3)

(207) Compound 20-2 (55 mg from last step) was dissolved in DMF (4.5 mL) and treated with diethylamine (0.6 ml) at rt for 10 min and immediately purified by RP-HPLC (Phenomenex Gemini NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) and lyophilized to obtain a white powder as TFA salt of compound 20-3 (24 mg, 24% from compound 20-1). .sup.1H-NMR (500 MHz, CDCl.sub.3/MeOH-d4) 8.52 (bs, 1H), 8.32 (d, J=8.5 Hz, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.56 (t, J=7.0 Hz, 1H), 7.45 (t, J=7.0 Hz, 1H), 7.08 (s, 1H), 6.95 (s, 1H), 4.73-4.72 (m, 2H), 4.28-4.20 (m, 3H), 4.04 (s, 3H), 3.99-3.96 (m, 1H), 3.90 (3, 3H), 3.89 (s, 3H), 3.69-3.65 (m, 1H), 3.21 (m, 2H). MS m/z 670 (M+H).sup.+.

Step C: tert-Butyl 2-(2-((((S)-1-(chloromethyl)-3-(5, 6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-1H-benzo[e]indol-5-yloxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)ethylamino)-2-oxoethoxycarbamate (20-4)

(208) A solution of 20-3 mono TFA salt (10 mg, 0.0128 mmol) in DMF (0.5 mL) was treated with 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylaminooxy)acetate (7.35 mg, 0.0256 mmol) and diisopropylethyl amine (8.9 L, 0.0512 mmol) at 0 C. for 10 min. The reaction mixture was purified by RP-HPLC (Phenomenex Gemini-NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to obtain a white powder as compound 20-4 (8.6 mg, 80%). MS m/z 843 (M+H).sup.+.

Step D: 2-(Aminooxy)-N-(2-((((S)-1-(chloromethyl)-3-(5,6,7-trimethoxy-1H-indole-2-carbonyl)-2,3-dihydro-H-benzo[e]indol-5-yloxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)ethyl)-acetamide (20-5)

(209) Compound 20-4 (8.5 mg, 0.010 mmol) was dissolved in dichloromethane (0.5 mL) and treated with TFA (0.1 mL) at rt for 15 min. The reaction mixture was concentrated in vacuum and co-evaporated with toluene (0.5 mL2). The residue was purified by RP-HPLC (Phenomenex Gemini-NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to obtain the target compound 20-5 as TFA salt (3.7 mg, 50%). .sup.1H-NMR (500 MHz, MeOH-d4) 8.53 (d, J=13.5 Hz, 1H), 8.3 (t, J=7.5 Hz, 1H), 7.84 (d, J=8.5 Hz, 1H), 7.60-7.57 (m, 1H), 7.47 (t, J=7.5 Hz, 1H), 7.12 (d, J=12.0 Hz, 1H), 6.99 (d, J=2.0 Hz, 1H), 4.76-4.74 (m, 2H), 4.58 (s, 1H), 4.37-4.34 (m, 1H), 4.25 (m, 1H), 4.20-4.14 (m, 2H), 4.06 (d, J=6.0 Hz, 3H), 4.02-4.00 (m, 1H), 3.90 (s, 6H), 3.76-3.70 (m, 1H), 3.45-3.44 (m, 2H). MS m/z 743 (M+H).sup.+.

Example 21

(210) This example shows the synthesis of duocarmycin comprising a diphosphate linker.

(211) ##STR00081##

Step A: (S)-methyl 8-(chloromethyl)-4-(phosphonooxy)-6-(5, 6,7-trimethoxy-1H-indole-2-carbonyl)-3, 6,7,8-tetrahydropyrrolo[3,2-e]indole-2-carboxylate (21-2)

(212) Duocarmycin 21-1 (85.0 mg, 0.165 mmol) was dissolved in THF/acetonitrile (3 mL/3 mL) and treated with phosphoryl chloride (0.139 mL, 1.49 mmol) and diisopropylethyl amine (0.144 mL, 0.825 mmol) with ice-water bath cooling. The reaction was kept at the same temperature for 1 h and sodium phosphate monobasic solution (1M, 10 mL) was added and kept the mixture at 4 C. overnight. The mixture was then subjected to the RP-HPLC purification (Phenomenex Gemini-NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to obtain compound 21-2 (55 mg, %). MS m/z 594 (M+H).sup.+.

Step B: (8S)-methyl 4-(((2-aminoethoxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)-8-(chloromethyl)-6-(5,6, 7-trimethoxy-1H-indole-2-carbonyl)-3, 6,7, 8-tetrahydropyrrolo[3, 2-e]indole-2-carboxylate (21-3)

(213) A solution of (9H-fluoren-9-yl)methyl 2-(phosphonooxy)ethylcarbamate (68.0 mg, 0.185 mmol) in DMF (1.5 mL) was treated with carbonyldiimidazole (90.0 mg, 0.558 mmol) and triethylamine (25.0 L, 0.186 mmol) at room temperature for 3 h. A drop of MeOH was added and stirred at room temperature for 10 min. The volatile was completely removed and co-evaporated with toluene. The residue was dissolved in DMF (1.85 mL) and compound 21-2 (55.0 mg, 0.093 mmol) was added. The mixture was kept room temperature for 19 h and then treated with diethylamine at room temperature for 4 min. The reaction was immediately purified by RP-HPLC (Phenomenex Gemini NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to obtain compound 21-3 TFA salt (19 mg, 14%). MS m/z 717 (M+H).sup.+.

Step C: (8S)-methyl 8-(chloromethyl)-4-(((2, 2-dimethyl-4, 8-dioxo-3, 6-dioxa-5, 9-diazaundecan-11-yloxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)-6-(5,6, 7-trimethoxy-1H-indole-2-carbonyl)-3, 6,7, 8-tetrahydropyrrolo[3, 2-e]indole-2-carboxylate (21-4)

(214) A solution of 21-3 (16 mg, 0.019 mmol) in dichloromethane (2 mL) was treated with 2,5-dioxopyrrolidin-1-yl 2-(tert-butoxycarbonylaminooxy)acetate (11.0 mg, 0.038 mmol) and triethylamine (10.1 L, 0.076 mmol) at 0 C. for 10 min. The reaction mixture directly purified by RP-HPLC (Phenomenex Gemini-NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to obtain a white powder 21-4 (14 mg, 83%). MS m/z 890 (M+H).sup.+.

Step D: (8S)-methyl 4-(((2-(2-(aminooxy)acetamido)ethoxy) (hydroxy)phosphoryloxy) (hydroxy)phosphoryloxy)-8-(chloromethyl)-6-(5, 6, 7-trimethoxy-1H-indole-2-carbonyl)-3, 6, 7, 8-tetrahydropyrrolo[3, 2-e]indole-2-carboxylate (21-5)

(215) Compound 21-4 (14 mg, 0.0157 mmol) was dissolved in dichloromethane (1 mL) and treated with TFA (0.2 mL) for 20 min. The reaction mixture was concentrated in vacuum and co-evaporated with toluene (0.5 mL2). The residue was purified by RP-HPLC (Phenomenex Gemini-NX C18 5 m 10030 mm; MeCN/water with 0.05% TFA) to obtain the target compound 21-5 (Duocarmycin-405) as TFA salt (5.5 mg, 39%). .sup.1H-NMR (500 MHz, CDCl.sub.3/MeOH-d4) 8.28 (br, 1H), 7.21 (s, 1H), 7.02 (s, 1H), 6.95 (s, 1H), 4.77 (2H buried in solvent peak), 4.58 (bs, 1H), 4.39 (m, 1H), 4.06-3.75 (m, 11H), 3.5 (m, 2H). MS m/z 790 (M+H).sup.+.

Example 22

(216) The solubility of exemplary drug-linker conjugates in aqueous solutions was evaluated. Linkers utilized in drug conjugates may have aqueous solubility to enable conjugation in aqueous media amenable to protein solubilization. Furthermore, linkers with aqueous solubility are considered hydrophilic, and may confer to the drug conjugate a reduced propensity to aggregate relative to standard hydrophobic linkers in the literature. The following exemplary drug-linkers were tested in an aqueous solution comprising 20% acetonitrile (MeCN/H.sub.2O) for solubility at a concentration of 10 mg/mL. As shown in Table 1, the exemplary linker-drug conjugates displayed high solubility, which may be a result of the contribution of the polarity and charge of the phosphate containing linker to the drug-linker conjugate.

(217) TABLE-US-00001 TABLE 1 Measured Solubility in Drug Linker 20% MeCN/H.sub.2O 1-4 >10 mg/mL 2-7 >10 mg/mL 3-4 >10 mg/mL 4-3 >10 mg/mL 5-3 >10 mg/mL

Example 23

(218) In Vitro Stability Studies of Exemplary Drug-Linkers in Blood and Lysosomal Lysates.

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

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

(221) TABLE-US-00002 TABLE 1 Time Course of Calculated Conc. for each compound in human whole blood (nM) Dexa- Ma- meth- trix Time asone 4-3 5-3 1.4 3-4 2-7 Matrix 2939 2821 n/a 3059 n/a 1059 spiking 0 m 1687 1539 1299 1617 1382 1040 5 m 1251 1325 10 m 15 m 1276 1398 20 m 2409 1861 1390 30 m 1111 1250 1 hr 2224 1619 937 1421 1141 1515 2 hr 1480 1703 3 hr 1502 1677 1899 6 hr 2792 2040 1400 123

(222) TABLE-US-00003 TABLE 2 Time Course of Calculated Conc. for dexamethasone in human whole blood (nM) Dexa- Ma- meth- trix Time asone 4-3 15-3 1.4 3-4 2-7 Matrix 2939 4 0 2 0 0 spiking 0 m 1687 0 0 0 0 0 5 m 0 0 10 m 15 m 0 0 20 m 2409 0 0 30 m 0 0 1 hr 2224 0 0 0 0 0 2 hr 0 0 3 hr 1502 1 0 6 hr 2792 3 0 0
General Experimental Procedure
Human Blood Incubation

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

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

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

(226) TABLE-US-00004 TABLE 3 Time Course of Calculated Conc. for each compound in rat lysosomal lysate (nM) Dexa- Ma- meth- trix Time asone 4-3 5-3 1.4 3-4 2-7 Matrix 2059.0 1590.7 2555.29 2487.3 3128.83 2228 spiking 0 m 1456.4 888.8 1394.08 1148.2 1252 1327 5 m 1851.2 1156.1 1295.86 158.2 501.28 56 10 m 1640.8 983.4 1278.4 19.0 149.18 512 15 m 1666.7 1032.6 1407.73 3.7 N/A 0 30 m 1602.6 980.1 1175.39 N/A N/A 37 1 hr 1576.8 882.4 970.65 N/A N/A 0 2 hr 1681.0 880.1 743.45 N/A N/A 0 3 hr 1689.7 903.8 500.41 N/A N/A 0 6 hr 1689.8 858.9 221.03 N/A N/A 0

(227) TABLE-US-00005 TABLE 4 Time Course of Calculated Conc. for dexamethasone in rat lysosomal lysate (nM) Ma- Dexameth- trix Time asone 4-3 5-3 1.4 3-4 2-7 Matrix 2059.0 N/A N/A N/A N/A 17 spiking 0 m 1456.4 0.9 N/A 4.7 15.74 44 5 m 1851.2 14.5 10.25 320.7 100.44 415 10 m 1640.8 14.2 21.56 559.4 209.07 682 15 m 1666.7 22.3 31.68 831.7 285.67 808 30 m 1602.6 28.9 78.6 1059.6 425.28 1039 1 hr 1576.8 41.6 162.45 1148.8 526.5 1159 2 hr 1681.0 72.5 290.66 854.5 534.84 1309 3 hr 1689.7 90.2 379.36 841.9 584.61 1207 6 hr 1689.8 141.9 550.43 826.6 561.88 1155
General Experimental Procedure
Rat Lysosome Incubation.

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

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

(230) LIQUID ChromatographyTandem Mass Spectrometry Analysis

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

Example 24

(232) Synthesis, Purification and Analysis of ADC Using Exemplary Drug-Linker 1-4

(233) To establish the chemical reactivity of this linker design to form a drug conjugate, exemplary drug-linker 1-4 was conjugated to an anti-mouse CD25 antibody (IgG1) (mCD25) to produce antibody-drug conjugate ADC 12-1 or anti-human CD70 antibody 2H5 antibody (hCD70) to produce ADC 12-2. Specifically, the drug-linker was conjugated to the unnatural amino acid para-azido-phenylalanine (pAF) replacing the alanine at position 1 of CH1 of the antibody using copper-free 3+2 cycloaddition chemistry as shown in FIG. 1. The amino acid sequence of the anti-mouse CD25 heavy chain comprising the pAF at position 115 is shown in SEQ ID NO:3 and the amino acid sequence of the light chain is shown in SEQ ID NO:4. The amino acid sequence of the anti-human CD70 heavy chain comprising pAF at position 119 is shown in SEQ ID NO:1 and the amino acid sequence of the light chain is shown in SEQ ID NO:2. Synthesis of antibodies containing an unnatural amino acid has been described in U.S. Pat. No. 7,632,924, incorporated herein by reference, and copper-free 3+2 cycloaddition chemistry has been described in U.S. Pat. No. 7,807,619, incorporated herein by reference. Conjugation, purification, and analysis confirmed synthesis of the Anti-CD25 antibody-drug conjugate ADC 12-1 and the anti-CD70 ADC 12-2.

(234) Experimental for Conjugation of Phosphate Linker 1-4 to Anti-Mouse CD25

(235) Antibodies were purified over protein A column (NovaSep) followed by SP 650S column (Tosoh Biosciences).

(236) Site Specific Conjugation Using Click (2+3) Chemistry.

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

(238) Site Specific Conjugation Using Oxime Chemistry.

(239) Para-acetyl phenylalanine (pAcF) containing antibodies were buffer exchanged into 50 mM sodium acetate; 2.5% trehalose; 0-20% Dimethylamine, pH 4.0-4.5 and concentrated to 1-20 mg/mL. 10-15 molar equivalents of amino-oxy drug-linker were added and reacted for 16-72 hours at 28-30 C. The antibody conjugates were purified over a SP 650S column (Tosoh Biosciences) to remove excess reagents. The conjugates were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; pH 6.0, 0.22 m filtered, and stored at 4 C.

(240) Conjugation Analysis.

(241) Conjugation efficiency and DAR values were determined by reversed phase HPLC. The ADC was run over a Zorbax 300SB-C3 column, 4.6150 mm (Agilent) at 80 C. and eluted with a linear gradient from 30% B to 90% B (A: water, 0.1% TFA; B: acetonitrile, 0.1% TFA). An Agilent 1200 series HPLC system and Chemstation software were used to resolve and quantify percentage of antibody conjugated with drug-linker.

Example 25

(242) Serum Stability of ADC 12-1

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

(244) In Vitro Stability Study Design in DBA1 Mice Serum

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

(246) In Vitro StabilityFree Payload Analysis

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

(248) In Vitro StabilityImmunoprecipitation Coupled to Intact Mass Analysis

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

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

(251) The deconvoluted spectra of the stock solution (FIG. 3) revealed that the antibody-drug conjugate 12-1 intact mass in its predominant DAR 2 form contained two GOF sugar motifs. Additional peaks at about 162 Da apart showed two other glycoforms containing G0F/G1F (peak at 148701 Da) and 2G1F (peak at 148863 Da). The glycan profile is typical of that for an IgG.

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

(253) TABLE-US-00006 TABLE 6 In vitro Stability MW of the ADC 12-1 Incubation (2xG0F form) Time 148539 1 hour 148539 8 hours 148539 2 days 148539 3 days 148540 7 days 148540 14 days 148540 21 days
In Vivo Stability of ADC 12-1

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

(255) In Vivo Pharmacokinetic (PK)/Stability Study

(256) General Experimental for In Vivo PK Study of ADC 12-1

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

(258) In vivo Pharmacokinetic (PK)/Stability StudyFree Payload Analysis

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

(260) In Vivo Pharmacokinetic (PK)/Stability StudyIntact Antibody Drug Conjugate Mass Analysis

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

(262) TABLE-US-00007 TABLE 7 In vivo PK/Stability Intact Mass Results MW of the 12-1 PK Time Sample (2xG0F motif) Group Point B1 148548 G2 1 hour C1 148547 G2 1 hour E1 148546 G2 2 hours F1 148548 G2 2 hours A2 148546 G2 6 hours C2 148546 G2 16 hours D2 148547 G2 16 hours F2 148545 G2 1 day G2 148548 G2 1 day A3 148546 G2 2 days D3 148548 G2 3 days G3 148550 G2 5 days H3 148542 G2 5 days B7 148542 G3 1 hour C7 148548 G3 1 hour E7 148547 G3 2 hours F7 148545 G3 2 hours A8 148547 G3 6 hours C8 148546 G3 16 hours D8 148546 G3 16 hours F8 148546 G3 1 day G8 148546 G3 1 day A9 148544 G3 2 days B9 148547 G3 2 days D9 148549 G3 3 days E9 148550 G3 3 days G9 148547 G3 5 days H9 148545 G3 5 days
In Vivo Pharmacokinetic (PK)/Stability StudyNaked/Total mAb Analysis

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

Example 26

(264) The payload-linkers made in Examples 20 and 21 were conjugated to an anti-human Her2 antibody (hHer2) comprising a para-acetyl phenylalanine (pAcF). Methods for making antibodies comprising an unnatural amino acid and conjugating molecules thereto is disclosed in U.S. Pat. No. 7,632,924 to Cho et al., which is incorporated by reference in its entirety.

(265) Site Specific Conjugation Using Oxime Chemistry.

(266) Para-acetyl phenylalanine (pAcF) containing antibodies were buffer exchanged into 50 mM sodium acetate; 2.5% trehalose; 0-20% Dimethylamine, pH 4.0-4.5 and concentrated to 1-20 mg/mL. 10-15 molar equivalents of amino-oxy drug-linker were added and reacted for 16-72 hours at 28-30 C. The antibody conjugates were purified over a SP 650S column (Tosoh Biosciences) to remove excess reagents. The conjugates were buffer exchanged into 50 mM Histidine; 100 mM NaCl; 2.5% Trehalose; pH 6.0, 0.22 m filtered, and stored at 4 C.

(267) Conjugation Analysis.

(268) Conjugation efficiency and DAR values were determined by reversed phase HPLC. The antibody-drug conjugate was run over a Zorbax 300SB-C3 column, 4.6150 mm (Agilent) at 80 C. and eluted with a linear gradient from 30% B to 90% B (A: water, 0.1% TFA; B: acetonitrile, 0.1% TFA). An Agilent 1200 series HPLC system and Chemstation software were used to resolve and quantify percentage of antibody conjugated with drug-linker.

(269) Cell Assay

(270) Her2 positive SKBR3 and Her2 negative MDA-MD-468 breast adenocarcinoma cell lines (ATCC Manassas, Va., USA) were cultured in DMEM medium supplemented with 10% FBS and 100 /mL penicillin, 100 g/mL streptomycin, or DMEM/F12 supplemented with 10% FBS and 100 /mL penicillin, 100 g/mL streptomycin respectively. The cells were incubated with serial dilutions of Duocarmycin-405 conjugated to anti-Her2 or a non-targeting antibody, Duocarmycin free drug and naked anti-Her2 controls for 6 days. Live cells were measured with CellTiter Glo luminescent cell viability assay kit (Promega, Madison, Wis., USA) and the percentage of live cells determined relative to untreated cells.

(271) The results are shown in FIGS. 8 and 9. FIG. 8 shows that Her2-Duo-405 was able to deliver its payload to Her2 expressing SKBR3 cells whereas a non-targeting antibody conjugated to Duo-405 was unable to. The Figure shows the specificity of the Her2-Duo-405 conjugate and its ability to effectively deliver the payload. FIG. 9 shows that Her2-Duo-405 had no activity on Her2 negative MDA-MD-468 cells.

Example 27

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

(273) Aggregation Assay

(274) An SE-HPLC method was used to conduct the aggregation/% monomer analysis.

(275) An isocratic gradient using 0.2 M potassium phosphate, 0.25 M potassium chloride pH 6.0 was used as the mobile phase at a flowrate of 0.5 mL/min. The column used was a Sepax Zenix-C SEC-300, 3 m, 300 A, 7.8300 mm (Cat#233300-7830). Detection of signal was monitored at 214 nm (280 for FIO). For a representative run, the analyte load was 10 g.

(276) The results are shown in Table 8.

(277) TABLE-US-00008 TABLE 8 Sample % High Molecular Weight % Name Drug-Linker (aggregate) Monomer mCD25 Phos-21Dex365 (1-4) 1.4 98.6 hCD70 Phos-21Dex365 (1-4) 0.9 99.1 hHer2 Phos-Duo405 (21-5) 0.5 99.5

Example 28

(278) ADC 12-2 (FIG. 1) is anti-CD70 antibody 2H5 conjugated to exemplary drug-linker 1-4 as described in Example 24. In vitro activity and targeted delivery of ADC 12-2, naked antibody, and anti-hexon conjugate control were assessed by transfecting into 786-O (renal cell) and then measuring glucocorticoid-induced leucine zipper (GILZ) mRNA, a widely expressed dexamethasone-induced mRNA transcript. As shown in FIG. 7, ADC 12-2 displayed potent in vitro activity (0.7 ug/ml IP value) in 786-O cells that were confirmed to express CD70. This activity reflects dexamethasone conjugation and targeted delivery as the nonconjugated IgG variant and anti-hexon controls did not induce and observable GILZ in this cell line.

(279) 786-O cells were plated at 30K cells/well overnight at 37 C. in RPMI Media as suggested by ATCC (+10% HI FBS). Cells were stimulated with ADCs for 2, 6, or 24 hours at 37 C. Cells were lysed using RLT and RNA is isolated using RNeasy 96 Kits. PCR was used to measure GAPDH, PER1, or TSC22D3

(280) Quantitation of Glucocorticoid-induced leucine zipper (GILZ) mRNA expression was determined as follows. Cellular quantitation of GILZ mRNA was conducted using the following method. Cells suspension were prepared in HBSS+2% FBS (assay buffer) and plated at 510.sup.4 cells per well. Dosing solutions for free drug, ADCs and parental antibodies were prepared by serial dilution of each stock solution using 1:3 in in HBSS+2% FBS supplemented with 1% final concentration of (50 mM Histidine, 100 mM NaCl, 5% Trehalose, pH 6.0), and incubated with cells final concentrations ranging from 20 to 0.002 g/ml and 100 to 0.0 ng dexamethasone/ml for 18 hours. Cell lysis, cDNA synthesis, and real-time PCR were performed according to manufacturer's instructions using TaqMan Gene Expression Cells-to-C.sub.T Kit (Invitrogen, Carlsbad, Calif.). Specific primers against human GILZ and GAPDH were purchased from the Life Technologies (Invitrogen, Carlsbad, Calif.). Real-time PCR reactions were performed on the Applied Biosystems 7900 HT Fast Real-Time PCR System. Thermal cycling conditions consisted of an initial UDG incubation hold (50 C., 2 min) denaturing and enzyme activation step (95 C., 2 min) followed by 40 cycles of denaturing (95 C., 15 s), annealing and extending (60 C., 1 min). The mRNA levels were normalized to GAPDH (internal control) using the formula A threshold cycle (CT)=CT targetCT reference. The differential expression signal were expressed as delta Ct (Ct) by subtracting the Ct values of the un-stimulated samples (containing only assay buffer or DMSO vehicle) from those of the stimulated samples and expressed as relative fold of change using the formula: 2.sup.CT.

(281) TABLE-US-00009 TableofSequences SEQID NO: Description AminoAcidSequence 1 Anti-CD70 QVQLVESGGGVVQPGRSLRLSC 2H5IgG1 AASGFTFSSYIMHWVRQAPGKG Xatposition LEWVAVISYDGRNKYYADSVKG 119is RFTISRDNSKNTLYLQMNSLRA para-azido- EDTAVYYCARDTDGYDFDYWGQ phenylalanine GTLVTVSSXSTKGPSVFPLAPS (pAF)(CDR1, SKSTSGGTAALGCLVKDYFPEP 2,and3bold VTVSWNSGALTSGVHTFPAVLQ type;Fc SSGLYSLSSVVTVPSSSLGTQT underlined) YICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQK SLSLSPG 2 Anti-CD70 EIVLTQSPATLSLSPGERATLS Kappalight CRASQSVSSYLAWYQQKPGQAP chain RLLIYDASNRATGIPARFSGSG (CDR1,2, SGTDFTLTISSLEPEDFAVYYC and3bold QQRTNWPLTFGGGTKVEIKRTV type) AAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC 3 Anti-murine QVKLLQSGAALVKPGASVKMSC CD25muIgG1 KASGYSFPDSWVTWVKQSHGKS D265A LEWIGDIFPNSGATNFNEKFKG Xatposition KATLTVDKSTSTAYMELSRLTS 115is EDSAIYYCTRLDYGYWGQGVMV para-azido- TVSSXKTTPPSVYPLAPGSAAQ phenylalanine TNSMVTLGCLVKGYFPEPVTVT (pAF) WNSGSLSSGVHTFPAVLQSDLY TLSSSVTVPSSTWPSETVTCNV AHPASSTKVDKKIVPRDCGCKP CICTVPEVSSVFIFPPKPKDVL TITLTPKVTCVVVAISKDDPEV QFSWFVDDVEVHTAQTQPREEQ FNSTFRSVSELPIMHQDWLNGK EFKCRVNSAAFPAPIEKTISKT KGRPKAPQVYTIPPPKEQMAKD KVSLTCMITDFFPEDITVEWQW NGQPAENYKNTQPIMDTDGSYF VYSKLNVQKSNWEAGNTFTCSV LHEGLHNHHTEKSLSHSPGK 4 Anti-murine DVVLTQTPPTLSATIGQSVSIS CD25muKappa CRSSQSLLHSNGNTYLNWLLQR PGQPPQLLIYLASRLESGVPNR FSGSGSGTDFTLKISGVEAEDL GVYYCVQSSHFPNTFGVGTKLE LKRADAAPTVSIFPPSSEQLTS GGASVVCFLNNFYPKDINVKWK IDGSERQNGVLNSWTDQDSKDS TYSMSSTLTLTKDEYERHNSYT CEATHKTSTSPIVKSFNRNEC

(282) 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.