EA045111B1 - STEROIDS AND THEIR ANTIBODY CONJUGATES - Google Patents

Description

Cross reference to related applications

This patent application claims priority to U.S. Provisional Patent Application No. 62/614,905, entitled Steroids and Antibody Conjugates thereof, which was filed January 2018. The contents of this provisional patent application are incorporated herein in their entirety by reference for all purposes.

List of sequences

This application contains a Sequence Listing that was provided in ASCII format via EFS-Web and is incorporated herein in its entirety by reference. The ASCII copy in question, created on January 3, 2019, is named 114581_00228_ST25.txt and is 11186 bytes in size.

Region

Disclosed herein are steroids, their protein conjugates (eg, antibody conjugates), as well as methods of treating diseases, disorders, and conditions involving the administration of steroids and conjugates.

Prior Art

Antibody-drug conjugates (ADCs) are antibodies that are covalently linked to biologically active small molecule drugs, thereby combining the targeting specificity of antibodies with the mechanism of action and activity of small molecule drugs. The therapeutic utility of ADCs has been confirmed in the treatment of cancer and is currently an important focus of research. ADCETRIS® (brentuximab vedotin) and KADCYLA® (ado-trastuzumab emtansine) are two ADCs approved for the treatment of certain types of cancer, and several other ADCs are currently in clinical trials.

Glucocorticoids (GC) are low molecular weight steroids that bind to glucocorticoid receptors (GR) and are used in anti-inflammatory and immunosuppressive therapy. However, due to the ubiquitous expression of glucocorticoid receptors in many cell types, a disadvantage of glucocorticoid therapy is toxicity to most organ systems. Thus, there is a need for new glucocorticoids and new therapies that reduce the side effects associated with glucocorticoid administration, especially those associated with activation of glucocorticoid receptors in non-target cells. The present description of the invention provides solutions to the above problems, as well as other outstanding problems in the field of technology to which this invention relates. Included herein are antibody-drug conjugates containing glucocorticoid loads.

Summary of the invention

Provided herein are compounds and methods useful for treating various diseases, disorders or conditions.

The first aspect is directed to the compound of formula (III)

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

R ⁴ represents C1-C ₆ alkyl;

both R ^x are hydrogen or both R ^x are fluorine; And

SP is -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₁₀ -alkylene)-NR ^50a -, where the terminal NR ^50a is attached to L;

R ⁵⁰ and R ^50a are independently hydrogen or C ₁ -C ₆ -alkyl;

z is an integer selected from 1 to 30 inclusive;

L is a linker of the formula -L1-L ² -(L ³ ) _0-1 -;

where L ¹ is connected to BA and L ³ is connected to SP;

L1 weight e oea o os special o” in bwa - ' eo

L represents a reactive group residue selected from or thereof

- 1 045111 o

regioisomer;

o _c or its regioisomer; or ' where S refers to the sulfur atom on

ABOUT

4 ^N X the cysteine residue through which the reactive group residue is attached to BA, and , where N refers to the nitrogen atom on the lysine residue through which the reactive group residue is attached to BA; L ² is a binding linker;

L ³ , if present, is a self-cleaving linker; and BA is an antibody, an antibody fragment, or an antigen-binding fragment thereof.

The second aspect is directed to the compound of formula (II):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

R ⁴ represents C1-C6 alkyl;

both R ^x are hydrogen or both R ^x are fluorine;

SP represents -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-αalkylene)-NR ^50a -, where the terminal NR ^50a is attached to (L ³ )0-1;

R ⁵⁰ and R ^50a are independently hydrogen or C1-C6 alkyl;

RG is a reactive group selected from

L ² represents a connecting linker; And

L ³ , if present, is a self-cleaving linker.

A compound of formula (III), wherein BA contains an antibody having the following structure

Ab-N N

L ^J Z ' where Ab is a monoclonal antibody, polyclonal antibody, antibody fragment or bispecific antibody; R represents C _2-4 -alkylene; and n is an integer selected from 2 to 4 inclusive.

A compound of formula (III), wherein BA contains an antibody or an antigen-binding fragment thereof that binds macrophage scavenger receptor 1 (MSR1).

The third aspect is directed to the compound of formula (I):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ⁴ represents -C ₁ -C6 alkyl;

a) both R ^x are hydrogen; And

R ³ represents -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)NR ^50a R ^50b ; or

b) both R ^x are fluorine; And

R ³ represents -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ^50b ; and in each case ^R50 , ^R50a and ^R50b are independently hydrogen or C1- _C6 alkyl.

A compound of one of the following formulas is also presented:

- 2 045111

or a mixture thereof;

Another aspect is directed to a method of treating a disease, disorder or condition associated with glucocorticoid receptor signaling, comprising administering to a patient with said disease, disorder or condition a therapeutically effective amount of a compound of formula (III).

Brief descriptions of figures

In fig. 1A, 1B and 1C and 2 show GR activation of steroid ADC conjugates as described in Example 7.

In fig. Figure 3 shows Scheme 1. Synthesis of spacer budesonide 1c-1e, 1g-1i and 1j-1l.

Detailed description

A. Definitions.

As used herein, alkyl means a monovalent and saturated hydrocarbon radical moiety. Unless otherwise stated, alkyl is unsubstituted and may be straight, branched or cyclic, i.e. cycloalkyl. Alkyl includes, but is not limited to, alkyls having 1-20 carbon atoms, i.e. C ₁ _ ₂₀ alkyl; 1-12 carbon atoms, i.e. C _1-12 alkyl; 1-8 carbon atoms, i.e. C^alkyl; 1-6 carbon atoms, i.e. C^alkyl; and 1-3 carbon atoms, i.e. S^alkyl. Cycloalkyl, which is a subgroup of alkyls, includes, but is not limited to, groups having 3-20 carbon atoms, i.e. C ₃ _ ₂₀ alkyl; 3-12 carbon atoms, i.e. C _{3_12} alkyl _; 3-10 carbon atoms, i.e. C _{3_10} alkyl _; 3-8 carbon atoms, i.e. C _{3_8} alkyl _; and 3-6 carbon atoms, i.e. C _{3_6} alkyl _. Examples of alkyl moieties

- 3 045111 include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl, i-butyl, pentyl moiety, hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, alkylene means divalent alkyl.

As used herein, aminoalkyl means an alkyl group substituted with one or two -NH2 groups.

As used herein, alkenyl means a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more non-aromatic carbon-carbon double bonds. Alkenyl is unsubstituted unless otherwise indicated and may be straight, branched or cyclic. Alkenyl includes, but is not limited to, alkenyls having 2-20 carbon atoms, i.e. C ₂ - ₂₀ alkenyl; 2-12 carbon atoms, i.e. C ₂ _ ₁₂ alkenyl; 2-8 carbon atoms, i.e. _C2 . ₈ αlkenyl; 2-6 carbon atoms, i.e. C ₂ - ₆ alkenyl; and 2-4 carbon atoms, i.e. _C2 . ₄ alkenyl. Cycloalkenyl, which is a subgroup of alkenyls, includes, but is not limited to, groups having 3-20 carbon atoms, i.e. C ₃ . ₂₀ alkenyl; 3-12 carbon atoms, i.e. C ₃ . ₁₂ alkenyl; 3-10 carbon atoms, i.e. C ₃ . ₁ alkenyl; 3-8 carbon atoms, i.e. C ₃ . ₈ alkenyl; and 3-6 carbon atoms, i.e. C ₃ . ₆ alkenyl. Examples of alkenyl moieties include, but are not limited to, vinyl, propenyl, butenyl and cyclohexenyl.

As used herein, alkenylene means divalent alkenyl.

As used herein, alkynyl means a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. Alkynyl is unsubstituted unless otherwise indicated and may be linear, branched or cyclic. Alkynyl includes, but is not limited to, alkynyls having 2-20 carbon atoms, i.e. C ₂ - ₂₀ alkynyl; 2-12 carbon atoms, i.e. C ₂ - ₁₂ alkynyl; 2-8 carbon atoms, i.e. _C2 . ₈ alkynyl; 2-6 carbon atoms, i.e. C ₂ - ₆ alkynyl; and 2-4 carbon atoms, i.e. C ₂ - ₄ alkynyl. Cycloalkynyl, which is a subgroup of alkynyls, includes, but is not limited to, groups having 3-20 carbon atoms, i.e. C ₃ . ₂₀ alkynyl; 3-12 carbon atoms, i.e. C ₃ . ₁₂ alkynyl; 3-10 carbon atoms, i.e. C ₃ . ₁₀ alkynyl; 3-8 carbon atoms, i.e. C ₃ . ₈ alkynyl; and 3-6 carbon atoms, i.e. C ₃ . ₆ alkynyl. Examples of alkynyl moieties include, but are not limited to, ethynyl, propynyl and butynyl.

As used herein, alkynylene means divalent alkynyl.

As used herein, alkoxy means a monovalent and saturated hydrocarbon radical moiety in which the hydrocarbon includes a single bond to an oxygen atom and in which the radical is located on the oxygen atom, for example, CH ₃ CH ₂ -O for ethoxy. Alkoxy substituents bond to the compound they replace through this oxygen atom of the alkoxy substituent. Alkoxy is unsubstituted unless otherwise indicated and may be linear, branched or cyclic, for example cycloalkoxy. Alkoxy includes, but is not limited to, alkoxy having 1-20 carbon atoms, i.e. C ₁ . ₂₀ alkoxy; 1-12 carbon atoms, i.e. C1_ ₁₂ alkoxy; 1-8 carbon atoms, i.e. C1_ ₈ alkoxy; 1-6 carbon atoms, i.e. C1_ ₆ alkoxy; and 1-3 carbon atoms, i.e. _C1-3 alkoxy. Cycloalkoxy, which is a subgroup of alkoxy, includes, but is not limited to, groups having 3-20 carbon atoms, i.e. C ₃ . ₂₀ alkoxy; 3-12 carbon atoms, i.e. C ₃ . ₁₂ αlkoxy; 3-10 carbon atoms, i.e. C ₃ . ₁₀ alkoxy; 3-8 carbon atoms, i.e. C ₃ . ₈ alkoxy; and 3-6 carbon atoms, i.e. C ₃ . ₆ alkoxy. Examples of alkoxy moieties include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, ibutoxy, pentyloxy moiety, hexyloxy moiety, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy.

As used herein, aryl means a monovalent moiety that is a radical of a monocyclic or polycyclic ring system containing at least one aromatic ring, where the ring atoms are carbon atoms. Aryl is optionally substituted. When aryl is monocyclic, the aryl is phenyl. If the aryl is polycyclic, for example bicyclic or tricyclic, at least one of the rings is aromatic and the other rings may be aromatic or partially unsaturated. Examples of aryl moieties include, but are not limited to, those containing from 6 to 20 ring carbon atoms, i.e. _C6 . ₂₀ aryl; from 6 to 15 carbon atoms of the ring, i.e. _C6 . ₁₅ aryl, and from 6 to 10 ring carbons, i.e. _C6 . ₁₀ aryl. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrenyl.

As used herein, arylene means a divalent aryl group, as defined herein. In some embodiments, the arylene is optionally substituted on one of the carbons, which does not provide an attachment point for the rest of the molecule. In some embodiments, the arylene is optionally not substituted. In some embodiments, the arylene is phenylene. In some embodiments, arylene is which is optionally substituted. In some embodiments, the arylene is

- 4 045111

which is optionally not replaced. In some embodiments, the phenylene in

o is optionally replaced.

In some embodiments, the phenylene in

o optionally not replaced.

As used herein, arylalkyl means a monovalent moiety which is a radical of an alkyl compound in which the alkyl compound is replaced by an aryl substituent, i.e. an aromatic compound includes a single bond with an alkyl group, where the radical is localized on the alkyl group. The arylalkyl group is linked to the structure shown via an alkyl group. In some embodiments, arylalkyl includes biensyl, phenylet-1-yl and phenylet-2-yl and the like. Unless otherwise stated, arylalkyl is unsubstituted. However, if arylalkyl is substituted, arylalkyl, i.e. an aryl group and/or an alkyl group is substituted as described herein. Examples of arylalkyl include, but are not limited to, benzyl.

As used herein, haloalkyl means an alkyl as defined herein, wherein the alkyl includes at least one substituent selected from halogen, such as F, Cl, Br, and I. In some embodiments, the alkyl is replaced with 1, 2, 3, or 4 halo atoms that are independently selected.

As used herein, haloalkoxy means alkoxy, as defined herein, where alkoxy includes at least one substituent selected from halogen, such as F, Cl, Br or I.

As used herein, heteroalkyl means an alkyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, heteroalkenyl means alkenyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, heteroalkynyl means an alkynyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen and sulfur atoms, which are independently selected. Heteroalkyl is unsubstituted unless otherwise noted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.

As used herein, heteroaryl means a monovalent moiety that is an aromatic radical wherein the ring atoms contain carbon atoms and at least one oxygen, sulfur, nitrogen or phosphorus atom. Heteroaryl includes monocyclic, fused bicyclic or fused tricyclic radicals, where the ring containing the monocyclic radical is aromatic and where at least one of the fused rings containing the bicyclic or tricyclic radical is aromatic. The fused bicyclic radical includes bridged ring systems. Examples of heteroaryl moieties include, but are not limited to, those having from 5 to 20 ring atoms; from 5 to 15 ring atoms; and 5 to 10 ring atoms. Heteroaryl is optionally unsubstituted. In some embodiments, the heteroaryl ring contains 1, 2, 3, or 4 (in some embodiments, 1, 2, or 3; in some embodiments, 1 or 2; in some embodiments, 1) oxygen, sulfur, nitrogen, and/or phosphorus atoms , which are chosen independently. In some embodiments, the heteroaryl is 1,2,4-triazolyl, 1,3,5-triazolyl, phthalimidyl, pyridinyl, pyrrolyl, imidazolyl, thienyl, furanyl, indolyl, 2,3-dihydro-N-indolyl (including, but not limited to , 2,3-dihydro-III-indol-2-or or 2,3-dihydro-III-indol-5-yl and the like), isoindolyl, indolinyl, isoindolinyl, benzimidazolyl, benzodioxol-4-yl, benzofuranyl, cinnolinyl, indolizinyl, naphthyridin-3-yl, phthalazin-3-yl, phthalazin-4-yl, pteridinyl, purinyl, quinazolinyl, quinoxalinyl, tetrazoyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, isooxazolyl, oxadiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, tetrahydroisoquinolinyl ( including, for example, tetrahydroisoquinolin-4-yl or tetrahydroisoquinolin-6-yl and the like), pyrrolo[3,2-c]pyridinyl (including, for example, pyrrolo[3,2c]pyridin-2-yl or pyrrolo[3,2 -c]pyridin-7-yl and the like), benzopyranyl, thiazolyl, isothiazolyl, thiadiazolyl, benzothiazolyl, benzothienyl.

As used herein, heteroarylene means divalent heteroaryl, as defined herein. In some embodiments, the heteroarylene is optionally substituted on one of the carbons or substituted nitrogens, which does not provide an attachment point for the rest of the molecule. In some embodiments, the heteroarylene is optionally not substituted. In some embodiments, the heteroarylene is pyridine-diyl.

- 5 045111

As used herein, heterocycloalkyl means a cycloalkyl, as defined above under alkyl, in which one or more carbon atoms are replaced by independently selected heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen and sulfur atoms. Unless otherwise stated, heterocycloalkyl is unsubstituted. Examples of heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl, tetrahydropyranyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, dioxolanyl, dithiolanyl, oxanyl, or tyanyl.

As used herein, hydroxyalkylene means an alkylene, as defined herein, where alkylene includes at least one hydroxy. In some embodiments, the alkylene is replaced with 1, 2, 3, or 4 hydroxy atoms.

As used herein, N-containing heterocycloalkyl means a cycloalkyl, as defined above under alkyl, in which one or more carbon atoms are replaced by heteroatoms and in which at least one heteroatom is a nitrogen atom. Suitable heteroatoms, which are independently selected, in addition to nitrogen, include, but are not limited to, oxygen and sulfur atoms. The N-containing heterocycloalkyl is optionally substituted. Examples of N-containing heterocycloalkyl moieties include, but are not limited to, morpholinyl, piperidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, or thiazolidinyl.

As used herein, the term optionally substituted when used to describe a radical moiety (e.g., optionally substituted arylene, optionally substituted heteroarylene, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkyl, and optionally substituted arylalkyl) means that such moiety is optionally linked to one or several substituents on any substituted atom in this fragment. Examples of such substituents include, but are not limited to, halo, cyano, nitro, haloalkyl, azido, epoxy, optionally substituted heteroaryl, optionally substituted heterocycloalkyl,

LLC ON

4-OR ^A -Fsr ^a -^-nr ^a r ^b -^OR ^a -^-L _nr a _r b _^ _nr cJ]_ _r a _^1L _nr a _r b

N R\

II Ng

4 ^- NR ^c -NR ^a R ^b -|-S(O)-R ^a 4 ^- S(O) ₂ - ^rA 4 ⁴ 4 or where R ^a , R ^b and R ^C heterocycloalkyl or R ^A and R ^B , together with the atoms to which they bond form a saturated or unsaturated heterocyclic ring (i.e., heterocycloalkyl or heteroaryl), where the ring is optionally substituted and where, in addition to the ring nitrogen atom, one or more ring atoms are optionally a heteroatom, independently selected from nitrogen, oxygen, sulfur and phosphorus. In some embodiments, when a radical moiety is optionally substituted with an optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated heterocyclic ring (heterocycloalkyl or heteroaryl), substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally substituted saturated or unsaturated heterocyclic ring (heter ocycloalkyl or heteroaryl), if substituted, are not substituted by substituents, which are then optionally substituted by additional substituents. In some embodiments, where a group described herein is optionally substituted, the substituent associated with that group is unsubstituted unless otherwise noted, i.e. the optionally substituted arylene as a substituent is not further replaced by a substituted arylene.

As used herein, a binding agent means any protein or peptide-based molecule capable of binding with specificity to a specified binding partner. In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the binding agent is not albumin.

As used herein, the term amide synthesis conditions means reaction conditions suitable for promoting the formation of an amide, for example by reacting a carboxylic acid, an activated carboxylic acid or an acyl halide with an amine. In some embodiments, amide synthesis conditions mean reaction conditions suitable for the formation of an amide bond between a carboxylic acid and an amine. In some of these embodiments, the carboxylic acid is first converted to an activated carboxylic acid before the activated carboxylic acid reacts with the amine to form an amide. Suitable conditions for amide formation include, but are not limited to, conditions under which reagents are used to promote the reaction between the carboxylic acid and the amine, including, but not limited to, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (benzotriazol-1-yloxy)tris(dimethylamino )phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyAOP), bromotripyrrolidinophosphonium hexafluorophosphate (PyBrOP), O-(benzotriazol-1-yl)-N ,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), 1

- 6 045111

[bis(dimethylamino)methylene]1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide (HATU), 2ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 1-ethyl -3-(3-dimethylaminopropyl)carbodiimide (EDC), 2-chloro-1,3-dimethylimidazolidinium hexafluorophosphate (CIP), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and carbonyldiimidazole ( CDI). In some embodiments, the carboxylic acid is first converted to an activated carboxylic acid ester before reacting with the amine to form an amide bond. In certain embodiments, the carboxylic acid reacts with the reagent. This reagent activates the carboxylic acid by deprotonating the carboxylic acid and subsequently complexing the product with the deprotonated carboxylic acid as a result of the nucleophilic attack of the deprotonated carboxylic acid on the protonated reagent. For certain carboxylic acids, such an activated ester is subsequently more susceptible to nucleophilic attack by the amine compared to the carboxylic acid prior to its conversion. This leads to the formation of an amide bond. In this regard, the carboxylic acid is described as activated. Examples of reagents are DCC and DIC.

As used herein, a therapeutically effective amount means an amount (of a compound) that is sufficient to provide a therapeutic effect to a patient in treating or controlling a disease or disorder or delaying or reducing one or more symptoms associated with that disease or disorder.

As used herein, a pharmaceutically acceptable salt means any salt suitable for administration to a patient. Suitable salts include, but are not limited to, those described in Berge et al., Pharmaceutical Salts, J. Pharm. Sci., 1977, 66:1, the contents of which are incorporated herein by reference. Examples of salts include, but are not limited to, salts derived from acids, salts derived from bases, organic, inorganic salts, amine salts, and alkali and alkaline earth metal salts, including, but not limited to, calcium salts, magnesium salts, potassium salts, sodium salts , salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid and the like.

Certain groups, moieties, substituents, and atoms are depicted with a wavy line that crosses or covers the bond or bonds to indicate the atom through which the groups, moieties, substituents, and atoms are bonded. For example, a phenyl group is replaced by a propyl group, which is represented as follows:

has the following structure:

As used herein, illustrations depicting substituents associated with a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through bonding between ring atoms indicate, unless otherwise indicated, that the cyclic group may be substituted such substituent at any ring position in a cyclic group or on any ring in a fused ring group, in accordance with methods set forth herein or known in the art to which this specification relates. For example, a group

in which the subscript q is an integer selected from 0 to 4, inclusive, and in which the positions of the substituent R ¹ are described in general, i.e. without direct attachment to any vertex of a non-linear communication structure, i.e. a specific ring carbon atom, includes the following non-limiting examples of groups in which the ^R1 substituent is bonded to a specific ring carbon atom:

- 7 045111

In cases where the name and structure of a compound conflict or are incompatible, the structure shall control, unless the context, such as NMR characterization data, may indicate otherwise.

As used herein, the phrase reactive group (RG) means a functional group or moiety that reacts with the reactive portion of an antibody, modified antibody, or antigen-binding fragment thereof. In certain embodiments, the reactive group is a functional group or moiety (eg, maleimide or NHS ester) that reacts with a cysteine or lysine residue of an antibody or antigen-binding fragment thereof. In certain embodiments, the reactive group is a functional group or moiety capable of undergoing a click chemistry reaction. In some embodiments of this click chemistry reaction, the reactive group contains an alkyne that is capable of undergoing a 1,3 cycloaddition reaction with the azide. Such suitable reactive groups include, but are not limited to, strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, such as cyclooctynes, benzannelated alkynes, and alkynes capable of undergoing 1,3 cycloaddition reactions with azides in the absence of copper-based catalysts. Suitable alkynes also include, but are not limited to, DIBAC (where the -C(O)CH ₂ CH ₂ C(O)- part of the DIBAC moiety is capped by L ² ), DIBO (where the -O- part

The DIBO moiety is capped by L ² ), BARAC (where the ' ^N portion of the BARAC moiety is capped by L ² ), DIFO (where the -O- moiety is capped by L ² ), substituted alkynes such as fluorinated alkynes, aza-cycloalkynes, BCN and derivatives thereof. Linker loads containing such reactive groups are useful for conjugating antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such a functionalized antibody is prepared by reacting an antibody containing at least one glutamine residue, such as a Q295 heavy chain (EU nomenclature), and a compound of the formula H ₂ N-LL-N ₃ , wherein LL is a divalent polyethylene glycol group , in the presence of the enzyme transglutaminase.

In some embodiments, the reactive group (RG) is an alkyne, for example, which can react via click chemistry with an azide, for example, n=n=n^ _g ', resulting in a click chemistry product, for example, 'its regioisomer or mixture thereof. In some embodiments, the reactive group is an alkyne, for example -8045111mer,

or

(where L ² covers -OCH ₂ C(O)-), which can react via click chemistry with an azide, e.g.

resulting in the formation of a click chemistry product, for example,

. In some embodiments, the reactive group is an alkyne, e.g. > which can react via click chemistry with an azide, e.g.

its regioisomer or mixture thereof. In some embodiments, the reactive group is a functional

nal group, for example ⁰ , which reacts with a cysteine residue on the antibody or

AB_

its antigen-binding fragment, resulting in the formation of a bond with it, for example, ° ’ where

Ab means an antibody or an antigen-binding fragment thereof, and S means the S atom on the cysteine residue through which the functional group binds to the Ab. In some embodiments, the rheo active group is a functional group, for example, ^o

• which reacts with a lysine residue on an antibody or its antigen-binding fragment, resulting in

AB^<

a connection is formed with it, for example, *. where Ab denotes an antibody or an antigen-binding fragment thereof, and N denotes the N atom on the lysine residue through which the functional group binds to the Ab.

As used herein, the phrase reactive moiety (-L ¹ -) means the reaction product of a functional group on a linker moiety with a reactive moiety of a coupling agent (BA), including a click chemistry product. The reactive group residue is formed by reacting the reactive group with the amino acid of the coupling agent and is attached to the coupling agent (eg, antibody) and to the connecting linker. In some embodiments, the reactive group moiety is a group that contains 1,2,3-tetrazole, i.e. formed through the reaction of an alkyne with an azide. In some embodiments, the remainder of the reactive group is

(a), where S denotes the S atom on the cysteine residue through which (a) binds to Ab, and which is formed by the reaction

with a cysteine residue on the antibody or its antigen _- binding fragment. In some embodiments, the reactive group residue is an N atom on a lysine residue and which is formed by reaction of a functional group, e.g.

’ with a lysine residue on the antibody or its antigen-binding fragment.

In some embodiments, the linker -L- contains and L ¹ is a reactive group moiety according to the following: 1

' its regioisomer or mixture thereof, where it represents a bond with a coupling agent. In some embodiments, the linker -L- contains and L ¹ represents a reactive group residue according to the following fragment:

N ' is its regioisomer or a mixture of them, where it represents a bond with a binding agent. In some embodiments, the linker -L- contains and L ¹ represents a reactive group residue according to the following fragment:

where -OCH ₂ C(O)- is covered by L ² , its regioisomer or a mixture thereof, where it represents a bond with a coupling agent. In some embodiments, the linker -L-contains and L ¹ is a reactive group moiety according to the following:

Qa ¹ i ' its regioisomer or mixture thereof, where * represents a bond to a coupling agent. In some embodiments, the linker -L-contains and L ¹ is a reactive group moiety according to the following:

O ' where represents a bond to a cysteine of an antibody or an antigen-binding fragment thereof. In some embodiments, the linker -L- contains and L ¹ represents a reactive group residue according to the following fragment:

Well I, where r represents a bond to a lysine of the antibody or its antigen-binding fragment. In these examples, the connection to the coupling agent is direct or via a linker. In certain embodiments, the coupling agent is modified with an azide to facilitate binding to the linker -L- or L ¹ , resulting in the formation of a reactive group residue.

As used herein, connecting linker (L ² ) means divalent groups that are cleavable or non-cleavable. Cleavable linkers are linkers that are cleaved through intracellular metabolism after internalization, such as cleavage through hydrolysis, reduction, or an enzymatic reaction. Non-cleavable linkers are linkers that release the attached load through lysosomal degradation of the antibody after internalization. Suitable connecting linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction-labile linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, linkers that are or contain glucuronides, succinimide thioesters, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, disulfide units (eg, -SS-, -SC(R ^1b R ^2b )-, where R ^1b and R ^2b are independently hydrogen or hydrocarbyl), carbamate units, para-amino-benzyl units (PAB), phosphate units, e.g. mono-, bis- or tris-phosphate units and peptide units, e.g. , peptide units containing two, three, four, five, six, seven, eight or more amino acid residues, including, but not limited to, valine-citrulline residue units. As used herein, caproyl means a -(CH ₂ ) ₅ -C(O)- group. In some embodiments, L ² contains cyclodextrin (CD). In some embodiments, L ² comprises -C(O)CH2CH2C(O)-, -C(O)CH ₂ CH ₂ CH2C(O)-, -OCH ₂ C(O)-,

-O- or

As used herein, a self-cleaving linker in (L ³ ) means any such

- 10 045111 group, known to specialists in this field. In specific embodiments, the self-cleaving group is p-aminobenzyl (PAB), i.e. on

or its derivative. By-

useful derivatives include p-aminobenzyloxy, i.e.

(RABC), i.e.

°/ ’ and p-aminobenzyloxycarbonyl. It is known to those skilled in the art that a self-cleaving group is capable of undergoing a chemical reaction that releases the remaining linker atoms from the load.

B. Steroid loads.

Steroids suitable for the conjugates described herein include budesonide and its prodrugs, budesonide analogs or derivatives (including fluorinated analogs and derivatives) and prodrugs thereof, and the steroids listed in Table 1. Oh, and their prodrugs. In some embodiments, the antibody-drug conjugates release budesonide, a prodrug of budesonide, a budesonide analog or derivative (including fluorinated analogs and derivatives), a prodrug of a budesonide analog or derivative (including fluorinated analogs and derivatives), a steroid in the table. A or prodrug of the steroid in the table. And after binding to the target antigen, antibodies. In table And the following compounds are connected to VA compounds of formula (III), formula (III-P), formula Ш-Р-1 or (3000) through hydroxy -C(O)CH ₂ OH groups, i.e. via -C(O)CH2-O-SP-L-, or via hydroxy Mapracorate, i.e. via -O-SP-L-; or bind to RG compounds of formula (II), formula (II-P), formula IIP-1 or (2000) through hydroxy -C(O)CH ₂ OH groups, i.e. through -C(O)CH2-O-SP-(L ³ ) ₀ _1-L ² - or through 3\ 2 hydroxy Mapracorate, i.e. via -O-SP-(L ) _0-1 -L -.

Table A

Tradename Structure Hydrocortisone butyrate Locoid® ^HVp . iXCO ⁰ XXCj ⁿ Halomethasone Sicorten®/ (C-48401-Ba) o io 1 .OH ..... Τ T F T N F Betamethasone Celestone®/Rinderon®/Diprosone® (NSC-39470; Sch-4831) BUT BUT . G? O Dr- J. b ABOUT'- - Fluchlorolone acetonide Cutanit®/T opi con® (RS-2252) b Ω / OdaL---S νΧχΧ O o T o-^

- 11 045111

Fluocinolone acetonide Flucort®/Fluoni d®/Huvi en®/Reti sert®/Sy nal ar®/Sy nal ar-HP®/Sy nemol ® (NSC-92339; DF-277) OH jXl i' O'.......·-..... Flunisolide (RS-3999; RS-1320) but P H.O. A\°C F Cloprednol (RS-4691) ABOUT HO tt he Cl Triamcinolone Ari stocort®/Kenacort® OH HO I .ΤηΊΓδ '"he A JT J Budesonide OH ......Xt^-· Flurandrenolide Cordran® y—OH ⁰ \oJ_HO. zxiy 1 iYhTJ'*° XX” j F

- 12 045111

Desoxymethasone Topicort® (DSXS) Ten- Betamethasone benzoate Uticort® (W-5975) ίο hundred 7 Che O Desonate® (D-2083) but P o%_ , 1 η 1 > ⁰ J 1 n J n Meprednisone (NSC-527579; Sch-4358) o io 1 he 1 X ⁿ G T n 1 n Prednisolone Delta-Cortef® (NCS-9120) θν^ΟΗ no^^-DTyun ιΎ ^η Τ/ T fn J th Triamcinolone acetonide but P , T n T u -° G Tm n

- 13 045111

Methylprednisolone Depo-Medrol; Medrol; Urbason® (NSC-19987) 0./-.. he has . Ύ n D y G D n J n Prednisone Decortin®/Deltasone®/Lodotra®/Meticort en®/Rayos® (NSC-10023) ABOUT . LP γ оН О J I ΌΗ -Up | [nH O' - Dexamethasone Decadron® (FT-4145; ENV-1105; Ш1-10090; ISV- 305; OTO-104) ^but >o ho^/xJUoh . T n T u..... G TfJ η Hydrocortisone valerate Westcort® but 'O iX ^h JO ° Г Th J n Mapracorat (BOL-242X; BOL-303242-X; ZK-245186; BAY-865319; BOL-303242-X) /=^ ΖΞ —(-U- ^O UL7 Benzodrocortisone BUT \?e 0 2 jX ^H X./ ⁰ G ThJ n i'

In some or any embodiments, the compound of formula (3000) or formula (2000) contains a prodrug of the steroid in the table. A, where SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-, -C(O)-N(C _1-6 αlkyl)-C ₁ -C ₁₀ -alkylene-X ^1b -, where X ^1b is attached to L in formula (3000) or (L ³ )0-1 in formula (2000), -C(O)-N(H)-(C1-C10-αalkylene)-X ^1b - , where X ^1b is attached to L in formula (3000) or (L ³ )0-1 in formula o (2000), where the point of attachment on the right side (i.e. on N) represents to L in formula (3000) or (L ³ ) _0-1 in formula (2000), -CH ₂ -NH-, where N is added to L in formula (3000) or

(L ³ )0-1 in formula (2000), ⁰ where N is added to L in formula (3000) or (L ³ )0-1 in formula (2000), -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to L in formula (3000) or (L ³ )0-1 in formula (2000), -C(O)-( C ₁ -C ₁₀ -αalkylene)-NR ⁵⁰ C(O)-(C1-C10-αalkylene)-NR ^50a -, where NR ^50a is added to L in formula (3000) or (L3)0-1 in formula (2000 ) and where in each case C1-C ₁₀ -αalkylene is independently and optionally replaced by one or more hydroxy, -C(O)-N(R ⁵ )-(C1-C10-alkylene)C(O)NH-X ² -, where X ² is added to L in formula (3000) or (L ³ )0-1 in formula (2000), or

- 14 045111

R ^{e R} V ^R ') _m >X<-X<° ⁷ 4 ..........3........

, where X ⁴ is added to L in formula (3000) or (L ³ ) _0-1 in formula (2000).

In some embodiments, a compound according to formula (I-P):

(I-P);

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof, wherein

R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl;

R ^e

R ³ represents -C(O)R ^z or '

R ^z represents -C _4-10 -alkylene-C(O)OH or NR ¹⁵ R ^15a ;

R ¹⁵ represents H or alkyl and R ^15a represents -(C _1-6 -alkylene)-C(O)NHNH ₂ ;

one of R ^c , R ^d , R ^e and R ^f is -CH2OR ⁸ and the others are independently -H, -OH, hydroxyalkyl, -C(O)OH or -CH2OR ⁸ where in each case R ^g is independently -(C _1-6 -alkylene)-C(O)OH or -(C _1-6 -alkylene)-C(O)O(alkyl); or one of R ^c , R ^d , R ^e and R ^f is hydroxyalkyl and the others are -H; and m represents 0 or 1.

In some embodiments, a compound according to formula (I-P-1):

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof, wherein: R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl;

R ^{e R} v4V XV.

R ³ represents -C(O)R ^Z or '

R ^z represents -C _4-10 -alkylene-C(O)OH or NR ⁵ R ^5a ;

R ¹⁵ represents H or alkyl and R ^15a represents -(C _1-6 -alkylene)-C(O)NHNH ₂ ;

one of R ^c , R ^d , R ^e and R ^f is -CH2OR ^g and the others are independently -H, -OH, hydroxyalkyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -(C _1- 6-alkylene)-C(O)OH or -(C _1- 6-alkylene)-C(O)O(alkyl); or one of R ^c , R ^d , R ^e and R ^f is hydroxyalkyl and the others are -H; and m represents 0 or 1.

In some embodiments, a compound of formula (I), formula (IP), or formula (IP-1) is provided herein, wherein R ⁴ is alkyl. In some embodiments, R ⁴ is non-cycloalkyl. In some of these embodiments, R ⁴ is straight or branched alkyl. In some of these embodiments, R ⁴ is aryl. In some of these embodiments, R ⁴ is arylalkyl. In some of these embodiments, R ⁴ is an N-containing heterocycloalkyl. In some embodiments, R ⁴ is alkyl, such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl. In some embodiments, R ⁴ is n-propyl. In some embodiments, R ⁴ is i-propyl. In some embodiments, R ⁴ is n-butyl. In some embodiments, R ⁴ is i-butyl. In some embodiments, R ⁴ is t-butyl. In some embodiments, R ⁴ is sec-butyl. In some embodiments, R ⁴ is pentyl. In some embodiments, R ⁴ is hexyl. In some embodiments, R ⁴ is heptyl. In some embodiments, R ⁴ is octyl or nonyl. In some variants

- 15 045111 implementation R ⁴ represents aryl, such as, inter alia, phenyl, phenol or naphthyl. In some embodiments, R ⁴ is phenyl. In some embodiments, R ⁴ is naphthyl. In some embodiments, R ⁴ is heteroaryl such as, but not limited to, thienyl. In some embodiments, R ⁴ is arylalkyl - such as, but not limited to, benzyl. In some embodiments, R ⁴ is an N-containing heterocycloalkyl, such as, but not limited to, piperidinyl. In some embodiments of formula (I), both R ^x are hydrogen. In some embodiments of formula (I), both R ^x are fluorine.

In some or any embodiments, a compound of formula (I) is provided wherein both R ^x is fluorine and R ³ is -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)NR50aR50b or -C(O)R ^z and R ^z represents NR ¹⁶ R ^16a or -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ alkylene)-NR ^50a R ^50b , where in each In the case of C ₁ -C ₁₀ -alkylene is independently and optionally replaced by one or more hydroxy.

In some embodiments of Formula (I), (IP) or (IP-1), R ^x is both hydrogen and R ³ is -C(O)R ^z . In some or any embodiments of Formula (I), (IP) or (IP-1), R ^z is -C4.10-alkylene-C(O)OH. In some or any embodiments of Formula (I), (IP), or (IP-1), R ^z is -C4. _6- alkylene-C(O)OH. In some or any embodiments of Formula (I), (IP) or (IP-1), R ^z is -C4-alkyleneC(O)OH. In some or any embodiments of Formula (I), (IP) or (IP-1), R ^z is -C5-alkylene-C(O)OH. In some or any embodiments of Formula (I), (IP) or (IP-1), R ^z is -C6-alkylene-C(O)OH. In some or any embodiments of Formula (I), (IP) or (IP-1), R ^z is NR ¹⁵ R ^15a . In some or any embodiments of Formula (I), (IP) or (IP-1), R ¹⁵ is H or C1_3-alkyl and R ^15a is -(C1.3-alkylene)-C(O)NHNH2. In some or any embodiments of Formula (I), (IP) or (IP-1), R ¹⁵ is H or methyl and R ^15a is -(C1. ₃ -alkylene)-C(O)NHNH ₂ . In some or any embodiments of Formula (I), (IP) or (IP-1), R ¹⁵ is H and R ^15a is -(C1.3-alkylene)-C(O)NHNH2. In some or any embodiments of Formula (I), (I-P) or (IP-1), R ¹⁵ is methyl and R ^15a is -(C1. ₃ -alkylene)C(O)NHNH ₂ . In some or any embodiments of Formula (I), (IP) or (IP-1), R ¹⁵ is H and R ^15a is -(C1-alkylene)-C(O)NHNH2. In some or any embodiments of Formula (I), (IP) or (IP-1), R ¹⁵ is methyl and R ^15a is -(C1-alkylene)-C(O)NHNH ₂ . In some or any embodiments of Formula (I), R ¹⁵ is H or C1-C6 alkyl and R ^15a is -(C1-C ₁₀ -alkylene)-SH. In some or any embodiments of Formula (I), R ¹⁵ is H or C1-C3 alkyl and R ^15a is -(C1-C ₁₀ -alkylene)-SH. In some or any embodiments of Formula (I), R ¹⁵ is H or CH3 and R ^15a is -(C1-C ₁₀ -alkylene)-SH. In some or any embodiments of formula (I), R ^z is -(C1-C10 alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ alkylene) _NR 50a _R 50b _, where in each case C ₁ - The C ₁₀ -alkylene is independently and optionally replaced by one or more hydroxy. In some or any embodiments of formula (I), R ^z is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ hydroxyalkylene)-NR ^50a R ^50b In some or any In embodiments of formula (I), R ^z is -(branched C ₁ -C ₆ alkylene)NR ⁵⁰ C(O)-(C1-C6 alkylene)-NR ^50a R ^50b

In some embodiments, a compound of formula (I) is provided, wherein

a) both R ^x are hydrogen; And

R ³ represents -C(O)R ^Z ; -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ^50b ; or R ^e

R ^c O / .

R ^z represents -C4-10-alkylene-C(O)OH; -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene) _NR 50a _R 50b _, where in each case the C ₁ - _{C 10} -alkylene is independently and optionally replaced by one or more hydroxy; or NR ¹⁵ R ^15a ;

R ¹⁵ represents H or C1-C6 alkyl and R ^15a represents -(C1-C ₁₀ -alkylene)-SH or -( _{C1.6 -} alkylene)-C(O)NHNH ₂ ; or

b) both R ^x are fluorine; And

R ³ represents -C(O)R ^z ; -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ^50b ; or

R ^e γΖ

RC^O^V .

R ^z represents -C4_10-alkylene-C(O)OH; -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene) _NR 50a _R 50b _, where in each case C ₁ -C ₁₀ -alkylene is independently and optionally replaced by one or more - 16 045111 how much hydroxy? or NR ¹⁶ R ^16a ;

R ¹⁶ represents H or C1-C6alkyl and R ^16a represents -(C1- _C10 -alkylene)-SH, -( _C1 - _C10alkylene ) _-NH2 , -(C1-C1 o-alkylene)-NH (C 1 - ₆ alkyl),

NH ' or -(C1 _- 6-alkylene)-C(O)NHNH2; and in each case R ⁵⁰ , R ^50a and R ^50b are independently hydrogen or C ₁ -C ₆ -alkyl;

one of R ^c , R ^d , R ^e and Rf is -CH2OR ⁸ and the others are independently -H, -OH, hydroxyalkyl, -C(O)OH or -CH2OR ⁶ where in each case R ^g is independently -(C ₁ . ₆ -alkylene)-C(O)OH or -(C ₁ _ ₆ -alkylene)-C(O)O(alkyl); or one of R ^c , R ^d , R ^e and Rf is hydroxyalkyl and the others are -H; and m represents 0 or 1.

In some embodiments of formula (I), both R ^x is fluorine and R ³ is

represents -C(O)R ^z . In some or any embodiments, R ^z is

In some or any embodiments of Formula (I), R ^z is -C4_10alkylene-C(O)OH. In some or any embodiments of Formula (I), R ^z is -C4-6-alkylene-C(O)OH. In some or any embodiments of Formula (I), R ^z is -C4-alkylene-C(O)OH. In some or any embodiments of Formula (I), R ^z is -C5-alkylene-C(O)OH. In some or any embodiments of Formula (I), R ^z is -C6-alkylene-C(O)OH. In some or any embodiments of Formula (I), R ^z is NR ¹⁶ R ^16a . In some or any embodiments of Formula (I), R ¹⁶ is H or C1-3-alkyl and R ^16a is -(C1-3-alkylene)-C(O)NHNH2. In some or any embodiments of Formula (I), R ¹⁶ is H or methyl and R ^16a is -(C1-3-alkylene)-C(O)NHNH ₂ . In some or any embodiments of Formula (I), R ¹⁶ is H and R ^16a is -(C1-3-alkylene)-C(O)NHNH2. In some or any embodiments of Formula (I), R ¹⁶ is methyl and R ^16a is -(C1_ ₃ alkylene)-C(O)NHNH ₂ . In some or any embodiments of Formula (I), R ¹⁶ is H and R ^16a is -(C1-alkylene)-C(O)NHNH2. In some or any embodiments of Formula (I), R ¹⁶ is methyl and R ^16a is -(C1-alkylene)C(O)NHNH2. In some or any embodiments of Formula (I), R ¹⁶ is H or C1-C6 alkyl and R ^16a is (C1-C ₁₀ alkylene)-SH. In some or any embodiments of Formula (I), R ¹⁶ is H or C1-C3 alkyl and R ^16a is -(C1-C ₁₀ alkylene)-SH. In some or any embodiments of Formula (I), R ¹⁶ is H or CH3 and R ^16a is -(C1-C ₁₀ -alkylene)-SH. In some or any embodiments of Formula (I), R ¹⁶ is H or C1-C6 alkyl and R ^16a is -(C1-C ₁₀ alkylene)-NH ₂ . In some or any embodiments of Formula (I), R ¹⁶ is H or C1-C3 alkyl and R ^16a is -(C1-C ₁₀ alkylene)-NH ₂ . In some or any embodiments of Formula (I), R ¹⁶ is H or CH3 and R ^16a is -(C1-C ₁₀ -alkylene)-NH ₂ . In some or any embodiments of formula (I), R ¹⁶ is H or C1-C6 alkyl and R ^16a is -(C1-C ₁₀ -alkylene)-NH(C _{1 -6} alkyl). In some or any embodiments of Formula (I), R ¹⁶ is H or C1-C3 alkyl and R ^16a is -(C1-C ₁₀ -alkylene)-NH(C _{1 -6} α-alkyl). In some or any embodiments of Formula (I), R ¹⁶ is H or CH3 and R ^16a is -(C 1 -C ₁₀ -alkylene)-NH(C ₁ . ₆ alkyl). In some or any embodiments of Formula (I), R ^z is -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ^50b , wherein in each case C1- The C10 alkylene is independently and optionally replaced by one or more hydroxy. In some or any embodiments of formula (I), R ^z is -(branched C1-C ₆ alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₆ -hydroxyalkylene)-NR ^50a R ^50b In some or all In any embodiment of formula (I), R ^z is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ alkylene)N _R - ^0a _R - ⁰ b

In some embodiments of Formula (I), (IP) or (IP-1), R ^x is both hydrogen and R ³ is -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ^50b . In some or any embodiments of formula (I), (IP) or (IP-1), R ³ is -(C1-C6 alkylene)NR ⁵⁰ C(O)-(C1-C ₆ alkylene)-NR ^50a R ^50b . In some or any embodiments of formula (I), (IP) or (IP-1), R ³ is -(C1-C3 alkylene)-NR ⁵⁰ C(O)-(C1-C ₃ alkylene)-NR ^50a R ^50b . In some or any embodiments of formula (I), (IP) or (IP-1), R ³ is -(linear C1C3 alkylene)-NR ⁵⁰ C(O)-(linear C1-C ₃ alkylene)-NR ^50a R ^50b .

In some embodiments of formula (I), both R ^x is fluorine and R ³ is -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ⁵⁰ . In some or any embodiments of formula (I), R ³ is -(C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ alkylene)NR ^50a R ^50b . In some or any embodiments of Formula (I), R ³ is -(C1C3-alkylene)-NR ⁵⁰ C(O)-(C1-C ₃ -alkylene)-NR ^50a R ^50b . In some or any embodiments

- 17 045111 of formula (I) R ³ represents -(linear C1-C3 alkylene)-NR ⁵⁰ C(O)-(linear C1-C ₃ alkylene)NR50aR50b.

In some embodiments of formula (I), (IP), or (IP-1), both R ^x are boyU

R^O^/ dorod and R ³ represents

In some embodiments of Formula (I), (IP), or (IP-1), m is 0. In some embodiments of Formula (I), (IP), or (IP-1), m is 1. In some embodiments, formula (I), (IP) or (IP-1) m represents 1 and R ^f represents -H or -OH. In some embodiments of Formula (I), (IP), or (IP-1), m is 1. In some embodiments of Formula (I), (IP), or (IP-1), m is 1 and one of R ^c , R ^d , R ^e and R ^f are -CH2OR ^g , and the others are independently -H, -OH, hydroxy alkyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently - (C _1-6 -alkylene)C(O)OH or -( _C1-6 -alkylene)-C(O)O(alkyl). In some embodiments of Formula (I), (IP), or (IP-1), m is 1 and R ^c is -CH2OR ^g , and others are, independently, -H, -OH, or hydroxyalkyl. In some embodiments of formula (I), (IP) or (IP-1), m is 1 and one of R ^c , R ^d , R ^e and R ^f is hydroxyalkyl and the others are H. In some embodiments of Formula (I), (IP), or (IP-1), m is 1 and R ^c is hydroxyalkyl and R ^d , R ^e , and R ^f are -H.

In some embodiments of formula (I), both R ^x is fluorine and R ³ is

In some embodiments of Formula (I), m is 0. In some embodiments of Formula (I), m is 1. In some embodiments of Formula (I), m is 1 and R ^f is -H or -OH. In some embodiments of formula (I), m is 1. In some embodiments of formula (I), m is 1 and one of R ^c , R ^d , R ^e and R ^f is -CH2OR ^g and the others are independently -H, -OH, hydroxyalkyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -(C _1-6 -alkylene)-C(O)OH or -(C _1-6 -alkylene)-C(O)O(alkyl). In some embodiments of Formula (I), m is 1 and R ^c is ^-CH2ORg , and others are independently -H, -OH, or hydroxyalkyl. In some embodiments of Formula (I), m is 1 and one of ^Rc , ^Rd , ^Re , and ^Rf is hydroxyalkyl and others is -H. In some embodiments of formula (I), m is 1 and R ^c is hydroxyalkyl and R ^d , R ^e and R ^f are -H.

In some embodiments of formula (I), both R ^x is fluorine and R ³ is

In some or any embodiments, a compound of formula (Z):

(Z) where R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl;

both R ^x are hydrogen or both R ^x are fluorine; And

SP is -CH2NH- where NH is combined with H or SP is

o where NH from SP combines with H in formula (Z), or SP represents

' where N from SP is combined with H in formula (Z). In some or any embodiments, both R ^x are hydrogen. In some or any embodiments

- 18 045111 both R ^x are fluorine. In some or any embodiments, R ⁴ is alkyl. In some of these embodiments, R ⁴ is linear alkyl. In some embodiments, R ⁴ is alkyl, such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl. In some embodiments, R ⁴ -n-propyl. In some embodiments, R ⁴ is i-propyl. In some embodiments, R ⁴ is n-butyl. In some embodiments, R ⁴ is i-butyl. In some embodiments, R ⁴ is t-butyl. In some embodiments, R ⁴ is sec-butyl. In some embodiments, R ⁴ is pentyl. In some embodiments, R ⁴ is hexyl. In some embodiments, R ⁴ is heptyl. In some embodiments, R ⁴ is octyl or nonyl. In some or any embodiments, SP is -CH2NH-, wherein NH is combined with H. In some or any

In any embodiments, SP is ⁰ , where NH is combined with H in formula (Z). In some or any embodiments, R ⁴ is alkyl, both R ^x being

are fluorine and SP is ⁰ , where NH is combined with H in formula (Z). In some embodiments, a compound of formula (Z) is

In some embodiments, a compound of formula (Z) is

In some embodiments, a compound of formula (Z) is

In some embodiments, a compound of formula (Z) is

Suitable steroids for the conjugates described herein include those in the following table.

- 19 045111

Table 1

Budesonide spacer (Budesonide-SP)

Connection no. Structure 1a (Budesonide) % / 0^0 \ \_Ui G t χ| w O' o 1s N Ng—, ° ° He 0 ID o AJ C ^X >C O ^X U ^X T ίΝ t 1e N P'Wt/ ⁿ /=\ ° n ⁰ ^\VA=> ⁰ ^γΛ ° ⁿ o 1g t o i ( ° 7 )— \—4-1 o ⁷ nJ \ Гзс/гш t χΙ t J SO o

- 20 045111

- 21 045111

100 HH 7_. o4l h / \ LH f >=\ ⁰ μν/Τνν ⁰ 101a ъ M ( ^ο 92ζ Οτιπ J ''Π V o 101b F Η Η H r-\ ^°ТЯi>° - Ν ΟθΧΚ-ν ¹ ^ Υ ^^0 ОН ¹ 0 101c / *1 Η ^Η /—\ ( JARI= /=\ Υ ⁴ —\ \~J ¥-/ >=ο 1 ο^/\_/ j\—/ ΗΝ^γθ^Ο 4 \ ⁰ lOld α °4 / Ο Γ\ οΆ ° ¹ рЧ о 102c -ξ Η/-, ⁴ —( 1 У~/ Η V-. °TV?tO=° Η ,ζ ⁰ ОН HS^V 0 102d '—\ X / 7\ Η V-. ^o jaVtQ=o 1 1 ° he HS^ ^N Y° 0

- 22 045111

or a stereoisomer or mixture of stereoisomers; and optionally a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

In some cases, n-propyl ^o in each of the above structures is in the R configuration, i.e. on the carbon shown with an asterisk. In some cases, the n-propyl in each of the above structures is in the S configuration, i.e. on carbon 23 045111 shown with an asterisk. In some cases n-propyl

in each of the above structures is a mixture of R- and S-configurations, i.e. on the carbon shown with an asterisk. In some cases, n-propyl oA in each of the above structures is a mixture of R and S configurations, i.e. on the carbon indicated by an asterisk, where the R:S ratio is about 1:1, about 2:1, about 3;1, about 4:1, about 5:1, about 6;1, about 7:1, about 8: 1, about 9:1 or about 10:1.

In some embodiments, formula (I) provides a compound of formula 1d, 1e, 1g, 1h, 101a, 101b, 101c, 101d, 102c, 102d, 102e, 102f, 103a, 103b, 104a, or 104b, or a pharmaceutically acceptable salt, solvate thereof or stereoisomer. In some embodiments, Formula (I) provides a compound of Formula 1d, 1e, 1g, or 1h, or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof. In some embodiments, the n-propyl compounds of formula 1d, 1e, 1g, 1h, 101a, 101b, 101c, 101d, 102c, 102d, 102e, 102f, 103a, 103b, 104a, and 104b are in the R configuration. In some embodiments, the n-propyl compounds of Formula 1d, 1e, 1g, 1h, 101a, 101b, 101c, 101d, 102c, 102d, 102e, 102f, 103a, 103b, 104a, and 104b are in the S configuration. In some embodiments, the n-propyl compounds of formula 1d, 1e, 1g, 1h, 101a, 101b, 101c, 101d, 102c, 102d, 102e, 102f, 103a, 103b, 104a, and 104b are a mixture of R and S configurations. In some embodiments, the n-propyl compounds of Formula 1d, 1e, 1g, 1h, 101a, 101b, 101c, 101d, 102c, 102d, 102e, 102f, 103a, 103b, 104a, and 104b are a mixture of R and S configurations , where the R:S ratio is about 1:1, about 2:1, about 3;1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9: 1 or about 10:1.

Included within the scope of the present disclosure are pharmaceutically acceptable salts, solvates, crystalline forms, amorphous forms, polymorphic forms, regioisomers, stereoisomers, metabolites and physiological adducts of the steroid loads described herein.

C. Protein steroid conjugates.

Protein conjugates of the steroids described herein are presented here. Such conjugates include proteins, for example, antibodies or antigen-binding fragments thereof, which are covalently linked, for example, by -L- and -L1-L ² -(L ³ ) _0-1 - described herein to the compounds described above in section B.

In some or any embodiments, a compound of formula (3000): BA-(L-SP-D)z or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof is provided, wherein

D is selected from

A)

5 ^x

o formula (a), where both R ^x in formula (a) represent hydrogen; R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; and SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-, -C(O)-N(C _1-6 alkyl)-C ₁ -C ₁₀ -alkylene-X ¹ - , where X ¹ is added to L in formula (3000), -C(O)-N(H)-(C1-C10-alkylene)-S-, where S is added to L in formula (3000), -C(O )-N(C _1-6 alkyl)-(C ₁ -C ₁₀ -alkylene)-S-, where S is added to L in formula (3000), where the point of addition is on the right side (i.e. on N ) is added to L in formula (3000), -CH2-NH-, where N is added to L in formula (3000),

wherein N is attached to L in formula (3000) and where Ar is an optionally substituted arylene (in some embodiments) or an optionally substituted heteroarylene, -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₁₀ alkylene)-NR ^50a -, where NR ^50a is added to L in formula (3000), -C(O)-(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1C ₁₀ -alkylene) -NR ^50a - where NR ^50a is added to L in formula (3000) and where in each case the C1-C10 alkylene is independently and optionally replaced by one or more hydroxy, -C(O)-N(R ⁵ )-C1-C ₁₀ alkylene -C(O)NH-X ² -,

- 24 045111 where X ² is added to L in formula (3000) or formula (3000); or

where X ⁴ is added to L in where both R ^x in formula (a) represent fluorine; R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; and SP represents -C(O)-C1-C ₁₀ -alkylene-C(O)-, -C(O)-N(C _1-6 alkyl)-C ₁ -C ₁₀ -alkylene-X ^1b -, where X ^1b is added to L in formula (3000), -C(O)-N(H)-(C ₁ -C ₁₀ -alkylene)-X ^1b -, where X ^1b is added to L in formula (3000),

where the point of addition on the right side (i.e. Ha N) represents L in formula (3000), -CH2-NH-, where N is added to L in formula (3000),

wherein N is attached to L in formula (3000) and where Ar is an optionally substituted arylene (in some embodiments) or an optionally substituted heteroarylene, -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₁₀ alkylene)-NR ^50a -, where NR ^50a is added to L in formula (3000), -C(O)-(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1C ₁₀ -alkylene)- NR ^50a - where NR ^50a is added to Lb of formula (3000) and where in each case C1-C10alkylene is independently and optionally replaced by one or more hydroxy, -C(O)-N( ^R5 )-(C1-C10alkylene)-C (O)NH-X ² -, where X ² is added to L in formula (3000) or formula (3000); And

, where X ⁴ joins L in

b) compounds in the above table. And where are the connections in the table? A are connected to BA Compounds of formula (3000) through hydroxy -C(O)CH ₂ OH groups, i.e. via -C(O)CH2-O-SP-L- or via hydroxy Mapracorate, i.e. via -O-SP-L-:

X ¹ represents -N(C _1-6 alkyl)-;

X ^1b represents -S-, -NH- or -N(C _1-6 alkyl)-;

X ² represents -NH-;

X ³ represents -CH2-, X ³ represents -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where

C(O) is added to X ⁴ or X ³ is -C(O)-; X ⁴ represents -O-;

R ⁵ represents H, -OH, -OCH ₃ or C _1-6 alkyl;

R ⁵⁰ and R ^50a are independently hydrogen or C1-C ₆ alkyl;

R ^d , R ^e and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -CH2C(O)OH or -CH ₂ C(O)O(alkyl); and m is 0 or 1;

z is an integer selected from 1-30, inclusive;

L represents a linker; and BA is a coupling agent.

In some or any embodiments, a compound of formula (W-P) is provided:

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof, wherein

R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl;

SP represents -C(O)-C1-C ₁₀ -alkylene-C(O)-, -C(O)-N(C _1-3 alkyl)-C ₁ -C ₁₀ -alkylene-X ¹ -, where X ¹ is added to L in formula (III), -C(O)-N(R ⁵ )-C ₁ -C ₁₀ -alkylene-C(O)NH-X ² -, where X ² is added to L

- 25 045111

R ^e in formula (III) or ^xx , _where χ ⁴ is added to L in formula (III); X ¹ represents

-№(C _1-3 alkyl)-; X ² represents -NH-; X ³ is -CH2-, X ³ is -CH2-O(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ or X ³ is -C(O )-; X ⁴ represents -O-;

R ⁵ represents H, -OH, -OCH3 or alkyl; R ^d , R ^e and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -CH2C(O)OH or -CH ₂ C(O)O(alkyl); and m is 0 or 1;

z is an integer selected from 1-30, inclusive; L represents a linker; and BA is a coupling agent.

In some or any embodiments, a compound of formula (W-R-1) is provided:

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof, wherein:

R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl;

SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-, -C(O)-N(C _1-3 alkyl)-C ₁ -C ₁₀ -alkylene-X ¹ -, where X ¹ is added to L in formula (III), -C(O)-N(R ⁵ )-C ₁ -C ₁₀ -alkylene-C(O)NH-X ² -, where X ² is added to L

R ^e

S-V'L

...... : χ* ^s ' · „ 4 .......

in formula (III) or , where X is added to L in formula (III);

X ¹ represents -N(C _1-3 alkyl)-;

X ² represents -NH-;

X ³ is -CH2-, X ³ is -CH2-O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ , or X ³ is -C (O)-;

X ⁴ represents -O-;

R ⁵ represents H, -OH, -OCH3 or alkyl;

R ^d , R ^e and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -CH2C(O)OH or -CH ₂ C(O)O(alkyl); and m is 0 or 1;

z is an integer selected from 1-30, inclusive; L represents a linker; and BA is a coupling agent.

In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein R ⁴ is alkyl. In some embodiments, R ⁴ is non-cycloalkyl. In some of these embodiments, R ⁴ is straight or branched alkyl. In some of these embodiments, R ⁴ is aryl. In some of these embodiments, R ⁴ is arylalkyl. In some of these embodiments, R ⁴ is an N-containing heterocycloalkyl. In some embodiments, R ⁴ is alkyl, such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl. In some embodiments, R ⁴ is n-propyl. In some embodiments, R ⁴ is i-propyl. In some embodiments, R ⁴ is n-butyl. In some embodiments, R ⁴ is i-butyl. In some embodiments, R ⁴ is tbutyl. In some embodiments, R ⁴ is sec-butyl. In some embodiments, R ⁴ is pentyl. In some embodiments, R ⁴ is hexyl. In some embodiments, R ⁴ is heptyl. In some embodiments, R ⁴ is octyl or nonyl. In some embodiments, R ⁴ is aryl, such as, but not limited to, phenyl, phenol, or naphthyl. In some embodiments, R ⁴ is phenyl. In some embodiments, R ⁴ is naphthyl. In some embodiments, R ⁴ is heteroaryl - such as, but not limited to, thienyl. In some embodiments, R ⁴ is arylalkyl - such as, but not limited to, benzyl. In some embodiments, R ⁴ is N-26045111 containing heterocycloalkyl, such as, but not limited to, piperidinyl. In some or any embodiments, both R ^x are hydrogen. In some or any embodiments, both R ^x are fluorine. In some embodiments, R ⁴ is in the R configuration. In some embodiments, R ⁴ is in the S configuration. In some embodiments, R ⁴ is a mixture of R and S configurations. In some embodiments, R ⁴ is a mixture of R and S configurations, wherein the R:S ratio is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6; 1, about 7:1, about 8:1, about 9:1 or about 10:1.

In some embodiments, a compound of formula (III) is provided wherein both R ^x are hydrogen; and SP is -C(O)-C1-C10-alkylene-C(O)-, -C(O)-N(C1_6alkyl)-C1Ci-alkylene-X ¹ -, where X ¹ is attached to L in the formula ( III), (III-P) or (Ш-Р-1); -C(0)-N(H)-(C1-C ₁₀ alkylene)-S-, where S is added to L in formula (III), (III-P) or (III-P-1); -C(O)-N(C ₁ _ ₆ alkyl)-(C ₁ -C ₁₀ alkylene)-S-, where S is added to L in formula (III), (III-P) or (III-P-1 ); -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to L in formula (III), (III-P) or (III- P-1); -C(0)-(C1-C ₁₀ alkylene)-NR ⁵⁰ C(O)-(C1-C10 alkylene)-NR ^50a -, where NR ^50a is added to L in formula (III), (III-P) or (IIIP-1), and wherein in each case the C1-C ₁₀ -alkylene is independently and optionally replaced by one or more hydroxy; -C(O)-N(R ⁵ )-C ₁ -C ₁₀ -alkylene-C(O)NH-X ² -, where X ² is added to L in formula (III), (III-P)

R ^e

Rt D7 ^R p y η 'm or (III-P-1); or where X ⁴ is added to L in formula (III), (III-P) or (III-P-1); or both R ^x are fluorine; and SP is -C(O)-N(C1_6alkyl)-C1-C10-alkylene-X ^1b -, where X ^1b is attached to L in formula (III), (III-P) or (III-P-1) ; -C(O)-N(H)-(C1-C ₁₀ -alkylene)-X ^1b -, where X ^1b

O is added to L in formula (III), (III-P) or (III-P-1); , where the attachment point on the right side (i.e. on N) represents to L in formula (III); -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-alkylene)NR ^50a -, where NR ^50a is added to L in formula (III), (III-P) or (III- P-1); -C(O)-(C1- _C10- alkylene)NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to L in formula (III), (III-P) or (III-P-1) and where in each case a C1-C ₁₀ -alkylene is independently and optionally replaced by one or more hydroxy, or -C(O)-N(R ⁵ )-(C ₁ -C ₁₀ -alkylene) -C(O)NH-X ² -, where X ² is added to L in formula (III), (III-P) or (III-P-1).

In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided wherein R ^x is both hydrogen and SP is -C(O)-C ₁ -C ₁₀ -alkylene -C(O)-. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is -C(O)-C ₂ -C ₅ -alkylene-C(O)-. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is -C(O)-CH ₂ CH ₂ c(0)-.

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x is fluorine and SP is -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-. In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-C ₂ -C ₅ alkylene-C(O)-. In some embodiments, provided herein is a compound of formula (II) wherein SP is -C(O)-CH ₂ CH ₂ -C(O)-.

In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided wherein R ^x is both hydrogen and SP is -C(O)-N(C1_6alkyl)-C1- C10alkylene-X ¹ -, where X ¹ is added to L ³ in formula (III), (III-P) or (III-P-1). In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided, wherein SP is -C(O)-N(C1_ ₃ alkyl)-C ₁ -C ₁₀ -alkylene -X ¹ -, where X ¹ is attached to L ³ in formula (III), (III-P) or (IIIP-1). In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein X ¹ is -N(C1_3alkyl)-. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is -C(O)-N(C1_3alkyl)C2-C5-alkylene-X1-. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is -C(O)-N(C1_3alkyl)-CH2CH2-X ¹ -. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is -C(O)-N(CH3)-C ₂ -C ₅ -alkylene-N (CH ₃ )-. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is -C(O)-N(CH ₃ )-CH ₂ CH ₂ -N(CH ₃ )-.

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x is fluorine and SP is -C(O)-N(C1_6alkyl)-C1-C10-alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (III) or -C(O)-N(H)-(C1-C ₁₀ -alkylene)-X ^1b -, where X ^1b is added to L in (III formula). In some embodiments, a compound of formula (III) is provided herein, wherein SP

- 27 045111 is -C(O)-N(H)-C ₁ -C ₁₀ -alkylene-X ^1b -, where X ^1b is added to L ³ in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-C1-C6-alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-C1C3-alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-CH2CH2-X ^1b -. In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-C ₂ -C ₅ -alkylene-N(CH ₃ )-. In some embodiments, provided herein is a compound of formula (III) wherein SP is -C(O)-N(H)-C ₂ -C ₅ -alkylene-N(CH ₂ CH ₃ )-. In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-CH2CH2-N(CH3)-.

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x are fluorine and SP is -C(O)-N(C1-6alkyl)-C1-C10-alkylene-X ^1b -, wherein X+ is attached to L ³ in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N( _C1.3alkyl ) _{-C1- C6-} alkylene- ^X1b- , wherein X+ is attached to ^L3 in the formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(C1.3alkyl)-C1-C3-alkylene-X ^1b -, wherein X+ is attached to L ³ in formula (III) . In some embodiments, a compound of formula (III) is provided herein, wherein Xlb is -N(C1-3alkyl)-. In some embodiments, provided herein is a compound of formula (III), wherein SP is -C(O)-N(C _1-3 alkyl)-C ₂ -C ₅ -alkylene-X ^1b -. In some embodiments, provided herein is a compound of formula (III) wherein SP is C(O)-N(C1.3alkyl)-CH2CH2-X ^1b -. In some embodiments, provided herein is a compound of formula (III) wherein SP is -C(O)-N(CH3)-C ₂ -C ₅ -alkylene-N(CH ₃ )-. In some embodiments, a compound of formula (III) is provided wherein SP is -C(O)N(CH ₂ CH ₃ )-C ₂ -C ₅ -alkylene-N(CH ₂ CH ₃ )-. In some embodiments, provided herein is a compound of formula (III) wherein SP is -C(O)-N(CH ₃ )-CH ₂ CH ₂ -N(CH ₃ )-.

In some embodiments, provided herein is a compound of formula (III) wherein R ^x O 4'^'Ν'^ι is both fluorine and SP is , where the point of attachment on the right side (i.e., on N) is L in formula (III).

In some embodiments, provided herein is a compound of formula (III) wherein R ^x is both hydrogen and SP is -C(O)-N(H)-(C ₁ -C ₁₀ -alkylene)-S-, wherein S is added to L in formula (III) or -C(O)-N(C _1-6 alkyl)-(C ₁ -C ₁₀ -alkylene)-S-, where S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-(C ₁ -C ₆ -alkylene)-S-, wherein S is added to L in formula (III) or -C(O)-N(C1 ₃ αlkyl)-(C ₁ -C ₆ -alkylene)-S-, where S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-(C1-C ₁₀ alkylene)-S-, wherein S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-(C ₁ -C ₆ -alkylene)-S-, wherein S is added to L in formula (III) . In some embodiments, a compound of formula (III) is provided, wherein SP-x-C(O)-N(C ₁ . ₆ alkyl)-(C ₁ -C ₁₀ -alkylene)-S-, wherein S is added to L in formula (III). In some embodiments, _{a compound of formula (III) is provided herein, wherein SP is -C(O)-N(C1.3αalkyl )} -( _C1- C6-alkylene)-S-, wherein S is added to L in the formula (III).

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x is fluorine and SP is -C(O)-N(H)-(C ₁ -C ₁₀ -alkylene)-S-, wherein S is added to L in formula (III) or -C(O)-N(C ₁ . ₆ alkyl)-(C ₁ -C ₁₀ -alkylene)-S-, where S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-(C ₁ -C ₆ -alkylene)-S-, wherein S is added to L in formula (III) or -C(O)-N(C1_ ₃ αlkyl)-(C ₁ -C ₆ -alkylene)-S-, where S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-(C1-C ₁₀ alkylene)-S-, wherein S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(H)-(C ₁ -C ₆ -alkylene)-S-, wherein S is added to L in formula (III) . In some embodiments, provided herein is a compound of formula (III), wherein SP -x -C(O)-N(C _1-6 alkyl)-(C ₁ -C ₁₀ -alkylene)-S-, wherein S is added to L in formula (III). In some embodiments, a compound of formula (III) is provided herein, wherein SP is -C(O)-N(C _1-3 αalkyl)-(C ₁ -C6-alkylene)-S-, wherein S is added to L in the formula (III).

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x are hydrogen and SP is -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)NR ^50a - where NR ^50a is added to L in formula (III). In some or any embodiments

- 28 045111 formula (I), SP represents -(C ₁ -C ₆ -alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₆ -alkylene)-NR ^50a -. In some or any embodiments of formula (III), SP is -(C ₁ -C ₃ -alkylene)-NR ⁵⁰ C(O)-(C ₁ C ₃ -alkylene)-NR ^50a -. In some or any embodiments of formula (III), SP is -(linear C ₁ -C ₃ -alkylene)-NR ⁵⁰ C(O)-(linear C ₁ -C ₃ -alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (III) wherein R ^x is both fluorine and SP is -(C1-C10 alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ alkylene)-NR ^50a -, where NR ^50a is added to L in formula (III). In some or any embodiments of Formula (I), SP is -(C1-C6-alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ -alkylene)-NR ^50a -. In some or any embodiments of formula (III), SP is -(C1-C3 alkylene)-NR ⁵⁰ C(O)-(C1-C ₃ alkylene)-NR ^50a -. In some or any embodiments of formula (III), SP is -(linear C1-C3 alkylene)-NR ⁵⁰ C(O)-(linear C1-C ₃ alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x are hydrogen and SP is -C(O)-(C1-C10 alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ alkylene )-NR ^50a -, where NR ^50a - is added to L in formula (III) and where in each case the C1-C10 alkylene is independently and optionally replaced by one or more hydroxy. In some or any embodiments of formula (III), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1C ₆ -hydroxyalkylene)-NR ^50a -. In some or any embodiments of formula (III), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (III) wherein R ^x is both fluorine and SP is -C(O)-(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ - alkylene)NR ^50a -, where NR ^50a - is added to L in formula (III) and where in each case the C1-C10 alkylene is independently and optionally replaced by one or more hydroxy. In some or any embodiments of formula (III), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ hydroxyalkylene)-NR ^50a -. In some or any embodiments of formula (III), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x are hydrogen and SP is -C(O)-N(R ⁵ )-C1-C10-alkylene-C(O)NH-X ² - , where X ² is added to L ³ in formula (III). In some embodiments, a compound of formula (III) is provided wherein SP is -C(O)-N(R ⁵ )-C1-C ₅ -alkylene-C(O)NH-NH-. In some embodiments, provided herein is a compound of formula (III) wherein SP is -C(O)N(R ⁵ )-CH ₂ -C(O)NH-NH-. In some embodiments, a compound of formula (III) is provided herein, wherein R ⁵ is H or alkyl. In some embodiments, a compound of formula (III) is provided herein, wherein R ⁵ is H or CH3.

In some embodiments, provided herein is a compound of formula (III) wherein both R ^x is fluorine and SP is -C(O)-N(R ⁵ )-C1-C10-alkylene-C(O)NH-X ² - , where X ² is added to L ³ in formula (III). In some embodiments, a compound of formula (III) is provided wherein SP is -C(O)-N(R ⁵ )-C1-C ₅ -alkylene-C(O)NH-NH-. In some embodiments, provided herein is a compound of formula (III) wherein SP is -C(O)N(R ⁵ )-CH2-C(O)NH-NH-. In some embodiments, a compound of formula (III) is provided herein, wherein R ⁵ is H or alkyl. In some embodiments, a compound of formula (III) is provided herein, wherein R ⁵ is H or CH3.

In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein R ^x is both hydrogen and SP is

where X ⁴ is added to L ³ in formula (III), (III-P) or (III-P-1). In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein R ^x is both fluorine and SP is

where X ⁴ is added to L ³ in formula (III), (III-P) or (III-P-1). In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP

and X ³ is -CH ₂ -. In some embodiments, the implementation here pre- 29 045111

R* ^R y% ^R 'L i X* X* is a compound of formula (III), (III-P) or (III-P-1), where SP is and

X ³ is -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ . In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein SP is R·

- „ bх<-Х»% / „3, „ „ represents and X ³ represents -C(O)-. In some embodiments, provided herein is a compound of formula (III), (III-P), or (III-P-1), wherein R ^d , R ^e , and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C (O)OH or -CH2OR ⁸ , where in each case R ^g is independently -CH ₂ C(O)OH or -CH ₂ C(O)O(alkyl). In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided herein, wherein R ^d and R ^e are independently -H or -OH. In some embodiments, herein is a compound of formula (III), (III-P), or (III-P-1), where m is 0. In some embodiments, herein is a compound of formula (III), (III-P), or (III-P-ι), where m is 1. In some embodiments, a compound of formula (III), (III-P), or (III-P-1), where m is 1 and R ^f is -N or -OH.

In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), wherein both R ^x are hydrogen and SP is -C(O)-N(C _1-3 -alkyl -C ₁ -C ₁₀ -alkylene-X ¹ -, where X ¹ is attached to L ³ in formula (III), (III-P) or (III-P-1); SP represents

R ^Ra /V ^R, L ' where X ³ represents -CH2-, m is 1, and R ^d , R ^e and R ^f represents -H;

R

VVl™... . _ „3... . ... ..... . . . , or SP represents ' where X ³ represents -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)C(O)-, where C(O) is added to X ⁴ , m represents 1, and R ^d , R ^e and R ^f -H. In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided, wherein SP is -

C(O)-N(CH3)-CH2CH2-N(CH3)-,

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided, wherein both R ^x is fluorine and SP is -C(O)-N(C _1-3 alkyl) -C ₁ -C ₁₀ -alkylene-X ¹ -, where

R

Rd D/ R ^f

X ¹ is attached to L ³ in formula (III), (III-P) or (III-P-1); SP represents ' where X ³ represents -CH2-, m is 1, and R ^d , R ^e and R ^f represents -H; or SP represents

R

Λ is ^e , where X ³ represents -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ , m represents 1, and R ^d . R ^e and R ^f represents -H. In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), where SP is C(O)-N(CH3)-CH2CH2-N(CH3)-,

or

In some embodiments, a compound of formula (III) is provided wherein both R ^x are fluorine or both R ^x are hydrogen or R ^x is as described in is

SP this paragraph;

(where both R ^x represent

- 30 045111 is fluorine), ⁰ (where both R ^x are fluorine),

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided, wherein z is 1. In certain embodiments, z is 2. In certain other embodiments, z is 3. In certain embodiments In some embodiments, z is equal to 4. In some embodiments, z is equal to 5. In some other embodiments, z is equal to 6. In certain embodiments, z is equal to 7. In some other embodiments, z is equal to 8. In certain embodiments, z is equal to 9. In some In some other embodiments, z is 10. In some other embodiments, z is 11. In some other embodiments, z is 12. In some other embodiments, z is 13. In some other embodiments, z is 14. In some other embodiments, z is 15. In some other embodiments, z is equal to 15. In some other embodiments, z is equal to 16. In some other embodiments, z is equal to 17. In some other embodiments, z is equal to 18. In certain other embodiments, z is equal to 19. In some other embodiments z is 20. In some other embodiments, z is 21. In some other embodiments, z is 22. In some other embodiments, z is 23. In some other embodiments, z is 24. In some other embodiments, z is 25 In some other embodiments, z is equal to 26. In some other embodiments, z is equal to 27. In some other embodiments, z is equal to 28. In some other embodiments, z is equal to 29. In some other embodiments, z is equal to 30. In some other embodiments, z is equal to 29. In some other embodiments, z is equal to 30. z is an integer selected from 1-15, inclusive. In certain embodiments, z is selected from 1-10. In certain embodiments, z is selected from 1-5. In certain embodiments, z is selected from 2-5. In certain embodiments, z is selected from 2-4.

In some embodiments, -L- or -L ¹ -L ² -(L ³ )0.1- in formula (III), (III-P) or (III-P-1) means any divalent group or moiety that binds or combines with a binding agent (eg, an antibody or an antigen-binding fragment thereof) with a loading compound provided herein (eg, a steroid). In general, -L- and -L ¹ -L ² -(L ³ )0 are suitable. ₁ - for the antibody conjugates described herein, are those that are sufficiently stable to utilize the circulating half-life of the antibody and at the same time capable of releasing their load after antigen-mediated internalization of the conjugate. Linkers can be cleavable or non-cleavable. Cleavable linkers are linkers that are cleaved through intracellular metabolism after internalization, such as cleavage through hydrolysis, reduction, or an enzymatic reaction. Non-cleavable linkers are linkers that release the attached load through lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction-labile linkers, self-cleavable linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, linkers that are or contain glucuronides, succinimide thioesters, polyethylene glycol (PEG) units, hydrazones, malcaproyl units, disulfide units (eg, -SS-, -SC(R ^1b R ^2b )- where R ^1b and R ^2b are independently hydrogen or hydrocarbyl), carbamate units, para-amino-benzyl units (PAB), phosphate units, e.g. mono-, bis- or tris-phosphate units, peptides, e.g. peptide units containing two, three, four, five, six, seven, eight or more amino acid units, including, but not limited to, valine-citrulline units, valine-alanine units, valine-arginine units, valine-lysine units, lysine-valine units citrulline and lysine-valine-alanine units. In some embodiments, -L- and -L ¹ -L ² -(L ³ )0.1- are obtained from the reaction of a reactive group (RG) of formula (II) (and embodiments thereof) with a reactive moiety of an antibody. A connecting group is any divalent moiety that connects the VA to the load. The connecting group can also be any trivalent moiety that connects the BA, the load, and the cyclodextrin moiety. In some embodiments, -L- and -L ¹ -L ² -(L ³ )0. ₁ - are trivalent and include a cyclodextrin moiety linked to a trivalent group (eg, a lysine residue) in a connecting group. -L- and -L ¹ -L ² -(L ³ ) ₀ . ₁ - contain a reactive group residue, which is a functional group or fragment that reacts with the reactive part of an antibody, modified antibody or antigen-binding fragment thereof.

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

- 31 045111

or a regioisomer or a mixture of isomers thereof;

where L and -L ¹ -L ² -(L ³ ) _o-1 - contain

ABOUT

or a stereoisomer or a mixture of stereoisomers thereof, where S is an S atom

on a cysteine residue through which the reactive group residue is attached to BA; or where N is the N atom on the lysine residue through which the reactive group residue is attached to BA.

In some embodiments, a compound of formula (III), (III-P) or (III-P-1) is provided, wherein L and -L ¹ -L ² -L ³ - contain

.

In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), Q-aa yCrb where L and -L ¹ -L ² -(L ³ ) _o-1 - contain ^ ^{= N} '' or regioisomer or mixture of isomers thereof;

or a stereoisomer or a mixture of stereoisomers thereof, where S is the S atom on the cysteine residue through which the reactive group residue is attached to BA; or

where N is the N atom on the lysine residue through which the reactive group residue is attached to BA; and L and -L ¹ -L ² -(L ³ ) _o-1 - contain

In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), where L represents -L ¹ -L ² -(L ³ ) _o-1 -. In some embodiments, L ³ is present. In some embodiments, L ³ is absent.

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

isomers;

where L represents -L ¹ -L ² -(L ³ ) _o-1 - and L ¹ is selected from

or a regioisomer or mixture thereof or a stereoisomer or mixture of stereoisomers thereof, where S is the S atom on the cysteine residue through which the reactive group residue is attached to

VA; And

where N denotes the N atom on the lysine residue through which the reactive group residue is attached to BA. In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided, wherein L is -L ¹ -L ² -(L ³ ) _o-1 - and L ¹ is

- 32 045111

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

where L represents -L ¹ -L ² -(L ³ ) _0-1 - and L ¹ represents

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

2 1 where L represents -L ¹ -L ² -(L ³ ) _0-1 - and L ¹ represents

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

where L represents -L ¹ -L ² -(L ³ ) _0-1 - and L ¹ represents

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

where L represents -L ¹ -L ² -(L ³ ) _0-1 - and L ¹ represents

In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), cL

N where L represents -L ¹ -L ² -(L ³ ) _0-1 - and L ³ represents

' where the NH group is attached to L ² when SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-; or L ³ represents

, where an NH group is attached to L ² when SP is -C(O)-N(C1-3alkyl)C1-C10-alkylene-X ¹ - or -C(O)-N( ^R5 )-C1-C ₁₀ -alkylene-C(O)NH-X ² -; or L ³ is not present when

. In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), where L represents -L1-L ² -(L ³ )- and L ³ represents H, where NH group attaches to L ² when SP is -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-. In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided, wherein L is -L ¹ -L ² -(L ³ ) _0-1 - and L ³ is

where an NH group is attached to L ² when SP is -C(O)-N(C1-3alkyl)-C1-C10-alkylene-X ¹ -or -C(O)-N( ^R5 )-C1- C ₁₀ -alkyleneC(O)NH-X ² -. In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), where L is -L ¹ -L ² -(L ³ ) _0-1 - and L ³ is not present, when SP represents

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

where L represents -L ¹ -L ² -(L ³ ) _0-1 - and L ² contains

- 33 045111

, -OCH ₂ C(O)where L represents -L ¹ -L ² -(L ³ ) _o _ ₁ - and L ² contains either a cyclodextrin residue (CD); or combinations thereof.

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided.

or CD or combinations thereof. In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), where L represents -L ¹ -L ² -(L ³ ) _{o_1 -} and L ² contains CD, where CD is selected from the group consisting of

- 34 045111

In some embodiments, the implementation is a compound of formula (III), (III-P) or (III-P-1), where L represents -Ε1-Ε ² -(Ε ³ )0-1- and -L ² -(L ³ )0-1- contain

- 35 045111

- 36 045111

- 37 045111

In some embodiments, a compound of formula (III), (III-P), or (III-P-1) is provided, wherein L ² contains

; L ^1a represents a reactive group residue; CD is a cyclodextrin; AA ⁴ is a trivalent linker; AA ⁵ is a peptide residue; PEG is polyethylene glycol; a is an integer that can be selected from 0 to 5, inclusive.

In some embodiments, AA ⁴ is an amino acid residue; AA ⁵ is a dipeptide residue, tripeptide residue, tetrapeptide residue or pentapeptide residue; PEG is a polyethylene glycol moiety; where Y denotes the atom through which the specified chemical group binds to adjacent groups in the formula, CD, in each case, is independently absent or is a cyclodextrin residue, where at least one CD is present, the subscript a is an integer, which is selected from 0 to 5, inclusive; in these examples, the index a is 0, 1, 2, 3, 4, or 5. In some embodiments, the index

- 38 045111 a is 0. In some embodiments, the index a is 1. In some embodiments, the index a is 2. In some embodiments, the index a is 3. In some embodiments, the index a is 4. In some embodiments, the index a is 5. In some embodiments, any of AA ⁴ or AA ⁵ contains, independently in each case, an amino acid that is selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine , glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or citrulline, a derivative thereof, or a combination thereof. In certain embodiments, AA ⁴ is an amino acid that is selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine , histidine or citrulline, a derivative thereof or a combination thereof. In certain embodiments, AA ⁴ is lysine or a lysine derivative. In certain embodiments, AA ⁴ is lysine. In certain embodiments, AA ⁴ is D-lysine. In certain embodiments, AA ⁴ is L-lysine. In certain embodiments, AA ⁵ is a dipeptide residue. In certain embodiments, AA ⁵ is valine-citrulline. In some embodiments, AA ⁵ is citrulline-valine. In some embodiments, AA ⁵ is valine-alanine. In some embodiments, AA ⁵ is alanine-valine. In some embodiments, AA ⁵ is valine-glycine. In some embodiments, AA ⁵ is glycine-valine. In certain embodiments, AA ⁵ is a tripeptide residue. In some embodiments, AA ⁵ is glutamate-valine-citrulline. In some embodiments, AA ⁵ is glutaminevaline-citrulline. In some embodiments, AA ⁵ is lysine-valine-alanine. In some embodiments, AA ⁵ is lysine-valine-citrulline. In some embodiments, AA ⁵ is glutamate-valine-citrulline. In some embodiments, AA ⁵ is a tetrapeptide residue. In some embodiments, AA ⁵ is glycine-glycine-phenylalanine-glycine. In some embodiments, AA ⁵ is a tetrapeptide residue. In some embodiments, AA ⁵ is glycine-glycine-glycine-glycine-glycine.

In certain embodiments, the reactive group residue is derived from the reaction of RG with a cysteine or lysine residue of an antibody or antigen binding fragment thereof. In certain embodiments, the reactive group moiety is derived from a click chemistry reaction. In some embodiments, said click chemistry reaction L ¹ is derived from a 1,3 cycloaddition reaction between an alkyne and an azide. Non-limiting examples of such a reactive moiety include residues derived from strained alkynes, e.g., moieties suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzannelated alkynes, and alkynes capable of undergoing 1,3 cycloaddition reactions with azides in the absence of copper-based catalysts. Suitable L ¹ also include, but are not limited to, fragments derived from DIBAC (where the -C(O)CH ₂ CH ₂ C(O)- portion of the DIBAC fragment is covered

L ² ), DIBO (where the -O- portion of the DIBO moiety is capped by L ² ), BARAC (where the BARAC moiety is capped by L ² ), substituted alkynes, eg fluorinated alkynes, azacycloalkynes, BCN and derivatives thereof. Conjugates containing such reactive group residues can be prepared from antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such a functionalized antibody is prepared by reacting an antibody containing at least one glutamine residue with a compound of the formula H2N-LLN3, where LL is a divalent polyethylene glycol group, in the presence of a transglutaminase enzyme, for example, microbial transglutaminase. Suitable antibody glutamine residues include Q295 or those generated by insertion or mutation, for example the N297Q mutation.

In some embodiments, the BA of the conjugates described herein is an antibody or an antigen-binding fragment thereof. In some embodiments, the conjugates described herein are derived from azido-functionalized antibodies. In certain embodiments, the VA of the conjugates described herein is:

where Ab is an antibody or an antigen-binding fragment thereof, n is an integer selected from 1 to 10, inclusive, z is the number of linker loads, and

- 39 045111 g represents a bond with L or -L ¹ -L ² -(L ³ ) _0- i-, for example, a bond with a fragment obtained from a reaction

1,3-cycloaddition between alkyne and azide. In certain embodiments, z is 3. In certain embodiments, z is 2 or 4, i.e. the conjugate contains 2 or 4 linker loads.

In some embodiments, -L- and -L ¹ -L ² -L ³ - is a divalent moiety of formula (LA)

-L ¹ -(SP ¹ )q-(A)z-(NR ^a ) _s- (B)t-(CH ₂ )u-(O) _v -( SP ² ) _Wi (L ^A );

where L ¹ corresponds to the definition given here;

A is an amino acid or a peptide;

R ^a represents H or alkyl;

B is aryl, heteroaryl or heterocycloalkyl, wherein aryl, heteroaryl or heterocycloalkyl is optionally replaced by alkyl, -OH or -NR ^a R ^b ;

SP ¹ and SP ² are independently spacer groups; and q, z, s, t, u, v and w, independently in each case, 0 or 1.

In some other embodiments, -L- and -L ¹ -L ² -(L ³ ) ₀ - ₁ - is a trivalent moiety with the formula (L ^B )

-iacspMaxnrvcb )t-(CH ₂ )u-(O)y-( sp ² ) _w (L ^B );

where L ¹ corresponds to the definition given here; A is a tripeptide or tetrapeptide, wherein at least one of the amino acids in the tripeptide or tetrapeptide binds directly or indirectly to a cyclodextrin moiety; R ^a represents H or alkyl;

B is aryl, heteroaryl or heterocycloalkyl, wherein aryl, heteroaryl or heterocycloalkyl is optionally replaced by alkyl, -OH or -NR ^a R ^b ; SP ¹ and SP ² are independently spacer groups; and q, z, s, t, u, v and w, independently in each case, 0 or 1.

In some embodiments, the cyclodextrin (CD) binds directly to an amino acid residue, such as a lysine amino acid residue. This means that the CD is one bond away from the covalent linker of the amino acid lysine. In some of these embodiments, the covalent linker is also directly coupled to the load. This means that the covalent linker is one bond away from the load, such as, but not limited to, the steroid load described herein. In some of these embodiments, the covalent linker also directly binds to the CD. This means that the covalent linker is one bond away from the CD, such as the CDs described herein. In some of these embodiments, the covalent linker is the amino acid lysine or a derivative thereof.

In some embodiments, the CD indirectly binds to a covalent linker in the connecting group (eg, L, -L ² -(L ³ )0-1- and -L ¹ -L ² -(L ³ ) _0-1 ). This means that the CD is more than one bond away from the covalent linker. This also means that the CD binds through another moiety to a covalent linker. For example, CD can bind to a maleimide group, which binds to a polyethylene glycol group, which binds to a covalent linker. In some of these embodiments, the covalent linker is also indirectly coupled to the load. This means that the covalent linker is more than one bond away from the load, such as, but not limited to, the steroid load described herein. This also means that the covalent linker binds through another moiety to the load. For example, the covalent linker may be linked to a dipeptide, such as, but not limited to, Val-Ala or Val-Cit, which may be linked to a para-amino benzoyl, which may be linked to a load. In some of these embodiments, the covalent linker also indirectly binds to the cyclodextrin. This means that the covalent linker is more than one bond away from the cyclodextrin, such as the cyclodextrins described herein. This also means that the covalent linker binds through another moiety to the cyclodextrin. For example, the covalent linker may be linked to a polyethylene glycol group, which may be linked to a reactive group with a cyclodextrin. In some of these embodiments, the covalent linker is the amino acid lysine or a derivative thereof.

In some embodiments, -L- and -L ¹ -L ² -(L ³ )0-1- is -L ¹ -(SP ¹ )q-(A) _z -. In some embodiments, -L- and -L ¹ -L ² -(L ³ )0-1- is -L ¹ -(SP ¹ )q-(A) ₂ -. In some embodiments, -L- and -L ¹ -L ² -(L ³ ) ₀ - ₁ - is a fragment with the formula (L ^A1 )

About R^ ¹

O(L ^A1 )

- 40 045111 where RAA ¹ and RAA ² represent in each case, independently, amino acid side chains. In some embodiments of formula LA ¹ , SP ¹ is a divalent polyethylene glycol group and L ¹ is a 1,3-cycloaddition reaction product between an alkyne and an azide.

In some embodiments, -L- and -L ¹ -L ² -(L ³ )0-1- is -L ¹ -(SP ¹ )q-(A)z-. In some embodiments, -L- and -L ¹ -L ² -(L ³ )0_1- is -L ¹ -(SP ¹ )q-(A) ₂ -. In some embodiments, -L- and -L ¹ -L ² -(L ³ ) ₀ - ₁ - is a fragment with the formula (L ^B1 )

AA1 AA2 ^(LB1) where RAA ¹ and RAA ² represent in each case, independently, amino acid side chains. R ^AA3 is an amino acid side chain that is linked directly or indirectly to cyclodextrin. In some embodiments of formula L ^B1 , SP ¹ is a divalent polyethylene glycol group and L ¹ is the product of a 1,3 cycloaddition reaction between an alkyne and an azide.

In some embodiments, -L- or -L ¹ -L ² -L ³ - has the following structure: -lACSP^qZ^-ZVi wherein L ¹ , SP ¹ are as defined herein;

q represents 0 or 1;

Z ¹ represents a polyethylene glycol or caproyl group;

Z ² is a dipeptide, tripeptide or tetrapeptide; And

Z ³ represents a PAB group.

In certain embodiments, L ¹ is derived from a click chemistry reactive group and Z ¹ is a polyethylene glycol group. In certain embodiments, L ¹ -(SP ¹ ) _q Z ¹ - is:

or a mixture thereof; or ^N

In some embodiments, the dipeptide is valine-citrulline or valinalanine. In some embodiments, the tripeptide is glutamate-valinecitrulline. In some embodiments, the tetrapeptide is glycine-glycine-phenylalanine-glycine.

In some embodiments herein, L ¹ is derived from a click chemistry reactive group. In some embodiments, L ¹

represents:

or a mixture thereof;

or a mixture thereof;

BA where represents the bond to the binding agent.

In certain other examples herein, L ¹ is selected from the group that reacts with a cysteine or lysine residue on an antibody or antigen binding fragment thereof. In some embodiments, L ¹ is

G' where ^g represents a bond to the cysteine of a coupling agent, such as an antibody. In some embodiments, L ¹

- 41 045111

represents

In some embodiments, SP ¹ is selected from:

ABOUT

• In some other embodiments, SP ¹ is, in other examples, SP ¹ is

In other examples, SP ¹ represents

In other examples, SP ¹ represents

ABOUT

In some other examples SP ¹ . In any of the above examples, the subscripts a, b, and c are independently, in each case, an integer that is selected from 1 to 20, inclusive.

HN

Ν

In some embodiments, RAA ³ is selected from

CD cyclodextrin fragment. In some embodiments, RAA ³

’ where CD represents selected from

In some embodiments, SP ¹ is selected from:

- 42 045111

O

In some embodiments, SP ¹ is °

In some embodiments, SP ¹ is

. In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

A°. In some embodiments, SP ¹ represents

BUT'' γ^ 'ΌΗ ^0- \ is OH. In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, -L ¹ -L ² - comprise:

or a mixture thereof;

- 43 045111

or a mixture thereof; or °, where b, c and d are independently, in each case, an integer that is chosen from 1 to 20, inclusive. In some of these embodiments, the subscripts b, c, and d are independently, in each case, an integer that is selected from 1 to 6, inclusive.

In some embodiments, -SP ¹ is selected from:

or mixtures thereof;

-44045111 or mixtures thereof;

In some embodiments, A is a peptide that is selected from valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine, serine-asparagine, asparagine- serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acidasparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulli on, alanine paragine or asparagine-alanine.

In some embodiments, A is valine-citrulline or citrulline valine.

In some embodiments, A is valine-alanine or alanine-valine.

In some embodiments, A is lysine-valine-alanine or alanine-valine-lysine.

In some embodiments, A is lysine-valine-citrulline or citrulline-valine-lysine.

In some embodiments, A is glutamate-valine-citrulline or citrulline-valine glutamate.

In some embodiments, A is valine. In some embodiments, A is alanine. In some embodiments, A is citrulline.

In some embodiments, A is

In some of these embodiments, RAA ¹ is an amino acid side chain and wherein RAA ² is an amino acid side chain.

O R ^M1 _w O

ΑΥϊΑ l . . RAA3 ^H A rAA2

In some embodiments, A is

- 45 045111

In some embodiments, RAA ¹ is an amino acid side chain,

RAA ² is an amino acid side chain and RAA ³ is an amino acid side chain that binds directly or indirectly to the cyclodextrin moiety.

nR^ ²

In some embodiments, A is

In some embodiments, A is

or

In some embodiments, A is

In some embodiments, A is a direct or indirect linkage to a cyclodextrin moiety.

In some embodiments, including any of the above, the CD is, independently in each case, selected from

- 46 045111

In some embodiments, the CD is

- 47 045111

In some embodiments, the CD is

In some embodiments, the CD is

- 48 045111

In some embodiments, the CD is

In some embodiments, A is

- 49 045111

or its isomer.

In some embodiments, R ^a is H.

In some embodiments, R ^a is alkyl.

In some embodiments, R ^a is methyl, ethyl, n-propyl, i-propyl, nbutyl, t-butyl, i-butyl, or pentyl.

In some embodiments, B is aryl.

In some embodiments, B is phenyl.

In some embodiments, B is phenyl or pyridinyl.

In some embodiments, here B is:

In these examples, R ¹⁰ is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylaryl, arylalkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, O

-|-OR« -i-SO ₂ RA JJLrA

NR ^a R ^b or azido. In these examples, the indices p and m are independently, in each case, selected from an integer that is selected from 0 to 4, inclusive.

In some embodiments herein, B is:

In these examples, p is 0, 1, 2, 3, or 4. In some of these embodiments, R ¹ is, in each case, alkyl, alkoxy, haloalkyl, or halo. In some embodiments, R ¹ is alkyl. In some embodiments, R ¹ is alkoxy. In some embodiments, R ¹ is haloalkyl. In some embodiments, R ¹ is a halo.

In some embodiments, the formula (L ^A ), -(NR ^a ) _s -(B) _t -(CH ₂ ) _u -(O) _v -(SP ² )w is:

In any embodiments of L or L ¹ - L ² -(L ³ )0-1 described herein, one or more enhancing groups may be connected to the side chain of any amino acid in L or L ¹ - L ² -(L ³ )0-1. Useful amino acids for linking enhancing groups include lysine, asparagine, aspartate, glutamine, glutamate and citrulline. The linkage to the enhancing group may be a direct linkage to the amino acid side chain, or the linkage may be indirect via a spacer and/or reactive group. Useful spacers and reactive groups include any described herein. The enhancing group can be any group that is considered useful by those skilled in the art. For example, an enhancing group can be any group that has a beneficial effect on a compound, load, linker load, or antibody conjugate, including, but not limited to, biological, biochemical, synthetic, solubility, visualization, detectability, and reactivity effects, and the like. . In certain embodiments, the reinforcing group is a hydrophilic group. In certain embodiments, the enhancing group is a cyclodextrin. In certain embodiments, the efforts

- 50 045111 the group is an alkyl, heteroalkyl, alkylenyl, heteroalkylenyl sulfonic acid, heteroalkylenyl taurine, heteroalkylenyl phosphoric acid or phosphate, heteroalkylenyl amine (for example, a quaternary ammonium compound) or heteroalkylenyl sugar. In certain embodiments, sugars include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. Examples of monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose and the like. In certain embodiments, sugars include sugar acids such as glucuronic acid, which further include conjugate forms such as glucuronides (ie, through glucuronidation). Examples of disaccharides include maltose, sucrose, lactose, lactulose, trehalose and the like. Examples of polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose and the like. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or mixtures thereof. In certain embodiments, the cyclodextrin is alpha cyclodextrin. In certain embodiments, the cyclodextrin is beta cyclodextrin. In certain embodiments, the cyclodextrin is a gamma cyclodextrin. In certain embodiments, the enhancing group is capable of improving the solubility of the remaining portion of the conjugate.

In certain embodiments, the enhancing group is an alkyl, heteroalkyl, alkylenyl, heteroalkylenyl sulfonic acid, heteroalkylenyl taurine, heteroalkylenyl phosphoric acid or phosphate, heteroalkylenyl amine (eg, a quaternary ammonium compound), or heteroalkylenyl sugar. In certain embodiments, sugars include, but are not limited to, monosaccharides, disaccharides, and polysaccharides. Examples of monosaccharides include glucose, ribose, deoxyribose, xylose, arabinose, mannose, galactose, fructose and the like. In certain embodiments, sugars include sugar acids such as glucuronic acid, which further include conjugate forms such as glucuronides (ie, through glucuronidation). Examples of disaccharides include maltose, sucrose, lactose, lactulose, trehalose and the like. Examples of polysaccharides include amylose, amylopectin, glycogen, inulin, cellulose and the like. The cyclodextrin can be any cyclodextrin known to those skilled in the art. In certain embodiments, the cyclodextrin is alpha cyclodextrin, beta cyclodextrin, or gamma cyclodextrin, or mixtures thereof. In certain embodiments, the cyclodextrin is alpha cyclodextrin. In certain embodiments, the cyclodextrin is beta cyclodextrin. In certain embodiments, the cyclodextrin is a gamma cyclodextrin. In certain embodiments, the enhancing group is capable of improving the solubility of the remaining portion of the conjugate. In certain embodiments, the alkyl, heteroalkyl, alkylenyl, or heteroalkylenyl sulfonic acid is substituted or unsubstituted. In certain embodiments, the alkyl, heteroalkyl, alkylenyl, or heteroalkylenyl sulfonic acid is -(CH2)1. ₅ SO ₃ H, -(CH2) _n -NH-(CH2)1.5SO ₃ H, -(CH ₂ )nC(O)NH-(CH ₂ )1. ₅ SO3H, <CT ₂ CT ₂ O)mC(O)NH-(CH ₂ )1. ₅ SO3H -(CH ₂ )nN((CH ₂ )1.5C(O)NH(CH ₂ )1.5SO3H) ₂ , -(CH ₂ VC(O)N((CH ₂ )i. ₅ C(O)NH (CH^ or

-(CH ₂ CH ₂ O) _m -C(O)N((CH ₂ ) ₁ _ ₅ C(O)NH(CH ₂ ) ₁ _ ₅ SO ₃ H) ₂ where n represents 1, 2, 3 , 4 or 5, and m is 1, 2, 3, 4 or 5. In one embodiment, the alkyl or alkylenyl sulfonic acid is -(CH ₂ )1. ₅ SO ₃ H. In another embodiment _, the heteroalkyl or heteroalkylenyl sulfonic acid is -(CH ₂ ) _n -NH-(CH ₂ ) _{1_5} SO ₃ H, where n is 1, 2, 3, 4 or 5. B In another embodiment, the alkyl, heteroalkyl, alkylenyl or heteroalkylenyl sulfonic acid is -(CH ₂ ) _n -C(O)NH-(CH ₂ ) _{1_5} SO ₃ H, where n is 1, 2, 3, ₄ or 5 In another embodiment, the alkyl, heteroalkyl, _alkylenyl or _{heteroalkylenyl sulfonic acid is -(CH2CH2O)m-C(O)NH-(CH2)1_5SO3H , where m is 1} , 2, 3, 4 or 5. In another embodiment _, the alkyl, heteroalkyl, alkylenyl or heteroalkylenyl _sulfonic acid is -(CH ₂ ) _n -N((CH ₂ ) _{1_5} C(O)NH(CH ₂ ) _{1_5} SO ₃ H) ₂ where n is 1, 2, 3, 4 or 5. In another embodiment, the alkyl, heteroalkyl, alkylenyl or heteroalkylenyl sulfonic acid is (CH ₂ ) _n -C(O)N((CH ₂ ) _{1_ 5} C(O)NH(CH ₂ ) ₁ _ ₅ SO ₃ H) ₂ where n is 1, 2, 3, 4 or 5. In another embodiment, the alkyl, heteroalkyl, alkylenyl or heteroalkylenyl sulfonic acid is (CH ₂ CH ₂ O) _m -C(O)N((CH ₂ ) ₁ _ ₅ C(O)NH(CH ₂ ) ₁ _ ₅ SO ₃ H) ₂ where m represents 1, 2, 3, 4 or 5 .

Presented here are antibody-drug conjugates containing an antibody or an antigen-binding fragment thereof conjugated to a compound selected from Table. 1. In some embodiments, the compound has formula 1d, 1e, 1g, or 1h.

Also provided herein are antibody-drug conjugates of formula Ab-L-SP-D or BA-L-SP-D, where D is a prodrug of budesonide or a prodrug of a budesonide analog or derivative (including fluorinated analogs and derivatives), and where Ab, BA , L and SP are as defined in any embodiment described herein. In some embodiments, 51 045111 D Ab-L-SP-D and BA-L-SP-D and

formulas (II) and (III) are selected from:

- 52 045111

In some embodiments, Ab-L-SP-D, BA-L-SP-D, and formula (III), an SP i moiety capable of releasing D or

In some embodiments, under physiological conditions, the bond between L and SP is cleaved, resulting in the release of a steroid prodrug, i.e. H-SP-D or

and the connection between SP and D or between SP and

is subsequently broken down, resulting in the release of the biologically active steroid. In some

In some embodiments, the n-propyl in each of the above structures is in the R configuration, i.e. on the carbon shown with an asterisk. In some embodiments, the n-propyl in each of the above structures is in the S configuration, i.e. on the carbon shown with an asterisk. In some embodiments, the n-propyl° in each of the above structures is a mixture of R and S configurations, „ - ω „ „ „ „ oA

those. on the carbon shown with an asterisk. In some embodiments, the n-propyl in each of the above structures is a mixture of R and S configurations, i.e. on the carbon indicated by an asterisk, where the R:S ratio is about 1:1, about 2:1, about 3;1, about 4:1, about 5:1, about 6;1, about 7:1, about 8 :1, about 9:1 or about 10:1.

- 53 045111

Table 3

Antibody-drug conjugates

Also presented here are antibody-steroid conjugates having the following formulas:

or a mixture thereof;

NH o^nh ₂ or a stereoisomer or a mixture of their stereoisomers;

or a mixture thereof;

- 54 045111

or a stereoisomer or a mixture of stereoisomers thereof;

or a stereoisomer or a mixture of stereoisomers thereof;

- 55 045111 or a mixture thereof;

or a mixture thereof;

AB—

where L1 is the residue of the reactive group containing a triazole fragment

- 56 045111 or a stereoisomer or a mixture of their stereoisomers;

or a mixture thereof;

or a mixture thereof or a mixture thereof

- 57 045111

or a mixture thereof;

- 58 045111

or a mixture thereof;

- 59 045111

or a mixture thereof;

- 60 045111

B.A.

where BA contains an antibody having the following structure or a mixture thereof;

where Ab is a monoclonal antibody, a polyclonal antibody, an antibody fragment or a bispecific antibody; R represents C _2-4 -alkylene; and n is an integer selected from 2 to 4, inclusive.

In some embodiments, n-propyl

in each of the above structures is in the R-configuration, i.e. on the carbon shown with an asterisk. In some embodiments, n-propyl OA in each of the above structures is in the S configuration, i.e. on the carbon shown with an asterisk. In some embodiments, npropyl °A in each of the above structures is a mixture of R and S configurations, i.e. on the carbon shown with an asterisk. In some embodiments, the n-propyl oA in each of the above structures is a mixture of R and S configurations, i.e. on the carbon indicated by an asterisk, where the R:S ratio is about 1:1, about 2:1, about 3;1, about 4:1, about 5:1, about 6;1, about 7:1, about 8 :1, about 9:1 or about 10:1.

In specific embodiments, BA is an antibody and z is an integer that is selected from 1-30, inclusive. In some embodiments, z is an integer that is selected from 1 to 4, inclusive. In some embodiments, z is 4. In some embodiments, z is 2.

Also provided herein are conjugates of a budesonide binder, a budesonide prodrug, a budesonide analog or derivative (including fluorinated analogs and derivatives), or a prodrug of a budesonide analog or derivative (including fluorinated analogs and derivatives).

Suitable coupling agents for any of the conjugates provided herein include, but are not limited to, antibodies, lymphokines, hormones, growth factors, viral receptors, interleukins, or any other cell-binding or peptide-binding molecules or substances.

In some embodiments, the binding agent is an antibody. The term antibody as used herein means any antigen-binding molecule or molecular complex containing at least one complementarity determining region (CDR) that specifically binds or interacts with a particular antigen. The term antibody includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated here as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, _CH 1, _CH 2 and _CH 3. Each light chain contains a light chain variable region (abbreviated here as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (V _L 1). The V _H and V _L regions can be further divided into regions of hypervariability, referred to as complementarity determining regions (CDRs), interspersed with regions that are more conserved, referred to as framework regions (FRs). Each V _H and V _L consists of three CDRs and four FRs, organized from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FR antibody (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs.

The term antibody as used herein also includes antigen-binding fragments of complete antibody molecules. The terms antigen-binding portion of an antibody, antigen-binding fragment of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding antibody fragments can be obtained, for example, from complete antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and/or can be easily obtained, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries) or can be synthesized. DNA can be sequenced or modified chemically or using molecular biology techniques, for example, by organizing one or more variable and/or constant domains in a suitable configuration or introducing codons, creating cysteine residues, changing, adding or deleting amino acids, etc.

The amino acid sequence of an antibody may be numbered using any known numbering schemes, including those described by Kabat et al. (Kabata nomenclature); AlLazikani et al., 1997, J. Mol. Biol., 273:927-948 (Chotia nomenclature); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (Contact nomenclature); Lefranc et al., Dev. Sotr. Immunol 2003, 27:55-77 (YamgT nomenclature); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (ANO nomenclature). If not specified

- 62 045111 other, the nomenclature used in this document is the Kabat nomenclature. EU nomenclature typically applies to a residue in the constant region of an antibody heavy chain (eg, as reported in Kabat et al., supra). However, the choice of nomenclature does not imply the existence of sequence differences in cases where they do not exist, and one of ordinary skill in the art can readily determine the sequence position by examining the amino acid sequence of one or more antibodies.

In some embodiments, the BA antibody is a functionalized antibody having the following structure:

In some embodiments, R is alkylene, alkenylene, or alkynylene. In some embodiments, R is alkylene. In some embodiments, R is C _2-4 alkylene. In certain embodiments, R is ethylene. In certain embodiments, n is 3. In certain embodiments, z is 2 or 4.

In some embodiments, the BA antibody is not an anti-PSMA antibody or an anti-CD70 antibody.

Non-limiting examples of BA antigen-binding fragments include: (i) Fab fragments; (ii) F(ab') ₂ fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable region of an antibody (eg, an isolated CDR, such as a CDR3 peptide) or a FR3-CDR3-FR4 peptide with limited conformational freedom. Other engineered molecules such as domain-specific antibodies, single-domain antibodies, camel antibodies (VhH fragments), domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, tri-bodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies, etc.), small modular protein (SMTP) immunopharmaceuticals and shark IgNAR variable domains are also included in the expression antigen binding fragment as used herein.

The antigen-binding fragment of an antibody typically contains at least one variable domain. This variable domain can be of any size and can have any amino acid composition and typically contains at least one CDR that is located adjacent to or in frame with one or more framework sequences. In antigen-binding fragments with a VH domain linked to a VL domain, the VH and VL domains may be positioned relative to each other in any suitable manner. For example, the variable region may be dimeric and may contain VH-VH, V _H V _L or V _L -V _L dimers. In another embodiment, the antigen-binding antibody fragment may comprise a monomeric V _H or V _L domain.

In certain embodiments, an antigen binding antibody fragment may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary variable and constant domain configurations that may be present in the antigen binding fragment of an antibody of the present invention include: (i) Vh-Ch1; (ii) Vh-Ch2; (iii) Vh-Ch3; (iv) Vh-Ch1-Ch2; (v) VH-Ch1-CH2-Ch3; (vi) Vh-Ch2-Ch3; (vii) Vh-Cl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-Ch2-Ch3; (xiii) Vl-Ch2-Ch3; and (xiv) Vl-Cl. In any configuration of variable and constant domains, including any of the exemplary configurations noted above, the variable and constant domains may either be directly connected to each other or may be connected by a full or partial hinge or linker portion. The hinge may consist of a minimum of 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and/or constant domains in a single polypeptide molecule. Moreover, the antigen binding fragment of an antibody of the present disclosure may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations described herein, in non-covalent association with each other and/or with one or more monomers. V _H or VL domain (eg, disulfide bond(s).

As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A multispecific antigen-binding antibody fragment typically contains at least two different variable domains, with each variable domain capable of specifically binding to a different antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the bispecific antibody formats described herein, can be adapted for use in the context of the antigen binding antibody fragment of the present invention using standard techniques in the art.

- 63 045111

Antibodies of the present disclosure may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). Complement-dependent cytotoxicity (CDC) means the lysis of antigen-expressing cells by the antibody of the present invention in the presence of complement. Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils, and macrophages) detect bound antibody on the target cell, thereby resulting in , to lysis of the target cell. CDC and ADCC can be measured by assays that are well known and available in the art. (See, for example, US Patent Nos. 5,500,362 and 5,821,337, and Clynes et al. (1998) Proc. Natl. Acad. Sci. (USA) 95:652-656). The constant region of an antibody is important for the ability of the antibody to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotope can be selected based on whether the antibody is desired to mediate cytotoxicity.

Antibodies useful for the compounds described herein include human antibodies. The term human antibody as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by germline human immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the CDR and, in particular, in CDR3. However, the term human antibody as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. The term human antibody does not include naturally occurring molecules that normally exist without modification or human intervention/manipulation in an unmodified living organism found in nature.

The antibodies of the present invention, in some embodiments, may be recombinant human antibodies. The term recombinant human antibody as used herein is intended to include all human antibodies that are prepared, expressed, generated, or released by recombinant means, such as antibodies expressed by a recombinant expression vector transfected into a host cell (described below), antibodies, antibodies isolated from a recombinant, combinatorial human antibody library (described below), antibodies isolated from an animal (eg, mouse) transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20:6287 -6295), or antibodies that are prepared, expressed, generated, or isolated by any other means that involve splicing human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from germline human immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies are subject to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and _VL regions of the recombinant antibodies are sequences which, if derived from or related to human germline VH and _VL sequences, would not naturally exist in the human germline antibody repertoire in vivo.

Human antibodies can exist in two forms, which are related to the heterogeneity of the hinge. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which the dimers are held together by an interchain disulfide bond between the heavy chains. In the second form, the dimers are not linked to each other through interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, consisting of covalently linked light and heavy chains (half-antibody). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in different isotypes of intact IgG is due, among other things, to structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the human IgG4 hinge region can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30:105) to levels typically observed with the human IgG1 hinge. The present invention covers antibodies having one or more mutations in the hinge, CH2 or CH3 region, which may be desirable, for example, in production to increase the yield of the desired form of the antibody.

Antibodies useful for the compounds described herein may be isolated antibodies. An isolated antibody as used herein means an antibody that has been identified and separated and/or obtained from at least one component of its environment. For example, an antibody that has been separated or removed from at least one component of an organ

- 64 045111 nism or from a tissue or cell in which the antibody naturally exists or is produced naturally is an isolated antibody for the purposes of the present invention. The isolated antibody also includes the antibody in situ within the recombinant cell. Isolated antibodies are antibodies that have undergone at least one purification or isolation step. In accordance with certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

Antibodies useful for the compounds described herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily identified by comparing the amino acid sequences described herein with germline sequences, for example, from publicly available antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences described herein, wherein one or more amino acids within one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence , from which the antibody was derived, or the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as germline mutations). One skilled in the art, starting with the heavy and light chain variable region sequences described herein, can produce a variety of antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all framework and/or CDR residues in the VH and/or _VL domains are mutated back to residues found in the original germinal sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original terminal sequence, for example, only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and/or CDR residues are mutated into the corresponding residues of a different germinal sequence (ie, a germinal sequence that is different from the germinal sequence from which the antibody was originally derived). Moreover, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework and/or CDR region, for example, where certain individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that differ from original germinal sequence are retained or mutate into the corresponding residue of another germinal sequence. Once produced, antibodies and antigen-binding fragments that contain one or more germline mutations can be tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on circumstances), decreased immunogenicity, etc.

In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, an antibody fragment (Fab, Fab' and F(ab) ₂ , minibody, diabody, tribody, and the like), or a bispecific antibody. The antibodies described herein can be humanized using the methods described in US Patent No. 6,596,541 and US Publication No. 2012/0096572, the contents of which are incorporated herein by reference in their entirety.

When the binding agent is an antibody, it binds to an antigen-binding partner, which is a polypeptide and may be a transmembrane molecule (eg, a receptor) or a growth factor, which may be glycosylated or phosphorylated.

Suitable targets to which the binding agent binds include any target/antigen for which selective delivery of a steroid is desired. In some embodiments, the binding agent is an antibody, a modified antibody, or an antigen-binding fragment thereof that binds to a target selected from: AXL, BAFFR, BCMA, list components

- 65 045111

BCR, BDCA2, BDCA4, BTLA,

BTNL2 BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD 11c, CD 137, CD 13 8, CD 14, CD 168, CD 177, CD 19, CD20, CD209 , CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56 , CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CRI, CR3, CRTAM, CSF1R, CTLA4, CXCR1/2 , CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HER2, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1 , IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG 3 , LIFR, MAG/Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PRLR, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5 , Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, components/asc. list TCR, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TNF-α, TROY, TSLPR, TYRO, VLDLR, VSIG4 and VTCN1.

In some embodiments, the binding agent is adalimumab or infliximab. In some embodiments, the binding agent is alemtuzumab, muromonab, rituximab, tosituzumab, or agonistic antibodies (where immune stimulation may be part of the desired mechanism of action).

Prolactin expression receptor, PRLR, and the like, as used herein, means the human prolactin receptor containing the amino acid sequence set forth in SEQ ID NO: 404 in WO2015026907, the contents of which are incorporated herein by reference in their entirety. PRLR expression includes both monomeric and multimeric PRLR molecules. As used herein, expression monomeric human PRLR means a PRLR protein or a portion thereof that does not contain or includes any multimerizing domains and that exists under normal conditions as a single PRLR molecule without direct physical connection to another PRLR molecule. An example of a monomeric PRLR molecule is the molecule referred to herein as hPRLR.mmh containing the amino acid sequence of SEQ ID NO: 401 in WO2015026907 (see, for example, Example 3 in WO2015026907). As used herein, dimeric human PRLR expression means a construct comprising two PRLR molecules linked to each other by a linker, a covalent bond, a non-covalent bond, or a multimerization domain, such as an antibody Fc domain. An example of a dimeric PRLR molecule is the molecule referred to herein as hPRLR.mFc containing the amino acid sequence of SEQ ID NO: 402 in WO2015026907 (see, for example, Example 3 in WO2015026907).

Examples of anti-PRLR antibodies are listed in Table. 1 in WO2015026907, which sets out amino acid sequence identifiers is or is substantially similar to a sequence identical to at least 90%, at least 95%, at least 98% or at least 99% identical to heavy chain variable regions (HCVRs), light chain variable regions (LCVR), heavy chain complementarity determining regions (HCDR1, HCDR2 and HCDR3) and light chain complementarity determining regions (LCDR1, LCDR2 and LCDR3) are examples of anti-PRLR antibodies. In table 2 in WO2015026907 provides nucleotide sequence identifiers that are at least 90% identical, at least 95% identical, at least 98% identical, or at least 99% identical to HCVR, LCVR, HCDR1, HCDR2 HCDR3, LCDR1, LCDR2 and LCDR3 examples of anti-PRLR antibodies.

In some embodiments, the antibody or antigen binding fragment thereof conjugated to the linker load or load may be an antibody targeting the human prolactin receptor (PRLR). Examples of antibodies against PRLR can be found, for example, in WO 2015/026907. In some embodiments, the anti-PRLR antibody comprises a heavy chain complementarity determining region (HCDR)-1 comprising SEQ ID NO: 17; HCDR2 containing SEQ ID NO: 19; HCDR3 containing SEQ ID NO: 20; light chain complementarity determining region (LCDR)-1 containing SEQ ID NO: 22; LCDR2 containing SEQ ID NO: 23; and LCDR3 comprising SEQ ID NO: 24. In some embodiments, the anti-PRLR antibody comprises a heavy chain variable region (HCVR) comprising SEQ ID NO: 17 and a light chain variable region (LCVR) comprising SEQ ID NO: 21. B In any of the above embodiments, an anti-PRLR antibody can be prepared by site-directed mutagage

- 66 045111 neza for the purpose of incorporating a glutamine residue into the site without disabling antibody function or binding. For example, in any of the above embodiments, the anti-PRLR antibody may contain an Asn297Gln (N297Q) mutation. Such antibodies having the N297Q mutation may also contain one or more additional natural glutamine residues in their variable regions that may be accessible to transglutaminase and therefore capable of conjugation to a load or linker load (Table B).

Table B Sequences of Exemplary Antibody H1H6958N (anti-PRLR)

SEP ID NO: Molecule / Antibody Region Subsequence 17 H1H6958N HCVR QVQLVESGGGVVQPGRSLRLSCGASGFTFRNYGMQWVRQGP GKGLEWVTLISFDGNDKYYADSVKGRFTISRDNSKNTLFLQM NSLRTEDTAVYYCARGGDFDYWGQGTLVTVSS 18 H1H6958N HCDR1 GFTFRNYG 19 H1H6958N HCDR2 ISFDGNDK 20 H1H6958N HCDR3 ARGGDFDY 21 H1H6958N LCVR DIQMTQSPSSLSSASVGDRVTITCRASQDIRKDLGWYQQKPGK APKRLIYAASSLHSGVPSRFSGSGSGTEFTLTISSLQPEDFATY YCLQHNSYPMYTFGQGTKLEIK 22 H1H6958N LCDR1 QDIRKD 23 H1H6958N LCDR2 A.A.S. 24 H1H6958N LCDR3 LQHNSYPMYT 25 hPRLR ecto- MMN MHRPRRRGTRPPPLALLAALLLAARGADAQLPPGKPEIFKCR SPNKETFTCWWRPGTDGGLPTNYSLTYHREGETLMHECPDYI TGGPNSCHFGKQYTSMWRTYIMMVNATNQMGSSFSDELYV DVTYIVQPDPPLELAVEVKQPEDRKPYLWIKWSPPTLIDLKTG WFTLLYEIRLKPEKAAEWEIHFAGQQTEFKILSL HPGQKYLVQ VRCKPDHGYWSAWSPATFIQIPSDFTMNDEQKLISEEDLGGE QKLISEEDLHHHHHH

In any of the present embodiments of the compound or conjugate, BA is an antibody or antigen-binding fragment thereof that binds PRLR. In any of the present embodiments of the compound or conjugate, BA is an antibody or an antigen-binding fragment thereof, and conjugation is accomplished through at least one Q295 residue. In any of the present embodiments of the compound or conjugate, BA is an antibody or an antigen-binding fragment thereof, and conjugation is accomplished through two Q295 residues. In any of the present embodiments of the compound or conjugate, BA is an N297Q antibody or an antigen-binding fragment thereof. In any of the present embodiments of the compound or conjugate, BA is an N297Q antibody or an antigen-binding fragment thereof, and conjugation occurs through at least one Q295 residue and at least one Q297 residue. In any of the present embodiments of the compound or conjugate, BA is antibody N297Q or an antigen binding fragment thereof, and conjugation is accomplished through two Q295 residues and two Q297 residues. In specific embodiments, the nomenclature corresponds to EU nomenclature.

In any of the above embodiments, the BA is an anti-MSR1 antibody. In certain embodiments, the BA is an anti-MSR1 antibody H1H21234N. In certain embodiments, the BA is an anti-MSR1 antibody H1H21234N N297Q. In certain embodiments, the BA is an anti-MSR1 antibody comprising HCVR according to SEQ ID NO: 2 and LCVR according to SEQ ID NO: 10. In certain embodiments, the BA is an anti-MSR1 antibody containing one, two, three, four, five or six HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 according to SEQ ID NOS: 4, 6, 8, 12, 14 and 16, respectively. In certain embodiments, the HCVR is encoded by SEQ ID NO: 1. In certain embodiments, the LCVR is encoded by SEQ ID NO: 9. In certain embodiments, one, two, three, four, five, or six HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are encoded by polynucleotide sequences SEQ ID NOS: 3, 5, 7, 11, 13 and 15, respectively. N297Q indicates that one or more residues 297 are mutated from asparagine (N) to glutamine (Q). Preferably, each residue 297 is mutated to Q. In preferred embodiments, the nomenclature follows EU nomenclature. In certain embodiments of this paragraph, z is 1 to 4. In certain embodiments, z is 1, 2, 3, or 4. In certain embodiments, z is

- 67 045111 is 2. In certain embodiments, ζ is 4.

SEP ID NO: Molecule / Antibody Region Subsequence 1 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc acctgcactg tcactggtgg ctccatcagt aggaactact ggagttggat ccggcagccc ccagggaagg gactggaatg gattggatat atctattaca gtgggagtat cgactacaat ccctccctca a gagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg aagctgagtt ctatgaccgc tgcggacacg gccgtatact actgtgcgag agatcggtgg aactggaaat acggtatgga cgtctggggc caagggacca cggtcatcgt ctcgtca 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Thr Gly Gly Ser lie Ser Arg Asn Tyr Trp Ser Trp lie Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp lie Gly Tyr lie Tyr Tyr Ser Gly Ser lie Asp Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Met Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Trp Asn Trp Lys Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val lie Val Ser Ser 3 ggtggctcca tcagtaggaa ctac 4 Gly Gly Ser lie Ser Arg Asn Tyr 5 atctattaca gtgggagtat c 6 lie Tyr Tyr Ser Gly Ser lie 7 gcgagagatc ggtggaactg gaaatacggt atggacgtc 8 Ala Arg Asp Arg Trp Asn Trp Lys Tyr Gly Met Asp Val 9 gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc ctctcctgca gggccagtca gactgttaga aacaactact tagcctggta ccaccagaaa cctggccagg ctcccaggct cctcatctat ggtgcatcca gcaggggccac tggcatccca gacaggttca gtggcagt gg gtctgggaca gacttcactc tcaccatcag cagactggag cctgaagatt ttacagtgta ttactgtcac cagtatggta actcaccttg gacgttcggc caagggacca aaatggaaat caaacga 10 Glu lie Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Thr Val Arg Asn Asn Tyr Leu Ala Trp Tyr His Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu lie Tyr Gly Ala Ser Ser Arg Ala Thr Gly lie Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu Pro Glu Asp Phe Thr Val Tyr Tyr Cys His Gln Tyr Gly Asn Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Met Glu lie Lys Arg AND cagactgtta gaaacaacta c 12 Gln Thr Val Arg Asn Asn Tyr 13 ggtgcatcc 14 Gly Ala Ser 15 caccagtatg gtaactcacc ttggacg 16 His Gln Tyr Gly Asn Ser Pro Trp Thr

A binding agent, for example, an antibody or antigen-binding molecule, may contain a linker linked to the binding agent through attachment at a specific amino acid within the antibody or antigen-binding molecule. Exemplary amino acid attachments that may be used in the context of this aspect of the present disclosure include, for example, lysine (see, for example, US 5,208,020; US 2010/0129314; Hollander et al., Bioconjugate Chem., 2008, 19:358-361 ; WO 2005/089808; US 5,714,586; US 2013/0101546; and US 2012/0585592), cysteine (see, for example, US 2007/0258987; WO 2013/055993; WO 2013/055990; WO 2013/ 053873;WO 2013 /053872; WO 2011/130598; US 2013/0101546; and US 7,750,116), selenocysteine (see, for example, WO 2008/122039; and Hofer et al., Proc. Natl. Acad. Sci., USA, 2008, 705 :12451-12456), formyl glycine (see, for example, Carrico et al., Nat. Chem. Biol., 2007, 3:321-322; Agarwal et al., Proc. Natl. Acad. Sci., USA, 2013, 770:46-51, and Rabuka et al., Nat. Protocols, 2012, 70:1052-1067), unnatural amino acids (see, for example, WO 2013/068874 and WO 2012/166559), and acidic amino acids ( see for example WO 2012/05982). Linkers can be conjugated via glutamine via transglutaminase-based chemoenzymatic conjugation (see, for example, Dennler et al., Bioconjugate Chem. 2014, 25, 569-578). Linkers can also be conjugated to an antigen-binding protein through attachment to carbohydrates (see, for example, US 2008/0305497, WO 2014/065661 and Ryan et al., Food & Agriculture Immunol, 2001, 73:127-130) and disulfide linkers (see, for example, WO 2013/085925, WO 2010/010324, WO 2011/018611, WO 2014/197854 and Shaunak et al., Nat. Chem. Biol, 2006, 2:312-313). In some embodiments, the binding agent is an anti-68 045111 body, and the antibody binds to the linker through a lysine residue. In some embodiments, the antibody binds to the linker via a cysteine residue.

In any of the above embodiments, the BA is an anti-PRLR antibody. In certain embodiments, the BA is an anti-PRLR antibody H1H6958N. In certain embodiments, the BA is an anti-PRLR antibody H1H6958N N297Q. In certain embodiments, BA is an anti-PRLR antibody comprising HCVR according to SEQ ID NO: 17 and LCVR according to SEQ ID NO: 21. In certain embodiments, BA is an anti-PRLR antibody containing one, two, three, four, five or six HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 according to SEQ ID NOS: 18, 19, 20, 22, 23 and 24, respectively. N297Q indicates that one or more residues 297 are mutated from asparagine (N) to glutamine (Q). Preferably, each residue 297 is mutated to Q. In preferred embodiments, the nomenclature follows EU nomenclature. In certain embodiments of this paragraph, k is 1 to 4. In certain embodiments, k is 1, 2, 3, or 4. In certain embodiments, k is 4.

D. Methods for preparing compounds.

The conjugates described herein can be synthesized by coupling the linker loads described herein with a coupling agent, such as an antibody, under standard conjugation conditions (see, e.g., Drug Deliv. 2016 Jun; 23(5):1662-6; AAPS Journal , Vol. 17, No. 2, March 2015; and Int. J. Mol. Sci. 2016, 17, 561, the contents of which are incorporated herein by reference in their entirety). Linker loads are synthetic intermediates containing the load of interest and a connecting moiety, which ultimately serves as the moiety (or part thereof) that connects the coupling agent to the load. Linker loads contain a reactive group that reacts with the coupling agent to form the conjugates described herein. When the linking agent is an antibody, the antibody may be linked to the linker load via one or more cysteine, lysine, or other residues of the antibody. Linker loads can be coupled to cysteine residues, for example, by exposing the antibody to a reducing agent, such as dithiothreitol, to cleave the disulfide bonds of the antibody, optionally purifying the reduced antibody, for example, by gel filtration, and subsequently reacting the antibody with the linker load containing a reactive moiety, for example a maleimido group. Suitable solvents include, but are not limited to, water, DMA, DMF and DMSO. Linker loads containing a reactive group, such as an activated ester or an acid halide group, can be linked to lysine residues. Suitable solvents include, but are not limited to, water, DMA, DMF and DMSO. The conjugates can be purified using known protein techniques including, for example, size exclusion chromatography, dialysis and ultrafiltration/diafiltration.

Linker-steroid intermediates.

Here are the linker steroids according to formula (2000),

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof that is useful in the preparation of antibody-drug conjugates, wherein D is selected from ^a)

^Rx

N

O of formula (a), where both R ^x in formula (a) represent hydrogen; R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; and SP represents -C(O)-C1-C10-alkylene-C(O)-, -C(O)-N(C1.6alkyl)-C1-C10-alkylene-X ¹ -, where X ¹ is attached to (L ³ )0.1 in formula (2000), -C(O)-N(H)-(C ₁ C ₁₀ -alkylene)-S-, where S is added to (L ³ ) _0.1 in formula (2000) , -C(O)-N(C ₁ . ₆ alkyl)-(C ₁ -C ₁₀ -alculene)-S-, o to n η where S is added to (L) _0-1 in formula (2000), , where the point of addition on the right side (i.e., on N) is to (L ³ )0.1 in formula (2000), -CH2-NH-, where N is added to (L ³ )0.1 in formula. n mule (2000), ⁰ ' where N is added to (L ³ ) ₀ . ₁ in formula (2000) and where Ar represents

- 69 045111 optionally substituted arylene (in some embodiments

V ^s ) or optionally substituted heteroarylene, -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to (L ³ )0.1 in the formula (2000), -C(O)-(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to (L ³ )0.1 in formula (2000) and where in each case C ₁ -C ₁₀ -alkylene is independently and optionally replaced by one or more hydroxy, -C(O)-N(R ⁵ )-C ₁ -C ₁₀ -alkylene-C(O)NH -X ² -, where X ² is added to (L ³ ) _0-1 in formula (2000) or ⁰ , where X ⁴ is added to (L ³ )<m in formula (2000); or where both R ^x in formula (a) represents fluorine; R ⁴ represents alkyl, aryl, arylalkyl or N containing heterocycloalkyl; and SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-, -C(O)-N(C ₁ . ₆ alkyl)-C ₁ -C ₁₀ -alkylene-X ^1b - , where X ^1b is added to (L ³ ) ₀ .1 in formula (2000), -C(0)-N(H)-(C ₁ -C ₁₀ o

k/Ny alkylene)-X ^1b -, where X ^1b is added to (L ³ ) ₀ -1 in formula (2000), 'where the point of addition on the right side (i.e. on N) is to (L ³ ) ₀ .1 in formula (2000), -CH2-NH-, where ^N is added to (L ³ ) ₀ -1 in formula (2000), ^O ' where N is added to (L ³ ) = in formula (2000) and where Ar is optionally substituted arylene (in some embodiments

or optionally substituted heteroarylene, -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to (L ³ )= in formula (2000) , -C(O)-(C1- _C10- alkylene)-NR ⁵⁰ C(O)-(C1-C10-αalkylene)-NR ^50a -, where NR ^50a is added to (L ³ )0.1 in formula (2000) and wherein in each case, C ₁ -C ₁₀ -alkylene is independently optionally replaced by one or more hydroxy, -C(O)-N(R ⁵ )-(C1-C10-alkylene)-C(O)NH-X ² -, where X ² is added to (L ³ )0.

R ^e in formula (2000) or A, where X ⁴ is added to (L ³ ) _0-1 ³ in formula (2000); And

b) compounds in the above table. And where are the connections in the table? And compounds with formula (2000) are connected to RG through hydroxy -C(O)CH ₂ OH groups, i.e. using -C(O)CH ₂ -O-SP-(L ³ )0.1- or through Mapracorate hydroxy, i.e. using -O-SP-(L ³ )0.1-:

X ¹ represents -N(C ₁ . ₆ alkyl)-;

X ^1b represents -S-, -NH- or -N(C ₁ . ₆ alkyl)-;

X ² represents -NH-;

X ³ is -CH2-, X ³ is -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ or X ³ is -C (O)-;

X ⁴ represents -O-;

R ⁵ represents H, -OH, -OCH ₃ or C1. ₆ alkyl;

R ⁵⁰ and R ^50a are independently hydrogen or C1-C ₆ alkyl;

R ^d , R ^e and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -CH2C(O)OH or -CH ₂ C(O)O(αalkyl); and m is 0 or 1;

RG is a reactive group;

L ² represents a connecting linker; And

L ³ , if present, is a self-cleaving linker.

Here are linker steroids according to formula (II)

which are useful in the preparation of antibody-drug conjugates; Where

- 70 045111

RG, L ² , L ³ , SP, R ⁴ and R ^x are as defined herein.

In some or any embodiments, a compound of formula (II-P) is presented below:

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof, wherein

R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; SP represents -C(O)-C1-C10-alkylene-C(O)-, -C(O)-N(C1-3alkyl)-C1-C10-alkylene-X ¹ -, where X ¹ is attached to L ³ in formula (II), -C(O)-N(R ⁵ )-C1-C ₁₀ -αalkylene-C(O)NH-X ² -, where X ² is added to L ³ in formula (II) or

, where X ⁴ is added to L ³ in formula (II);

X ¹ represents -N(C _1-3 alkyl)-; X ² represents -NH-;

X ³ is -CH2-, X ³ is -CH2-O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ or X ³ is -C( O)-;

X ⁴ represents -O-;

R ⁵ represents H, -OH, -OCH3 or alkyl; R ^d , R ^e and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -CH2C(O)OH or -CH ₂ C(O)O(alkyl); and m is 0 or 1;

RG is a reactive group;

L ² represents a connecting linker; And

L ³ , if present, is a self-cleaving linker.

In some or any embodiments, a compound of formula (II-P-1) is presented below:

or a pharmaceutically acceptable salt, solvate, stereoisomer or derivative thereof, wherein

R ⁴ represents alkyl, aryl, arylalkyl or N-containing heterocycloalkyl;

SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-, -C(O)-N(C _1-3 alkyl)-C ₁ -C ₁₀ -alkylene-X ¹ -, where X ¹ is added to L ³ in formula (II), -C(O)-N(R ⁵ )-C ₁ -C ₁₀ -alkylene-C(O)NH-X ² -, where X ² is added to L ³

in formula (II) or *, where X ⁴ is added to L ³ in formula (II);

X ¹ represents -N(C _1-3 alkyl)-;

X ² represents -NH-;

X ³ is -CH2-, X ³ is -CH2-O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ or X ³ is -C( O)-;

X ⁴ represents -O-;

R ⁵ represents H, -OH, -OCH3 or alkyl;

R ^d , R ^e and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C(O)OH or -CH2OR ^g , where in each case R ^g is independently -CH2C(O)OH or -CH ₂ C(O)O(alkyl); and m is 0 or 1;

RG is a reactive group;

L ² represents a connecting linker; And

L ³ , if present, is a self-cleaving linker.

In some embodiments, provided herein is a compound of formula (II) wherein both R ^x are hydrogen. In some or any embodiments, both R ^x are fluorine.

In some embodiments, a compound of formula (II), (II-P) and (II-P- 71 045111) is provided herein

1), where R ⁴ represents alkyl. In some embodiments, R ⁴ is non-cycloalkyl. In some of these embodiments, R ⁴ is straight or branched alkyl. In some of these embodiments, R ⁴ is aryl. In some of these embodiments, R ⁴ is arylalkyl. In some of these embodiments, R ⁴ is an N-containing heterocycloalkyl. In some embodiments, R ⁴ is alkyl, such as, but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, or nonyl. In some embodiments, R ⁴ is methyl. In some embodiments, R ⁴ is ethyl. In some embodiments, R ⁴ is n-propyl. In some embodiments, R ⁴ is i-propyl. In some embodiments, R ⁴ is n-butyl. In some embodiments, R ⁴ is i-butyl. In some embodiments, R ⁴ is tbutyl. In some embodiments, R ⁴ is sec-butyl. In some embodiments, R ⁴ is pentyl. In some embodiments, R ⁴ is hexyl. In some embodiments, R ⁴ is heptyl. In some embodiments, R ⁴ is octyl or nonyl. In some embodiments, R ⁴ is aryl, such as, but not limited to, phenyl, phenol, or naphthyl. In some embodiments, R ⁴ is phenyl. In some embodiments, R ⁴ is naphthyl. In some embodiments, R ⁴ is heteroaryl - such as, but not limited to, thienyl. In some embodiments, R ⁴ is arylalkyl - such as, but not limited to, benzyl. In some embodiments, R ⁴ is an N-containing heterocycloalkyl, such as, but not limited to, piperidinyl. In some embodiments, provided herein is a compound of formula (II) wherein both R ^x are hydrogen. In some or any embodiments, both R ^x are fluorine. In some embodiments, R ⁴ is in the R configuration. In some embodiments, R ⁴ is in the S configuration. In some embodiments, R ⁴ is a mixture of R and S configurations. In some embodiments, R ⁴ is a mixture of R and S configurations, where the R:S ratio is about 1:1, about 2:1, about 3;1, about 4:1, about 5:1, about 6:1, around 7:1, around 8:1, around 9:1 or around 10:1.

In some embodiments, a compound of formula (II) is provided wherein both R ^x are hydrogen; and SP is -C(O)-C1-C10-alkylene-C(O)-, -C(O)-N(C1-6alkyl)-C1C10-alkylene-X1-, where X ¹ is attached to L in the formula (II), -C(O)-N(H)-(C1-C ₁₀ -alkylene)-S-, where S is added to L in formula (II), -C(O)-N(C _1-6 alkyl)-(C ₁ -C ₁₀ -alkylene)-S-, where S is added to L in formula (II), -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10- alkylene)-NR ^50a -, where NR ^50a is added to L in formula (II), C(O)-(C1-C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a - where NR ^50a is added to L in formula (II) and where in each case the C1-C ₁₀ alkylene is independently and optionally replaced by one or more hydroxy, -C(O)-N(R ⁵ )-C ₁ -C ₁₀ -alkylene-C(O)NH-X ² -, where X ² is added to L in formula (II) or

where X ⁴ is added to L in formula (II); or both R ^x are fluorine; and SP is -C(O)-N(C _1-6 alkyl)-C ₁ -C ₁₀ -alkylene-X ^1b -, where X ^1b is added to L in formula (II), o

-C(O)-N(H)-(C ₁ -C ₁₀ -alkylene)-X ^1b -, where X ^1b is added to L in formula (II), V where the point of attachment is on the right side (i.e. . on N) is to L in formula (II), -(C1- _C10- alkylene)NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to L in the formula (II), -C(O)-(C1- _C10- alkylene)NR ⁵⁰ C(O)-(C1-C10-alkylene)-NR ^50a -, where NR ^50a is added to L in formula (III) and where in each case, C1-C ₁₀ -alkylene is independently and optionally replaced by one or more hydroxy or -C(O)N(R ⁵ )-(C ₁ -C ₁₀ -alkylene)-C(O)NH-X ² -, where X ² is added to L in formula (II).

In some embodiments, a compound of formula (II), (II-P), or (IIP-1) is provided herein, wherein both R ^x is hydrogen and SP is -C(O)-C ₁ -C ₁₀ -alkylene-C (O)-. In some embodiments, a compound of formula (II), (II-P) or (I-P-1) is provided herein, wherein SP is -C(O)-C ₂ -C ₅ -alkylene-C(O)-. In some embodiments, a compound of formula (II), (II-P) or (I-P-1) is provided herein, wherein SP is -C(O)-CH ₂ CH ₂ -C(O)-.

In some embodiments, a compound of formula (II), (II-P) or (IIP-1) is provided herein, wherein both R ^x is fluorine and SP is -C(O)-C ₁ -C ₁₀ -alkylene-C (O)-. In some embodiments, a compound of formula (II), (II-P) or (I-P-1) is provided herein, wherein SP is -C(O)-C ₂ -C ₅ -alkylene-C(O)-. In some embodiments, a compound of formula (II), (II-P) or (I-P-1) is provided herein, wherein SP is -C(O)-CH ₂ CH ₂ -C(O)-.

In some embodiments, a compound of formula (II), (II-P) or (IIP-1) is provided herein, wherein both R ^x is hydrogen and SP is -C(O)-N(C _1-6 alkyl)- From ₁ to C ₁₀

- 72 045111 alkylene-X ¹ -, where X ¹ is added to L ³ in formula (II), (II-P) or (II-P-1). In some embodiments, a compound of formula (II), (II-P) or (P-P-1) is provided herein, wherein SP is C(O)-N(C1.3alkyl)-C1-C10-alkylene-X ¹ -, where X ¹ is attached to L ³ in formula (II), (II-P) or (II-P-1). In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein X ¹ is -N(C1-3alkyl)-. In some embodiments, a compound of formula (II), (II-P), or (II-P-1) is provided, wherein SP is -C(O)-N(C ₁ . ₃ αlkyl)-C ₂ -C ₅ -alkylene-X ¹ -. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is -C(O)-N(C1.3alkyl)-CH2CH2-X ¹ -. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is -C(O)-N(CH3)-C ₂ C5-alkylene-N(CH ₃ )-. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is -C(O)-N(CH ₃ )-CH ₂ CH ₂ -N(CH ₃ )-.

In some embodiments, provided herein is a compound of formula (II) wherein both R ^x is fluorine and SP is -C(O)-N(C1.6alkyl)-C1-C1o-alkylene-X ^1b -, wherein X ^1b is added to L ³ in formula (II) or -C(O)-N(H)-(C1-C ₁₀ -alkylene)-X ^1b -, where X ^1b is added to L ³ in formula (II) in formula (II) . In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-C1-C10-alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-C1-C ₆ -alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (II) . In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-Ci-C3-alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)CH2CH2-X ^1b -. In some embodiments, a compound of formula (II) is provided wherein SP is -C(O)-N(H)-C2-C ₅ -alkylene-N(CH ₃ )-. In some embodiments, provided herein is a compound of formula (II), wherein SP is -C(O)-N(H)-C ₂ -C ₅ -alkyleneN(CH ₂ CH ₃ )-. In some embodiments, provided herein is a compound of formula (II) wherein SP is -C(O)-N(H)-CH ₂ CH ₂ -N(CH ₃ )-.

In some embodiments, provided herein is a compound of formula (II) wherein both R ^x is fluorine and SP is -C(O)-N(C1.6alkyl)-C1-C10-alkylene-X ^1b -, wherein X ^1b is added to L ³ in formula (II). In some embodiments, provided herein is a compound of formula (II) wherein SP is -C(O)-N( _C1.3 alkyl)-C ₁ -C ₆ -alkylene-X ^1b - wherein X ^1b is attached to L ³ in formula (II). In some embodiments, provided herein is a compound of formula (II), wherein SP is -C(O)-N(C1.3αalkyl)-C1-C3-alkylene-X ^1b -, wherein X ^1b is attached to L ³ in formula (II ). In some embodiments, a compound of formula (II) is provided herein, wherein X ^1b is -N( _C1.3 alkyl)-. In some embodiments, a compound of formula (II) is provided wherein SP is -C(O)-N(C ₁ . ₃ alkyl)-C ₂ -C ₅ -alkylene-X ^1b -. In some embodiments, provided herein is a compound of formula (II) wherein SP is -C(O)-N(C1-3alkyl)-CH2CH2-X ^1b -. In some embodiments, provided herein is a compound of formula (II) wherein SP is -C(O)-N(CH3)-C ₂ -C ₅ -alkylene-N(CH ₃ )-. In some embodiments, provided herein is a compound of formula (II), wherein SP is -C(O)-N(CH ₂ CH ₃ )-C ₂ -C ₅ alkylene-N(CH ₂ CH ₃ )-. In some embodiments, provided herein is a compound of formula (II) wherein SP is -C(O)-N(CH ₃ )-CH ₂ CH ₂ -N(CH ₃ )-.

In some embodiments, a compound of formula (II) is provided herein, wherein both R ^x o

H N D represent fluorine and SP represents = where the point of attachment on the right side (eg on N) represents to L in formula (II).

In some embodiments, provided herein is a compound of formula (II) wherein R ^x is both hydrogen and SP is -C(O)-N(H)-(C1-C10-alkylene)-S-, wherein S is attached to L ³ in formula (II) in formula (II) or -C(O)-N(C1. ₆ αlkyl)-(C ₁ -C ₁₀ -alkylene)-S-, where S is added to L ³ in formula (II) . In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-(C1-C6-alkylene)-S-, wherein S is attached to L ³ in formula (II) or -C(O)-N(C1. ₃ alkyl)-(C ₁ -C ₆ -alkylene)-S-, where S is added to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-(C1C1o-alkylene)-S-, wherein S is added to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-(C1-C6-alkylene)-S-, wherein S is attached to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(C1.6alkyl)-(C1-C1o-alkylene)-S-, wherein S is attached to L ³ in formula (II ). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N( _C1.3αalkyl )-( _{C1- C6alkylene} )-S-, wherein S is attached to ^L3 in formula (II).

In some embodiments, a compound of formula (II) is provided herein, wherein both R ^x

- 73 045111 are fluorine and SP is -C(O)-N(H)-(C ₁ -C ₁₀ -alkylene)-S-, where S is added to L ³ in formula (II) or -C(O )-N(C1.6alkyl)-(C1-C10-alkylene)-S-, where S is added to L ³ in formula (II)in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-(C1- _C6 -alkulene)-S-, wherein S is added to ^L3 in formula (II) or C(O)-N(C1.3alkyl)-(C1-C6-alkylene)-S-, where S is added to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-(C1C10-alkylene)-S-, wherein S is added to L ³ in formula (II). In some embodiments, a compound of formula (II) is provided herein, wherein SP is -C(O)-N(H)-(C1-C6-alkylene)-S-, wherein S is attached to L ³ in formula (II). In some embodiments, provided herein is a compound of formula (II), wherein SP is -C(O)-N(C1-6alkyl)-( _C1 - _C1o -alkylene)-S-, wherein S is attached to ^L3 in formula (II). In some embodiments, a _{compound of formula (II) is provided herein, wherein SP is -C(O)-N(C1.3αalkyl )} -(C1 _- C6-alkylene)-S-, wherein S is attached to ^L3 in formula (II).

In some embodiments, provided herein is a compound of formula (II) wherein both R ^x are hydrogen and SP is -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)NR ^50a - where NR ^50a is added to L ³ in formula (II). In some or any embodiments of formula (I), SP is -(C1-C6-alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ -alkylene)-NR ^50a -. In some or any embodiments of Formula (II), SP is -(C1-C3-alkylene)-NR ⁵⁰ C(O)-(C1C ₃ -alkylene)-NR ^50a -. In some or any embodiments of formula (II), SP is -(linear C1-C3 alkylene)-NR ⁵⁰ C(O)-(linear C1-C ₃ alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (II) wherein R ^x is both fluorine and SP is -(C1-C10 alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ alkylene)-NR ^50a -, where NR ^50a is attached to L ³ in formula (II). In some or any embodiments of formula (I), SP is -(C1-C6-alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ -alkylene)-NR ^50a -. In some or any embodiments of formula (II), SP is -(C1-C3 alkylene)-NR ⁵⁰ C(O)-(C1-C ₃ alkylene)-NR ^50a -. In some or any embodiments of formula (II), SP is -(linear C1-C3 alkylene)-NR ⁵⁰ C(O)-(linear C1-C ₃ alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (II) wherein R ^x is both hydrogen and SP is -C(O)-(C1-C10 alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ alkylene )-NR ^50a -, where NR ^50a - is added to L ³ in formula (II) and where in each case the C1-C10 alkylene is independently and optionally replaced by one or more hydroxy. In some or any embodiments of formula (II), SP is -(branched C1-C6-alkylene)-NR ⁵⁰ C(O)-(C1C ₆ -hydroxy-alkylene)-NR ^50a -. In some or any embodiments of formula (II), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ alkylene)-NR ^50a -.

In some embodiments, provided herein is a compound of formula (II) wherein R ^x is both fluorine and SP is -C(O)-(C1-C10 alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)NR ^50a -, where NR ^50a - is added to L ³ in formula (II) and where in each case the C1-C10 alkylene is independently optional and replaced by one or more hydroxy. In some or any embodiments of formula (II), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ hydroxyalkylene)-NR ^50a -. In some or any embodiments of formula (II), SP is -(branched C1-C6 alkylene)-NR ⁵⁰ C(O)-(C1-C ₆ alkylene)-NR ^50a -.

In some embodiments, a compound of formula (II), (II-P) or (IIP-1) is provided herein, wherein both R ^x are hydrogen and SP is -C(O)-N(R ⁵ )-C1-C10 -alkyleneC(O)NH-X ² -, where X ² is added to L ³ in formula (II), (II-P) or (II-P-1). In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is C(O)-N(R ⁵ )-C1-C ₅ -alkylene-C( O)NH-NH-. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is -C(O)-N(R ⁵ )-CH2-C(O)NH- NH-. In some embodiments, a compound of formula (II), (II-P), or (II-P-1) is provided herein, wherein R ⁵ is H or alkyl. In some embodiments, a compound of formula (II), (II-P), or (II-P-1) is provided herein, wherein R ⁵ is H or CH3.

In some embodiments, a compound of formula (II), (II-P), or (IIP-1) is provided, wherein both R ^x is fluorine and SP is -C(O)-N(R ⁵ )-C1-C10 -alkylene-C(O)NHX ² -, where X ² is added to L ³ in formula (II). In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is -C(O)-N( ^R5 )-C1- _C5 -alkyleneC(O )NH-NH-. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is -C(O)-N(R ⁵ )-CH2-C(O)NH- NH-. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein R ⁵ is H or alkyl. In some embodiments, a compound of formula (II), (II-P) or (IIP-1) is provided herein, wherein ^R5 is H or CH3.

In some embodiments, a compound of formula (II), (II-P) or (II-74 045111) is provided herein

P-1), where both R ^x represent hydrogen and SP represents

where X ⁴ is added to L ³ in formula (II), (II-P) or (II-P-1). In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein both R ^x is fluorine and SP is R·, wherein X ⁴ is attached to L ³ in the formula ( II), (II-P) or (II-P-1). In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is

and X ³ is -CH2-. In some embodiments

R*

m here represents a compound of formula (II), (II-P) or (II-P-1), where SP and X ³ represents -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)-C(O)- , where C(O) is added to X ⁴ . In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided herein, wherein SP is

and X ³ is -C(O)-. In some embodiments, provided herein is a compound of formula (II), (II-P), or (II-P-1), wherein R ^d , R ^e , and R ^f are independently -H, -OH, hydroxyalkyl, alkoxycarbonyl, -C (O)OH or -CH2OR ^g , where in each case R ^g is independently -CH ₂ C(O)OH or -CH ₂ C(O)O(alkyl). In some embodiments, a compound of formula (II), (II-P) or (P-P-1) is provided herein, wherein R ^d and R ^e are independently -H or OH. In some embodiments, a compound of formula (II), (II-P), or (II-P1) is provided, wherein m is 0. In some embodiments, a compound of formula (II), (II-P), or (P- P-1), where m is 1. In some embodiments, a compound of formula (II), (II-P), or (P-P-1), where m is 1 and R ^f is -H or - HE.

In some embodiments, the implementation is a compound of formula (II), (II-P) or (P-P-1), wherein both R ^x are hydrogen and SP is -C(O)-N(C ₁ - ₃ alkyl )-C ₁ -C ₁₀ -alkylene-X ¹ -, R* where X ¹ where X ³ . 3......_ - b ^x *- ^x ''° is added to L ³ in formula (II), (II-P) or (P-P-1); SP is -CH2-, m is 1, and ^Rd , ^Re and ^Rf are -H; or SP represents

R* m

is ' where X ³ represents -CH ₂ -O-(C ₁ -C ₁₀ -alkylene)-C(O)-, where C(O) is added to X ⁴ , m represents 1, and R ^d , R ^e and R ^f is -H. In some embodiments, the implementation is a compound of formula (II), (II-P) or (II-P-1), where SP is -C(O)ζ. G . V ⁰

N(CH3)-CH2CH2-N(CH3)-, ^v

or

In some embodiments, a compound of formula (II), (II-P), or (P-P-1) is provided, wherein both R ^x is fluorine and SP is -C(O)-N(C ₁ . ₃ alkyl )-C ₁ -C ₁₀ -alkylene-X ¹ -, where

R*

*) / ^-X*-X ^J

X ¹ is added to L ³ in formula (II), (II-P) or (P-P-1); SP represents

X ³ represents -CH2-, m is 1, and R ^d , R ^e , and R ^f represents -H; or SP represents

- 75 045111

I'-X> -o, „ _ _ „ _____ _ ' where X ³ represents -CH ₂ -O-(C1-C ₁₀ -alkylene)-C(O)-, where C(O) is added to

X ⁴ , m is 1, and R ^d , R ^e and Rf are -H. In some embodiments,

R* represents a compound of formula (II), (II-P) or (I-P-1), where SP is -C(O)-N(CH ₃ )CH2CH2-N(CH ₃ )-,

or

SP represents

In some embodiments, a compound of formula (II) is provided wherein both R ^x are fluorine or both R ^x are hydrogen, or R ^x is as defined herein;

(where fluorine), (where both R ^x are fluorine),

In some embodiments, a compound of formula (II), (II-P) or (IIRG is an active group, both R ^x is p-1) is provided herein, wherein

In some embodiments, a compound of formula (II), (II-P), or

(IIP-1), where RG represents

In some embodiments, a compound of formula (II), (II-P), or

(IIP-1), where RG represents

In some embodiments, a compound of formula (II), (II-P) or (II-

P-1), where RG represents

In some embodiments, a compound of formula (II), (II-P) or (II-

P-1), where RG represents ^o

In some embodiments, -L2-(L ³ )0-1- in formula (II), (II-P) or (I-P-1) means any divalent group or moiety that connects or binds a reactive group (RG in formula (II), (II-P) or (I-P-1) with a loading compound represented herein (e.g., a steroid).In general, suitable -L ² -(L ³ )0-1- are those , which are stable enough to utilize the circulating half-life of the antibody and at the same time capable of releasing their load after antigen-mediated internalization of the conjugate. is L2-(L ³ ) _0-1 can be cleavable or non-cleavable linkers. Cleavable linkers are linkers that are cleaved by intracellular metabolism after internalization, such as cleavage through hydrolysis, reduction, or an enzymatic reaction.

- 76 045111 linkers are linkers that release the attached load through lysosomal degradation of the antibody after internalization. Suitable linkers include, but are not limited to, acid-labile linkers, hydrolysis-labile linkers, enzymatically cleavable linkers, reduction-labile linkers, self-cleavable linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, linkers that are or contain glucuronides, succinimide thioesters, polyethylene glycol (PEG) units, hydrazones, malcaproyl units, disulfide units (eg, -SS-, -SC(R ^1b R ^2b )- where R ^1b and R ^2b are independently hydrogen or hydrocarbyl), carbamate units, para-amino-benzyl units (PAB), phosphate units, e.g. mono-, bis- or tris-phosphate units, peptides, e.g. peptide units containing two, three, four, five, six, seven, eight or more amino acid units, including, but not limited to, valine-citrulline units, valine-alanine units, valine-arginine units, valine-lysine units, lysine-valine units citrulline and lysine-valine-alanine units. In some embodiments, -L ² -(L ³ )0-1- is trivalent and includes a cyclodextrin moiety linked to a trivalent group (eg, a lysine residue) in -L ² -(L ³ )0-1-. In some embodiments, L ³ is present. In some embodiments, L ³ is absent.

In some embodiments, a compound of formula (II), (II-P) or (II-P-ι) is provided, wherein L ² contains

or a cyclodextrin residue (CD); or combinations thereof. In some embodiments, a compound of formula (II), (II-P) or (II-P-1) is provided, wherein L ² contains

or CD or combinations thereof. In some embodiments, the implementation is a compound of formula (II), (II-P) or (II-P-1), wherein L ² contains CD, where CD is selected from the group consisting of

- 77 045111

In some embodiments, the implementation is a compound of formula (II), (II-P) or (II-P-1), wherein -L ² -(L ³ ) _0-1 - contains

- 78 045111

- 79 045111

In some embodiments, the implementation is a compound of formula (II), (II-P) or (II-P-1),

where L ³ represents ⁿ where the NH group is attached to L ² when SP represents

itself -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-; or L ³ represents ' where an NH group is attached to L ² when SP represents -C(O)-N(C ₁ . ₃ alkyl)-C ₁ -C ₁₀ -alkylene-X ¹ -or -C(O) -N(R ⁵ )-

C ₁ -C ₁₀ -alkylene-C(O)NH-X ² -; or L ³ is missing when SP - ·

In some embodiments, the implementation is a compound of formula (II), (II-P) or (H-P-1),

where L ³ represents ⁿ , where an NH group is attached to L ² when SP represents -C(O)-C ₁ -C ₁₀ -alkylene-C(O)-. In some embodiments, a connection is provided

- 80 045111 formula (II), (II-P) or (II-P-1), where L ³ represents

where an NH group is attached to L ² when SP is -C(O)-N(C1-3alkyl)-C1-C10-alkylene-X ¹ - or -C(0)-N( ^R5 )-C1-C ₁₀ alkylene-C(O)NH-X ² -. In some embodiments, a compound of formula (II), (II-

P) or (II-P-1), where L ³ is absent when SP Compounds having the following structures are also presented here.

table 2

Budesonide spacer linker reactive group

- 81 045111

- 82 045111

- 83 045111

- 84 045111

- 85 045111

- 86 045111

Binding agents, such as antibodies, can also be conjugated via click chemistry. In some embodiments of said click chemistry reaction, the linker-load contains a reactive group, such as an alkyne, that is capable of undergoing a 1,3 cycloaddition reaction with an azide. Such suitable reactive groups include, but are not limited to, strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, such as cyclooctynes, benzannelated alkynes, and alkynes capable of undergoing 1,3 cycloaddition reactions with azides in the absence of copper-based catalysts. Suitable alkynes also include, but are not limited to, DIBAC, DIBO, BARAC, DIFO, substituted alkynes, eg fluorinated alkynes, aza-cycloalkynes, BCN. Linker loads containing such reactive groups are useful for conjugating antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azidopolyethylene glycol groups. In certain embodiments, such a functionalized antibody is prepared by reacting an antibody containing at least one glutamine residue, such as a Q295 heavy chain, and a compound of the formula H ₂ N-LL-N ₃ , where LL is a divalent polyethylene glycol group, in the presence of a transglutaminase enzyme. For convenience, in certain formulas presented herein, an Ab antibody is a modified antibody with one or more covalently linked -LL-N ₃ groups or residues thereof. Preferably, in each case, -LL-N3 is covalently linked to the amino acid side chain of the glutamine residue of the antibody. Also preferably, -LL-N3 reacts or can react with the reactive group of RG, resulting in the formation of a covalent bond with the linker load. Again, for convenience, in certain formulas presented here, -LL-N3 groups are presented in explicit form.

Disclosed herein are methods for synthesizing the conjugates described herein, involving contact between a coupling agent, such as an antibody, and a linker load described herein. In certain embodiments, the linker load includes a cyclodextrin moiety.

Compounds of formula (II), (II-P) or (II-P-1) are linker loads that are useful synthetic intermediates in the synthesis of the conjugates described herein. These linker loads contain a reactive group that reacts with the antibody to form the conjugates described herein.

In some embodiments of Formula (II), (II-P), or (II-P-1), R ⁴ is alkyl. In some embodiments of Formula (II), (II-P), or (II-P-1), R ⁴ is methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, t-butyl , i-butyl, pentyl moiety, hexyl moiety, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments of Formula (II), (II-P), or (II-P-1), R ⁴ is alkyl. In some embodiments of Formula (II), (II-P), or (II-P-1), R ⁴ is methyl, ethyl, n-propyl, i-propyl, n-butyl, sbutyl, t-butyl, i- butyl, pentyl moiety or hexyl moiety. In some embodiments of Formula (II), (II-P), or (II-P-1), R ⁴ is n-propyl.

In certain embodiments, RG-L ² -(L3) _0-1 - is rgspMpeg)—sp^aa'aaMa Usn ₂ o—|

CD CD _; _ 'NV is aryl or heteroaryl; RG is a reactive group as defined below; SP ¹ and SP ² are each, independently in each example, absent or a residue of a spacer group, and wherein SP ¹ contains a trivalent linker; AA ¹ is a trivalent linker containing an amino acid residue; AA ² is a peptide residue; PEG is polyethylene glycol

Λ remainder; where ^x denotes the atom through which the specified chemical group binds to neighboring groups in the formula, CD is independently in each case, absent or is a cyclodextrin residue where at least one CD is present, subscript q is an integer that is selected from 0 to 5 , inclusive. In these examples, the index q is 0, 1, 2, 3, 4, or 5. In some embodiments, the index q is 0. In some embodiments, the index q is 1. In some embodiments, the index q is 2. In some embodiments, the index q is 3. In some embodiments, the q index is 4. In some embodiments, the q index is 5. In some embodiments, either AA ¹ or AA ² contains, independently in each case, an amino acid that is selected from alanine, valine, leucine , isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or citrulline, a derivative thereof, or a combination thereof. In certain embodiments, AA ¹ is an amino acid that is selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine , histidine or citrulline, a derivative thereof or a combination thereof. In certain embodiments, AA ¹ is lysine or a lysine derivative. In certain embodiments, AA ¹ is lysine. In certain embodiments, AA ¹ is D-lysine. In certain embodiments, AA ¹ is L-lysine. In certain embodiments, AA ² is a dipeptide residue, a tripeptide residue, a tetra-peptide residue, or a penta-peptide residue. In certain embodiments, AA ² is a dipeptide residue. In certain embodiments, AA ² is valine-citrulline. In some embodiments, AA ² is citrulline-valine. In some embodiments, AA ² is valine-alanine. In some embodiments, AA ² is alanine-valine. In some embodiments, AA ² is valine-glycine. In some embodiments, AA ² is glycine-valine. In certain embodiments, AA ² is a tripeptide residue. In some embodiments, AA ² is glutamate-valine-citrulline. In some embodiments, AA ² is glutamate-valine-citrulline. In some embodiments, AA ² is lysine-valinalanine. In some embodiments, AA ² is lysine-valine-citrulline. In some embodiments, AA ² is glutamate-valine-citrulline. In some embodiments, AA ² is a tetrapeptide residue. In some embodiments, AA ² is glycine-glycine-phenylalanine-glycine. In some embodiments, AA ² is a pentapeptide residue. In some embodiments, AA ² is glycine-glycine-glycine-glycine-glycine. In some embodiments, SP ¹ is independently selected in each instance from the group consisting of C1-6alkylene, -NH-, -C(O)-, (-CH2-CH2O)e, -NH-CH2-CH2-(-O- CH2-CH2)eC(O)-, -C(O)-(CH2)uC(O)-, -C(O)-NH-(CH2)v- and their combinations, where the index e is an integer, which is selected from 0 to 4, inclusive, index and is an integer that is selected from 1 to 8, inclusive, and index v is an integer that is selected from 1 to 8, inclusive. In some embodiments, SP ² is independently selected in each case from the group consisting of C1_ ₆ alkylene, -NH-, -C(O)-, (-CH ₂ -CH ₂ -O) _e , NH-CH2-CH2-( -O-CH2-CH2)eC(O)-, -C(O)-(CH2)uC(O)-, -C(O)-NH-(CH2)v- and their combinations, where the subscript e represents an integer that is selected from 0 to 4, inclusive, index u is an integer that is selected from 1 to 8, inclusive, and index v is an integer that is selected from 1 to 8, inclusive.

In certain embodiments, provided herein is a compound having the structure of formula (IIIc-R):

^with 1 5 RG

CD

Formula (IIIc-R)

RG is a reactive group;

CD is a cyclodextrin;

SP ¹ is a spacer group;

AA ⁴ is an amino acid residue;

AA ⁵ is a dipeptide residue;

PEG is polyethylene glycol;

q is an integer that can be selected from 0 to 5, inclusive;

x is an integer that can be selected from 0 to 30, inclusive;

R ⁴ is as defined here;

SP ¹ and SP ² are independently in each case absent or a residue of a spacer group and where SP ¹ contains a trivalent linker; AA ⁴ is a trivalent linker containing an amino acid residue; AA ⁵ is a dipeptide residue; PEG is a polyethylene glycol moiety; where denotes the atom through which the specified chemical group binds to neighboring groups in the formula, CD is independently absent in each case or is a cyclodextrin residue, where at least one CD is present, the index q is an integer that is selected from 0 to 5, inclusive; in these examples, the index q is 0, 1, 2, 3, 4, or 5. In some embodiments, the index q is 0. In some embodiments, the index q is 1. In some embodiments, the index q is 2. In some embodiments, the index q is 3. In some embodiments, the q index is 4. In some embodiments, the q index is 5. In some embodiments, either AA ⁴ or AA ⁵ contains, independently in each case, an amino acid that is selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or citrulline, a derivative thereof, or a combination thereof. In certain embodiments, AA ⁴ is an amino acid that is selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine , histidine or citrulline, a derivative thereof or a combination thereof. In certain embodiments, AA ⁴ is lysine. In certain embodiments, AA ⁴ is lysine or a lysine derivative. In certain embodiments, AA ⁵ is valine-citrulline. In some embodiments, AA ⁵ is citrulline-valine. In some embodiments, AA ⁵ is valine-alanine. In some embodiments, AA ⁵ is alanine-valine. In some embodiments, AA ⁵ is valine-glycine. In some embodiments, AA ⁵ is glycine-valine. In some embodiments, AA ⁵ is glutamate-valine-citrulline. In some embodiments, AA ⁵ is glutamine-valine-citrulline. In some embodiments, AA ⁵ represents whether

- 89 045111 zine-valine-alanine. In some embodiments, AA ⁵ is lysine-valinecitrulline. In some embodiments, AA ⁵ is glutamate-valine-citrulline. In some embodiments, SP ¹ is independently selected from the group consisting of C ₁ in each case. ₆ alkylene, -NH-, -C(O)-, (-CH ₂ -CH ₂ -O) _e , -NH-CH ₂ -CH ₂ -(-O-CH ₂ -CH ₂ ) _e -C(O )-, -C(O)-(CH ₂ ) _u -C(O), -C(O)-NH-(CH2) _v - and their combinations, where the index e is an integer that is selected from 0 to 4, inclusive, index u is an integer that is selected from 1 to 8, inclusive, and index v is an integer that is selected from 1 to 8, inclusive. In some embodiments, SP ² is independently selected in each case from the group consisting of C1-6alkylene, -NH-, -C(O)-, (-SCH-SCH-OU -NH-CH2-CH2-(-O-CH2 -CH2)eC(O)-, -C(O)-(CH2)uC(O)-, -C(O)-NH-(CH2)v- and their combinations, where the index e is an integer that is selected from 0 to 4, inclusive, index and is an integer that is selected from 1 to 8, inclusive, and index v is an integer that is selected from 1 to 8, inclusive. Reactive group (RG) is a functional group or moiety that reacts with a reactive moiety of an antibody, a modified antibody, or an antigen-binding fragment thereof.In certain embodiments, the reactive moiety is a functional group or moiety (e.g., maleimide or NHS ester) that reacts with a cysteine or lysine residue of an antibody or antigen binding fragment thereof.In certain embodiments, a reactive group is a functional group or moiety capable of click chemistry. In some embodiments of this click chemistry reaction, the reactive group contains an alkyne that is capable of undergoing a 1,3 cycloaddition reaction with the azide. Such suitable reactive groups include, but are not limited to, strained alkynes, such as those suitable for strain-promoted alkyne-azide cycloadditions (SPAAC), cycloalkynes, such as cyclooctynes, benzannelated alkynes, and alkynes capable of undergoing 1,3 cycloaddition reactions with azides in the absence of copper-based catalysts. Suitable alkynes also include, but are not limited to, DIBAC, DIBO, BARAC, DIFO, substituted alkynes, eg fluorinated alkynes, aza-cycloalkynes, BCN. Linker loads containing such reactive groups are useful for conjugating antibodies that have been functionalized with azido groups. Such functionalized antibodies include antibodies functionalized with azido-polyethylene glycol groups. In certain embodiments, such a functionalized antibody is prepared by reacting an antibody containing at least one glutamine residue, such as a Q295 heavy chain, and a compound of the formula H2N-LL-N3, wherein LL is a trivalent polyethylene glycol group that includes a polyethylene glycol and a cyclodextrin moiety , in the presence of the enzyme transglutaminase. In some embodiments, the reactive group is an alkyne, for example, 'which may

N=N=K reacts via click chemistry with an azide, e.g., resulting in a click chemistry product, e.g.

’ its regioisomer or mixture thereof. In some embodiments, the reactive group is an alkyne, e.g.

action via click chemistry with an azide, e.g.

’ which can enter into relic chemistry, for example,

resulting in the formation of a product. In some embodiments, the reactive group is an alkyne, such as ^CH , which can react via click chemistry with

N=N=N _ azide, for example, ’ resulting in the formation of a click chemistry product, for example,

regioisomer or mixture thereof. In some embodiments, the reactive group is

- 90 045111 functional group, for example,

’ which reacts with a cysteine residue on an antibody or its antigen-binding fragment, resulting in the formation of a bond with it, for example, AB„

^υ > where Ab denotes an antibody or an antigen-binding fragment thereof, and S denotes the S atom on the cysteine residue through which the functional group binds to the Ab. In some embodiments, the reactive group is a functional group, e.g.

which reacts with a lysine residue on an antibody or an antigen-binding fragment thereof to form a bond with it, for example, where Ab is the antibody or an antigen-binding fragment thereof and N is the N atom on the lysine residue through which the functional group binds to Ab. In some embodiments, the N atom on the lysine residue through which the functional group binds is indicated herein by the letter N above the bond, e.g., O

In some embodiments, RG-L ² -(L ³ ) _0-1 is a monovalent moiety of formula (LA);

RG-(SPV(A)z-(NR ^a )s-(BHCH2)u-(OM SP ² )W(L ^A );

where RG represents a reactive group; A is an amino acid or a peptide; R ^a represents H or alkyl;

B is aryl, heteroaryl or heterocycloalkyl, wherein aryl, heteroaryl or heterocycloalkyl is optionally replaced by alkyl, -OH or -NR ^a R ^b ; SP ¹ and SP ² are independently spacer groups; and q, z, s, t, u, v and w, independently in each case, 0 or 1.

In some embodiments, RG-L ² -(L ³ ) _0-1 - is RG-(SP1) _q -(A) _z -. In some embodiments, RG-L -(L) _0-1 - is RG-(SP) _q -(A) ₂ -. In some embodiments, RG-L ² -(L ³ ) _0-1 is a fragment of the formula (LA ¹ )

RG— (sp ¹ )—I

o (L ^A1 ) where RAA ¹ and RAA ² represent in each case, independently, amino acid side chains. In some embodiments of formula LA ¹ SP ¹ is a divalent polyethylene glycol group and RG is an alkyne containing group that is capable of undergoing a 1,3-cycloaddition reaction with an azide.

In some embodiments, RG-L ² -(L ³ )0-1- has the following structure: RG-(SP ¹ )qZ ¹ Z ² -Z ³ oi-, where

RG, SP ¹ and q are as defined herein;

Z ¹ represents a polyethylene glycol or caproyl group;

Z ² is a dipeptide; And

Z ³ represents a PAB group.

In some other embodiments, -L1-L ² -(L ³ ) _0-1 - is a trivalent moiety with the formula (L ^B );

-L ¹ -(SP ¹ )q-(A)z-(NR ^a )s-(B)t-(CH2)u-(O) _v -(SP ² ) _W (L ^B );

where L ¹ corresponds to the definition given here;

A is a tripeptide or tetrapeptide, wherein at least one of the amino acids in the tripeptide or tetrapeptide binds directly or indirectly to a cyclodextrin moiety;

R ^a represents H or alkyl;

B is aryl, heteroaryl or heterocycloalkyl, wherein aryl, heteroaryl or heterocycloalkyl is optionally replaced by alkyl, -OH or -NR ^a R ^b ;

SP ¹ and SP ² are independently spacer groups; and in the formula L ^B , q, z, s, t, u, v and w, independently in each case represent 0 or 1.

In some embodiments, the cyclodextrin (CD) binds directly to an amino acid residue, such as a lysine amino acid residue. This means that the CD is one bond away from the covalent linker of the amino acid lysine. In some of these embodiments, the covalent linker is also directly coupled to the load. This means that the covalent linker is one bond away from the load, such as, but not limited to, the steroid load described herein. In some of these embodiments, the covalent linker also directly binds to the CD. This means that the covalent linker is one bond away from the CD, such as the CDs described herein. In some of these embodiments, the covalent linker is the amino acid lysine or a derivative thereof.

In some embodiments, the CD indirectly binds to a covalent linker in the connecting group (eg, L, -L ² -(L ³ )0-1- and -L ¹ -L ² -(L ³ )0-1). This means that the CD is more than one bond away from the covalent linker. This also means that the CD binds through another moiety to a covalent linker. For example, CD can bind to a maleimide group, which binds to a polyethylene glycol group, which binds to a covalent linker. In some of these embodiments, the covalent linker is also indirectly coupled to the load. This means that the covalent linker is more than one bond away from the load, such as, but not limited to, the steroid load described herein. This also means that the covalent linker binds through another moiety to the load. For example, the covalent linker may be linked to a dipeptide, such as, but not limited to, Val-Ala or Val-Cit, which may be linked to a para-amino benzoyl, which may be linked to a load. In some of these embodiments, the covalent linker also indirectly binds to the cyclodextrin. This means that the covalent linker is more than one bond away from the cyclodextrin, such as the cyclodextrins described herein. This also means that the covalent linker binds through another moiety to the cyclodextrin. For example, the covalent linker may be linked to a polyethylene glycol group, which may be linked to a reactive group with a cyclodextrin. In some of these embodiments, the covalent linker is the amino acid lysine or a derivative thereof.

In some embodiments, -L ¹ -L ² -(L ³ )0-1- is -L ¹ -(SP ¹ )q-(A) _z -. In some embodiments, -L ¹ -L ² -(L ³ )0-1- is -L ¹ -(SP ¹ )q-(A) ₂ -. In some embodiments, -L ¹ -L ² -(L ³ ) _{0-1 -} is a fragment of the formula (L ^B1 )

(L ^B1 ) where RAA ¹ and R ^AA2 represent in each case, independently, amino acid side chains. RAA ³ is an amino acid side chain that is linked directly or indirectly to cyclodextrin. In some embodiments of formula L ^B1 , SP ¹ is a divalent polyethylene glycol group and L ¹ is the product of a 1,3 cycloaddition reaction between an alkyne and an azide.

О R ^M1 _ы О AWA □ААЗ ^П q RAA2

In some embodiments, A is

In some embodiments, RAA ¹ is an amino acid side chain, R ^AA2 is an amino acid side chain, and R ^AA3 is an amino acid side chain that binds directly or indirectly to the cyclodextrin moiety.

tt___________ ___________ _________________ l____________A^CD ___jwwCD____

In some embodiments, A is ’ where represents a direct or indirect linkage to the cyclodextrin moiety.

In some embodiments, including any of the above, the CD is, independently in each case, selected from

- 92 045111

In some embodiments, the CD is

- 93 045111

In some embodiments, the CD is

In some embodiments, the CD is

In some embodiments, the CD is

- 94 045111

In some embodiments, the CD is

In some embodiments, A is

In some embodiments herein, the RG is selected from a click chemistry reactive group.

In certain other examples herein, the RG is selected from the group that reacts with a cysteine or lysine residue on an antibody or antigen binding fragment thereof.

- 95 045111

In some embodiments, RG is

(where -OCH2C(O)- is covered by -L ² -),

In some embodiments, RG is

In other examples, RG is

In some other examples, RG is .O

In some embodiments, RG is ⁰

In other examples, RG is

In other examples, RG is

In some embodiments ^{, SP 1 can} be selected from:

, where -OCH ₂ C(O)- is covered by -L ² -. covered by -L ² -. In some embodiments,

In some other examples, SP ¹ represents

ABOUT

In some embodiments, SP ¹ is

In other examples, SP ¹ represents

In other examples, SP ¹ represents

In some other examples, SP ¹ represents

In any of the above examples, the subscripts a, b, and c are independently, in each case, an integer that is selected from 1 to 20, inclusive.

In some embodiments, SP ¹ is covered by -L ² -. In any of the SP ¹ connections

- 96 045111

In some embodiments, SP ¹ is covered by -L ² -. In some embodiments,

implementation

SP ¹ some variants SP ¹ B

B represents some options

implementation

SP ¹ is

In some embodiments, SP ¹ is

. In some embodiments, SP ¹ is *. In some embodiments, SP ¹ is ⁰

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some variants

implementation

SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

In some embodiments, SP ¹ is covered by -L ² -. In some embodiments, SP ¹ is

In some embodiments, SP ¹ is

- 97 045111

IN

IN

IN

IN

In some embodiments, SP ^1, some embodiments, SP ^1, some embodiments, SP ^1, some embodiments, SP ¹ is is is is is is is

In some embodiments, RG-SP ¹ (where SP ¹ is covered by -L ² -) can be selected from the group consisting of:

where b, c and d are independently, in each case, an integer from 1 to 20, inclusive. In some of these embodiments, the subscripts b, c, and d are independently, in each case, an integer that is selected from 1 to 6, inclusive.

In some embodiments, RG-SP ¹ - is

In some embodiments, RG-SP

represents

In some embodiments, RG-SP ¹

In some embodiments, RG-SP ¹ is

In some embodiments, RG-SP ¹ is

- 98 045111

In some embodiments, RG-SP ¹ is

In some embodiments, RG-SP ¹ is

in some embodiments, RG-SP ¹ is In any of the connections RG-SP ¹ -:

In some embodiments, A is a peptide that is selected from valine-citrulline, citrulline-valine, lysine-phenylalanine, phenylalanine-lysine, valine-asparagine, asparagine-valine, threonine-asparagine, asparagine-threonine, serine-asparagine, asparagine- serine, phenylalanine-asparagine, asparagine-phenylalanine, leucine-asparagine, asparagine-leucine, isoleucine-asparagine, asparagine-isoleucine, glycine-asparagine, asparagine-glycine, glutamic acidasparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulli na, alanine asparagine or asparagine-alanine.

In some embodiments, A is valine-citrulline or citrulline valine.

In some embodiments, A is valine-alanine or alanine-valine.

In some embodiments, A is glutamate-valine-citrulline or cytrulline-valine-glutamate.

In some embodiments, A is valine. In some embodiments, A is alanine. In some embodiments, A is citrulline.

- 99 045111

In some embodiments, A is

In some of these embodiments, RAA ¹ is an amino acid side chain and wherein RAA ² is an amino acid side chain.

In some embodiments, A is

In some embodiments, A is

In some embodiments, R ^a is H. In some embodiments, R ^a is alkyl.

In some embodiments, R ^a is methyl, ethyl, n-propyl, i-propyl, nbutyl, t-butyl, i-butyl, or pentyl.

In some embodiments, B is aryl.

In some embodiments, B is phenyl.

In some embodiments, B is phenyl or pyridinyl.

In some embodiments, here B is:

(R ¹⁰ ),

or

In these examples, R ¹⁰ is alkyl, alkenyl, alkynyl, alkoxy, aryl, alkylaryl, arylalkyl, halo, haloalkyl, haloalkoxy, heteroaryl, heterocycloalkyl, hydroxyl, cyano, nitro, O

-|-or ^a -|-so ₂ r ^; ' NR ^a R ^b or azido. In these examples, the indices p and m are independently, in each case, selected from an integer that is selected from 0 to 4, inclusive. In some embodiments herein, B is:

In these examples, p is 0, 1, 2, 3, or 4. In some of these embodiments, R ¹ is, in each case, alkyl, alkoxy, haloalkyl, or halo. In some embodiments, R ¹ is alkyl. In some embodiments, R ¹ is alkoxy. In some embodiments, R ¹ is haloalkyl. In some embodiments, R ¹ is a halo.

In some embodiments of formula (LA), -(NR ^a ) _s -(B) _t -(CH ₂ ) _u -(O) _v -(SP ² )w represents

by yourself:

Also provided herein are linker loads of budesonide, a budesonide prodrug, a budesonide analog or derivative (including fluorinated analogs and derivatives), or a prodrug of a budesonide analog or derivative (including fluorinated analogs and derivatives). In some embodiments, a load linker with the following structure is provided here:

- 100 045111 where RG, R ⁴ , L ² , L ³ and SP correspond to the definitions given here.

E. Pharmaceutical compositions and methods of treatment.

Provided herein are methods of treating diseases, conditions or disorders, such as inflammatory diseases and autoimmune disorders, or controlling symptoms thereof, comprising administering a therapeutically effective amount of one or more compounds provided herein. Included are any diseases, disorders or conditions associated with the glucocorticoid receptor, glucocorticoid binding and/or glucocorticoid receptor signaling. Such methods include administering a steroid load or a protein conjugate thereof, as provided herein, to a patient. Thus, included herein are methods of treating a disease, disorder or condition associated with the glucocorticoid receptor, comprising administering a compound of formula (I), formula I-P, formula I-P-1, formula (III-P), formula III-P-1 , formula (3000) or (III) to a patient who has said disease, disorder or condition.

The present description of the invention includes methods for the prevention of certain diseases or conditions, including the administration of a therapeutically effective amount of one or more compounds provided herein (ie, prophylactic use). Examples include, but are not limited to, prevention of CD3 bispecific cytokine release syndrome and adoptive cell therapies such as CAR T cells, systemic administration of IL-2, prevention of graft-versus-host disease, and postoperative nausea and vomiting. Examples also include, but are not limited to, therapeutic antibodies such as alemtuzumab, muromonab, rituximab, tosituzumab, and agonistic Abs, where immune stimulation may be part of the required mechanism of action.

In some embodiments, such disease, disorder, or condition is an allergic condition, including, but not limited to, asthma, atopic dermatitis, contact dermatitis, allergic dermatitis, drug hypersensitivity, anaphylactic rhinitis, perennial or seasonal allergic rhinitis, and serum sickness; dermatological diseases and conditions, including, but not limited to, pruritus, seborrheic dermatitis, neurodermatitis, eczema, bullous dermatitis herpetiformis, exfoliative erythroderma, mycosis fungoides, pemphigus and erythema multiforme severe (Stevens-Johnson syndrome); endocrine disorders including, but not limited to, primary or secondary adrenocortical insufficiency, congenital adrenal hyperplasia, hypercalcemia associated with cancer, and De Quervain's granulomatous goiter; gastrointestinal diseases; hematological disorders, including, but not limited to, acquired (autoimmune) hemolytic anemia, congenital (erythroid) hypoplastic anemia (Diamond-Blackfan anemia), adult idiopathic thrombocytopenic purpura, true red cell aplasia and secondary thrombocytopenia; trichinosis; tuberculous meningitis with subarachnoid block or developing block; neoplastic diseases including, but not limited to, leukemias and lymphomas; disorders of the nervous system, including, but not limited to, severe exacerbations of multiple sclerosis, cerebral edema associated with a primary or metastatic brain tumor, craniotomy or head trauma; ophthalmic diseases, including, but not limited to, sympathetic ophthalmia, temporal arteritis, uveitis, xerophthalmia, and inflammatory conditions of the eye not responding to topical corticosteroids; renal disease, including, but not limited to, to induce diuresis or remission of proteinuria in idiopathic nephrotic syndrome or due to erythematous lupus; respiratory diseases, including, but not limited to, berylliosis, progressive or disseminated pulmonary tuberculosis when used concomitantly with appropriate anti-tuberculosis chemotherapy, idiopathic eosinophilic pneumonia, symptomatic sarcoidosis; and rheumatic disorders, including, but not limited to, use as adjunctive therapy for short-term administration (to overcome an acute episode or exacerbation in a patient) for acute gouty arthritis, acute rheumatic carditis, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, including juvenile rheumatoid arthritis, and for use in dermatomyositis, polymyositis, stomatitis and systemic lupus erythematosus.

In some embodiments, provided herein is a method of treating a disease, disorder, or condition that is selected from the following: an autoimmune disease, allergy, arthritis, asthma, respiratory disorder, blood disorder, cancer, collagen disease, connective tissue disorder, dermatologic disease, eye disease , endocrine disorder, immunological disease, inflammatory disease, intestinal disorder, gastrointestinal disease, neurological disorder, transplantation conditions, rheumatoid disorder, skin disease, edema condition, wound healing condition, and combinations thereof, including administration of a steroid load or a conjugate presented herein.

In some embodiments, the autoimmune disorder is selected from multiple sclerosis, autoimmune hepatitis, herpes zoster, systemic lupus erythematosus (ie, lupus), myasthenia gravis, Duchenne muscular dystrophy, and sarcoidosis. In some embodiments, the disorder

- 101 045111 breathing quality is selected from asthma, chronic respiratory disease, chronic obstructive pulmonary disease, bronchial inflammation and acute bronchitis. In some embodiments, the cancer is selected from leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), and multiple myeloma. In some embodiments, the collagen disease is systemic lupus erythematosus. In some embodiments, the eye disease is keratitis. In some embodiments, the endocrine disorder is selected from Addison's disease, adrenal insufficiency, adrenal dysfunction, adrenocortical hyperplasia, and congenital adrenal hyperplasia. In some embodiments, the inflammatory disease is selected from inflammation following cataract surgery, joint inflammation, immune inflammation, ligamentous inflammation, bursitis, epicondylitis, Crohn's disease, inflammatory bowel disease, lipoid pneumonitis, thyroiditis, urticaria, pericarditis, nephrotic syndrome, and uveitis. In some embodiments, the intestinal disorder is selected from collagenous colitis, ulcerative colitis, Crohn's disease, and inflammatory bowel disease. In some embodiments, the rheumatoid disorder is selected from rheumatoid arthritis, polymyalgia rheumatica, psoriatic arthritis, ankylosing spondylitis, and systemic lupus erythematosus. In some embodiments, the skin condition is selected from psoriasis, eczema, and dermatitis resulting from exposure to poison ivy. In some embodiments, the neurological disorder is chronic inflammatory demyelinating polyradiculoneuropathy.

In some embodiments, the compounds described herein are administered to a patient for the treatment of an acute inflammatory event, including, but not limited to, shock, cerebral edema, and graft-versus-host disease. In some embodiments, the compounds described herein are administered to treat lympholytic effects, including, but not limited to, effects associated with hematologic malignancies, such as leukemias, lymphomas, and myelomas.

In some embodiments, provided herein is a method of reducing inflammation in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a steroid or a conjugate thereof described herein. In some embodiments, provided herein is a method of modulating the immune system in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a steroid or a conjugate thereof described herein. In some embodiments, provided herein is a method of modulating cortisol levels in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a steroid or a conjugate thereof described herein. In some embodiments, provided herein is a method of reducing lymphocyte migration in a subject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a steroid or a conjugate thereof described herein. In some embodiments, provided herein is a method of treating hypercalcemia due to cancer, Meniere's syndrome, migraines, cluster headaches, severe canker sores, laryngitis, severe tuberculosis, Herxheimer's reaction to syphilis, decompensated heart failure, allergic rhinitis, or nasal polyps, comprising administering to a subject in need thereof, a therapeutically effective amount of a steroid or a conjugate thereof described herein. In some embodiments, the compounds provided herein can be used to treat inflammatory bowel disease, Crohn's disease, or ulcerative colitis. In some embodiments, said disease, disorder, or condition is a chronic inflammatory condition, including, but not limited to, asthma, skin infections, and eye infections. In some embodiments, the compounds provided herein are used for immunosuppression in patients undergoing organ transplantation.

In some embodiments, the steroid loads and conjugates thereof described herein are administered to a patient for the treatment of a nervous system disorder associated with GH signaling, including, but not limited to, psychiatric disorders such as schizophrenia, drug addiction, post-traumatic neuropsychiatric disorder, and disorders mood, alcohol or substance abuse, stress and anxiety.

In some embodiments, the steroid loads and their conjugates described herein are administered to a patient to treat a disorder of the visual system, including, but not limited to, ocular inflammation (eg, conjunctivitis, keratitis, uveitis), macular edema, and macular degeneration. In some embodiments, the steroid loads and their conjugates described herein are administered to a patient for the treatment of a cardiovascular disease. In some embodiments, steroid loads and their conjugates described herein are administered to a patient to treat a glucose disorder and/or a liver metabolic disorder. In some embodiments, steroid loads and their conjugates described herein are administered to a patient to treat a musculoskeletal disorder. In some embodiments, the steroid loads and their conjugates described herein are administered to a patient to treat skin inflammation, such as eczema and psoriasis.

- 102 045111

The protein conjugates described herein provide a means for targeting the delivery of a steroid load to specific cells or organ systems, thereby reducing or preventing the side effects associated with the administration of a free, unconjugated steroid load. Examples of such potential side effects that may be desired to be reduced or prevented include those listed in the approved drug label for Decadron® (dexamethesone), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the side effect to be reduced or prevented is selected from increased blood pressure; sodium retention; water/fluid retention (edema, angioedema, pulmonary edema); increased excretion of potassium; reversible suppression of the hypothalamic-pituitary-adrenal system; potential corticosteroid deficiency after completion of treatment; susceptibility to infections; exacerbation of systemic fungal infections; increasing the severity of chickenpox in pediatric and adult patients; increasing the severity of measles in pediatric and adult patients; posterior subcapsular cataract; glaucoma with possible damage to the optic nerves; increased manifestation of secondary eye infections caused by exposure to bacteria, fungi or viruses; an increase in new episodes of optic neuritis; Kaposi's sarcoma; drug-induced secondary adrenocortical insufficiency; increased risk of perforation in the presence of active or latent peptic ulcers, diverticulitis, new enteroenterostomy and ulcerative colitis; peritoneal irritation after gastrointestinal perforation; decreased osteogenesis; increased bone resorption; inhibition of osteoblast function; inhibition of bone growth in pediatric patients; development of osteoporosis at any age; acute myopathy (possibly affecting the muscles of the visual or respiratory system and possibly leading to quadriparesis); increased creatinine kinase; mental disorders ranging from euphoria, insomnia, mood swings, personality changes and severe depression to severe manifestations of psychosis; worsening existing emotional instability or psychotic tendencies; increased intraocular pressure; bradycardia; cardiac arrest; cardiac arrhythmias; heart enlargement; vascular collapse; chronic heart failure; fat embolism; hypertension; hypertrophic cardiomyopathy in premature newborns; myocardial perforation after a recent myocardial infarction; syncopation; tachycardia; thromboembolism; thrombophlebitis; vasculitis; acne; allergic dermatitis; dry scaly rashes; ecchymosis and petechiae; erythema; impaired wound healing; increased sweating; rash; striae; suppression of reactions to skin tests; decreased skin tissue turgor; thinning hair on the head; hives; decreased tolerance to carbohydrates and glucose; development of Cushingoid condition; hyperglycemia; glycosuria; hirsutism; hypertrichosis; increased need for insulin or oral antidiabetic agents in diabetes (insulin resistance); manifestations of latent diabetes mellitus; menstrual irregularities; secondary adrenocortical and pituitary unresponsiveness (especially during stress; as in trauma; surgery or disease); growth retardation in pediatric patients; chronic heart failure in susceptible patients; fluid retention; hypokalemic alkalosis; decreased potassium levels; sodium retention; bloating; increases in serum liver enzyme levels (usually reversible upon discontinuation of dosing); hepatomegaly; increased appetite; nausea; pancreatitis; peptic ulcer with possible perforation and hemorrhage; perforation of the small and large intestine (especially in patients with inflammatory bowel disease); ulcerative esophagitis; negative nitrogen balance due to protein catabolism; aseptic necrosis of the heads of the femur and humerus; loss of muscle mass; muscle weakness; osteoporosis; pathological fracture of long bones; steroid myopathy; tendon rupture; compression fractures of the spine; convulsions; depression; emotional instability; euphoria; headache; increased intracranial pressure with papilledema (benign intracranial hypertension), usually after cessation of treatment; insomnia; mood changes; neuritis; neuropathy; paresthesia; personality changes; psychiatric disorders; vertigo; exophthalmos; glaucoma; increased intraocular pressure; posterior subcapsular cataracts; abnormal fat deposits; decreased resistance to infection; hiccups; increased or decreased motility and sperm count; general malaise; moon-shaped face and weight gain; as well as side effects associated with drug-drug interactions. In some embodiments, the side effects that are desired to be reduced or prevented are those associated with drug-drug interactions. In some embodiments, the side effect sought to be reduced or prevented is associated with drug-drug interactions from the use of a corticosteroid with aminoglutethimide, including a decrease in adrenal suppression by corticosteroids; injection of amphotericin B and potassium-non-sparing drugs, including the development of hypokalemia, heart enlargement and chronic heart failure; antibiotics, including a significant decrease in corticosteroid clearance; anticholinesterase drugs, including the development of severe weakness in patients with myasthenia gravis; oral anticoagulants, including inhibition of response to warfarin; antidiabetic drugs, including increased con

- 103 045111 blood glucose concentration; anti-tuberculosis drugs, including reduced serum concentrations of isoniazid; cholestyramine, including increased clearance of corticosteroids; cyclosporine, including increased activity of both cyclosporine and corticosteroids and the frequency of convulsions; the impact of the dexamethasone suppression test, including false-negative results in patients treated with indomethacin; digitalis glycosides, including an increased risk of arrhythmias due to hypokalemia; ephedrine, including an increase in the metabolic clearance of corticosteroids, which leads to decreased blood levels and decreased physiological activity; estrogens, including oral contraceptives, including decreased hepatic metabolism of certain corticosteroids and a corresponding increase in their exposure; hepatic enzyme inducers, inhibitors and substrates (drugs that induce the enzyme activity of cytochrome P450 3A4 (CYP 3A4), for example, barbiturates, phenytoin, carbamazepine, rifampin), including increased metabolism of corticosteroids; drugs that inhibit CYP 3A4 (eg, ketoconazole, macrolide antibiotics such as erythromycin), including the possibility of increasing plasma concentrations of corticosteroids; drugs that are metabolized by CYP 3A4 (eg, indinavir, erythromycin), including an increase in their clearance, which leads to reduced plasma concentrations; ketoconazole, including reduced metabolism of certain corticosteroids by up to 60%, which leads to an increased risk of corticosteroid side effects, and inhibition of adrenal corticosteroid synthesis, which may cause adrenal insufficiency during corticosteroid withdrawal; nonsteroidal anti-inflammatory drugs (NSAIDS), including an increased risk of gastrointestinal side effects and increased clearance of salicylates; phenytoin, including increases or decreases in phenytoin levels, changes in seizure control; skin tests, including suppression of reactions to skin tests; thalidomide, including toxic epidermal necrolysis; and vaccines, including reduced response to toxoids and live or inactivated vaccines due to inhibition of antibody response or potentiation of replication of some organisms contained in live attenuated vaccines).

Thus, provided herein are methods of treating a disease, disorder or condition associated with a glucocorticoid receptor, comprising administering a conjugate of formula (III) to a patient with said disease, disorder or condition, wherein the side effects associated with administration of a free steroid load of said conjugate are reduced. Moreover, provided herein are methods of delivering a compound of formula (III-P), formula III-P-1, formula (3000) or formula (III) to a cell, comprising exposing said cell to a protein conjugate of a compound of formula (III-P), formula Ш-Р-1, formula (3000) or formula (III), when the protein conjugate contains an antibody or an antigen-binding fragment thereof that binds to a surface antigen of said cell.

The compounds described herein may be administered alone or together with one or more additional drugs. Such one or more drugs may be administered immediately before, simultaneously with, or shortly after administration of the compounds described herein. The present disclosure includes pharmaceutical compositions containing any of the compounds described herein in combination with one or more drugs, and methods of treatment comprising administering such combinations to subjects in need thereof.

Suitable additional medications include, but are not limited to: a second glucocorticoid, an autoimmune drug, a hormone, a biologic, or a monoclonal antibody. Suitable drugs also include, but are not limited to, any pharmaceutically acceptable salts, acids or derivatives of a compound herein.

The compounds described herein may also be administered and/or combined with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotective agents, metal chelators, IFN-gamma and/or non-steroidal anti-inflammatory drugs.

In some embodiments of the methods described herein, multiple doses of a compound described herein (or a pharmaceutical composition containing a combination of a compound described herein and any of the additional drugs mentioned herein) may be administered to a subject over a period of time. In accordance with this aspect of the invention, such methods involve sequentially administering multiple doses of a compound described herein to a subject. As used herein, sequential administration means that each dose of a compound is administered to a subject at different points in time, eg, on different days separated by a predetermined interval (eg, hours, days, weeks, or months). The present invention includes methods that include sequentially administering to a patient one initial dose of a compound described herein, followed by one or more secondary doses of the compound, and then optionally one or more tertiary doses of the compound.

The terms initial dose, secondary doses and tertiary doses refer to the time sequence of administration of the compounds described herein. Thus, the initial dose is the dose that is administered at the beginning of the course of treatment (also called the basal dose); secondary doses

- 104 045111 represents these doses that are administered after the initial dose; and tertiary doses are doses that are administered after secondary doses. The initial, secondary and tertiary doses may all contain the same amount of the compound described herein, but typically differ from each other in terms of frequency of administration. In some embodiments, the amount of compound included in the primary, secondary and/or tertiary doses varies (eg, increases or decreases as appropriate) over the course of treatment. In some embodiments, two or more (eg, 2, 3, 4, or 5) doses are administered at the beginning of treatment as loading doses, followed by subsequent doses that are administered at less frequent intervals (eg, maintenance doses).

In certain illustrative embodiments of the present invention, each secondary and/or tertiary dose is administered through 1 is 26 (e.g., 1, _11/2 , 2, 21/2, 3, 31/2, ₄ , 41/2, 5, 51 /2, 6, 61/2, 7, 71/2, 8, 81/2, 9, 91/2, 10, 101/2, 11, 111/2, 12, 121/2, 13, 131/2 , 14, 141/2, 15, 151/2, 16, 161/2, 17, 171/2, 18, 181/2, 19, 191/2, 20, 201/2, 21, 211/2, 22 , 221/2, 23, 231/2, 24, 241/2, 25, 251/2, 26, 261/2 or more) weeks after the immediately preceding dose. The phrase immediately preceding dose as used herein means, in a sequence of multiple administrations, the dose of a compound that is administered to a patient before administration of the next dose in the sequence without intervening doses.

Methods in accordance with this aspect of the invention may include administering to a patient any number of secondary and/or tertiary doses of the compound. For example, in some embodiments, only one secondary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses are administered to the patient. Likewise, in some embodiments, only one tertiary dose is administered to the patient. In other embodiments, two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) tertiary doses are administered to the patient. The administration regimen may be continued indefinitely throughout the life of the subject or until such treatment is no longer therapeutically required or beneficial.

In embodiments involving multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Likewise, in embodiments involving tertiary doses, each tertiary dose may be administered at the same frequency as other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In some embodiments, the frequency with which secondary and/or tertiary doses are administered to a patient may vary throughout the course of treatment. The frequency of administration may also be adjusted throughout treatment by the physician depending on the needs of the individual patient after a clinical examination.

The present invention includes administration regimens in which 2 to 6 bolus doses are administered to a patient at a first frequency (e.g., weekly, biweekly, triweekly, monthly, bimonthly, etc.) , after which the patient is given two or more maintenance doses at less frequent intervals. For example, in accordance with this aspect of the invention, if loading doses are administered at a frequency of once a month, then the patient may be given maintenance doses every six weeks, every two months, every three months, etc.

The present invention includes pharmaceutical compositions and/or conjugates described in the present invention, for example, compounds of formula (I), formula I-P, formula I-P-1, formula (IIIP), formula III-P-1, formula (3000) and formula ( III), for example, compositions containing a compound described herein, a salt, stereoisomer, polymorph thereof, as well as a pharmaceutically acceptable carrier, diluent and/or excipient. Examples of suitable carriers, diluents and excipients include, but are not limited to, buffers to maintain the proper pH of the composition (e.g., citrate buffers, succinate buffers, acetate buffers, phosphate buffers, lactate buffers, oxalate buffers, and the like), carrier proteins (e.g. human serum albumin), saline solution, polyols (eg, trehalose, sucrose, xylitol, sorbitol and the like), surfactants (eg, polysorbate 20, polysorbate 80, polyoxolate and the like), antimicrobials and antioxidants.

In some embodiments, provided herein is a method of treating a disease, disorder, or condition comprising administering to a patient with said disorder a therapeutically effective amount of a compound of Formula (I), Formula I-P, Formula I-P-1, Formula (III-P), Formula III-P -1, formula (3000) or formula (III) or a pharmaceutical composition thereof.

In some embodiments, provided herein is a method of treating a disease, disorder, or condition comprising administering to a patient with said disorder a therapeutically effective amount of a compound of the formula provided herein or a pharmaceutical composition thereof.

In some embodiments, provided herein is a method of treating a disease, disorder or condition selected from the group consisting of an immunological disorder, an autoimmune disease, inflammation, asthma or inflammatory bowel disease, a disease

Crohn's, ulcerative colitis.

In some embodiments, provided herein is a method of treating a disease, disorder, or condition by targeting an antigen, such as a cell surface antigen, for which steroid delivery may provide a therapeutic effect, including administration of the conjugates described herein. In some embodiments, such antigen is AXL, BAFFR, BCMA, components of the list

BCR, BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8,BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CDllc, CD 137, CD 13 8, CD 14, CD 168, CD 177 , CD 19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, CD40, CD44, CD45, CD47, CD46, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2, CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1/2, CXCR4, CXCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA class II, HVEM, ICOSLG, IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL4R, IL6R, IL5R, IL7R, IL9R, Integrins, LAG3, LIFR, MAG/Siglec-4, MMR, MSR1, NCR3LG1, NKG2D, NKp30, NKp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec-1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR-list components/assoc, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT , TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4 or VTCNL

In some embodiments, such antigen is IL2R-y.

In some embodiments, provided herein is a method of treating a disease, disorder, or condition that is selected from an immunological disease, an autoimmune disease, an inflammatory disease, a dermatologic disease, a dermatologic disease, or a gastrointestinal disease.

In some embodiments, the disease is Crohn's disease, ulcerative colitis, Cushing's syndrome, adrenal insufficiency, or congenital adrenal hyperplasia.

In some embodiments, the disease is inflammation, asthma, or inflammatory bowel disease.

In some embodiments, such a disease is an autoimmune disease selected from multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, psoriasis, or eczema.

In some embodiments, provided herein is a method of reducing or alleviating the side effects of chemotherapy, comprising administering to a patient having the disease a therapeutically effective amount of a compound or composition described herein.

In some embodiments, provided herein is a method of reducing or alleviating the side effects of immunosuppressive therapy, comprising administering to a patient having the disease a therapeutically effective amount of a compound or composition described herein.

In some embodiments, provided herein is a method of treating cancer, comprising administering to a patient having the disease a therapeutically effective amount of a compound or composition described herein. In some embodiments, such cancer is selected from acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or multiple myeloma, among others.

F. Examples.

Certain embodiments are illustrated by the following non-limiting examples.

Reagents and solvents were obtained from commercial sources such as Sinopharm Chemical Reagent Co. (SCRC), Sigma-Aldrich, Alfa or other suppliers unless expressly stated otherwise.

¹ H NMR and other NMR spectra were recorded on a Bruker AVIII 400 or Bruker AVIII 500. Data were processed using Nuts software or MestReNova software measuring proton shifts in parts per million (ppm) downfield from the internal

- 106 045111 of the reference tetramethylsilane.

HPLC-MS measurements were performed on an Agilent 1200 HPLC/6100 SQ System using the following conditions:

Method A for HPLC-MS measurement included, as the mobile phase: A: Water (0.01% trifluoroacetic acid (TFA)) and B: acetonitrile (0.01% TFA). The gradient phase was 5% B, which was increased to 95% B over a time period of 15 min and at a flow rate of 1.0 ml/min. The column used was SunFire C18, 4.6x50 mm, 3.5 µm. The column temperature was 50°C. Detectors included an ADC evaporative light scattering detector (hereafter referred to as ADC ELSD), DAD (diode array detector, 214 nm and 254 nm), and electrospray ionization-atmospheric pressure ionization (ES-API).

Method B for HPLC-MS measurements included, as mobile phase: A: Water (10 mM NH4HCO3) and B: acetonitrile. The gradient phase was 5% B, which was increased to 95% B over a time period of 15 min and at a flow rate of 1.0 ml/min. The column used was XBridge C18, 4.6x50 mm, 3.5 µm. The column temperature was 50°C. Detectors included ADC ELSD, DAD (214 nm and 254 nm) and a mass selective detector (MSD ES-API).

LC-MS measurements were performed on an Agilent 1200 HPLC/6100 SQ System using the following conditions:

Method A for LC-MS measurements was implemented using a WATERS 2767 instrument. The column was a Shimadzu Shim-Pack, PRC-ODS, 20x250 mm, 15 µm, two in series. The mobile phase was A: Water (0.01% TFA) and B: acetonitrile (0.01% TFA). The gradient phase was 5% B, which was increased to 95% B over a time period of 3 min and at a flow rate of 1.8-2.3 ml/min. The column used was SunFire C18, 4.6x50 mm, 3.5 µm. The column temperature was 50°C. Detectors included ADC ELSD (Evaporative Light Scattering Detector), DAD (Diode Array Detector) (214 nm and 254 nm) and ES-API.

Method B for LC-MS measurements was implemented using a Gilson GX-281 instrument. The speaker used was Xbridge Prep C18 10 um OBD, 19x250 mm. The mobile phase was A: Water (10 mM NH ₄ HCO ₃ ) and B: Acetonitrile. The gradient phase was 5% B, which was increased to 95% B over a time period of 3 min and at a flow rate of 1.8-2.3 ml/min. The column used was XBridge C18, 4.6x50 mm, 3.5 µm. The column temperature was 50°C. Detectors included ADC ELSD, DAD (214 nm and 254 nm) and mass selective detector (MSD) (ES-API).

Preparative high-pressure liquid chromatography (Prep-HPLC) was performed using a Gilson GX-281 instrument. Two solvent systems were used, one acidic and one basic. The acid solvent system included a Waters SunFire 10 µm C18 column (100 A, 250x19 mm). Solvent A for prep-HPLC was 0.05% TFA in water, and solvent B was acetonitrile. The elution condition was a linear gradient that increased solvent B from 5% to 100% over a period of 20 minutes and at a flow rate of 30 ml/min. The primary solvent system included a Waters Xbridge 10 µm C18 column (100 A, 250 x 19 mm). Solvent A for prep-HPLC was 10 mM ammonium bicarbonate (NH4HCO3) in water, and solvent B was acetonitrile. The elution condition was a linear gradient that increased solvent B from 5% to 100% over a period of 20 minutes and at a flow rate of 30 ml/min.

Flash chromatography was carried out using a Biotage device with an Agela Flash Column silica-CS quartz flash column. Reversed phase flash chromatography was performed using a Biotage instrument, with Boston ODS or Agela C18, unless explicitly stated otherwise.

The following abbreviations are used in the examples and throughout the specification:

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Reduction Term ADC Antibody-drug conjugate Aglycosylated antibody Antibody that has no glycans water Water BARAC Biarylazacyclooctynone BCN (1A,8A95)-bicyclo[6.1,0]non-4-in-9-yl Vos N-tert-butoxycarbonyl VirN™ Thermo Scientific Prod# 28372, containing 100 mM sodium phosphate and 150 mM sodium chloride, no potassium, pH adjusted from 7.2 to 7.6-7.8 MQ unless otherwise noted. CD Cyclodextrin SOT Cyclooctynol Yes Dalton DAR Drug-antibody relationship. DCM Dichloromethane DIBAC Dibenz[/>/| Azocine, 11,12-didehydro-5,6-dihydro-; dibenzocyclooctyne; or dibenz[b,1]azocine-5(6H)-butanoic acid, 11,12-didehydro DIBAC-Suc Dibenz[b,1]azocine-5(6H)-butanoic acid, 11,12-didehydro DIBACT ZA-Benzo[s]-1,2,3-triazolo[4,5-e][1]benzazocin, 8,9-dihydro- DIBO Dibenzocyclooctyne DIFO Cyclooctyne bifluoride DIPEA Diisopropylethylamine DMA Ν,Ν-dimethyl acetamide DMAP 4-O1-methylaminopyridine DMF N,N-dimethylformamide DMSO Dimethyl sulfoxide ESI Electrospray ionization G gram HATU 2-(7-Aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate

- 108 045111

NS Immunoglobulin heavy chain NEK Human embryonic kidney cells HPLC High performance liquid chromatography h hour L.C. Immunoglobulin light chain L.C. Liquid chromatography MS Maleimidocaproyl mg milligram min minute ml milliliter mm millimolar MMAE Monomethyl auristatin E MS Mass spectrometry MsCl Methanesulfonyl chloride MSD Mass selective detector MTG Microbial transglutaminase M.B. Molecular weight ncADC Noncytotoxic antibody-drug conjugate NHS N-hydroxy succinimide nM nanomolar NMR Nuclear magnetic resonance NOESY Spectroscopy of the nuclear Overhauser effect PAB Para-aminobesyloxy(carbonyl) PBS 10 mM sodium phosphate buffer and 150 mM sodium chloride PBSg 10 mM phosphate, 150 mM sodium chloride, 5% glycerol P.E.G. Polyethylene glycol PNP p-nitrophenol PPm Parts per million (chemical shift) R.P. Reversed phase KT Room temperature SDS-PAGE Polyacrylamide gel electrophoresis using sodium dodecyl sulfate

- 109 045111

SEC Size exclusion chromatography Sue succinic acid TSER Tris(2-carboxyethyl)phosphine hydrochloride TEA Triethylamine T.F.A. Trifluoroacetic acid TG Transglutaminase THF Tetrahydrofuran VP Time of flight UPLC Ultra-performance liquid chromatography UV Ultraviolet V.A. Valine-aniline V.C. Valine-citrulline MKL microliter mkM micromolar

Cooking methods

Example 1.

In diagram 1 in Fig. Figure 3 shows the synthesis of budesonide spacers containing reactive groups, succinic acid (compound 1c), carbamate analogs (1d, 1e), THP analogs (1g and 1h), glucose analogs (1i and 1j), phosphate analogs (1k and 1l) and commercial phosphate analog (1m)

Intermediate 3a.

1-[({2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo [10.8. 0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy} carbonyl)oxy]pyrrolidine2,5-dione (3 a)

To a solution of budesonide 1a (0.10 g, 0.26 mmol) in DCM (1 ml) was added bis(2,5-dioxopyrrolidin-1yl) carbonate (71 mg, 0.30 mmol), triethylamine (47 mg, 0.52 mmol) and DMAP (3.0 mg , cat.). The reaction mixture was stirred at 15-25°C for 12 hours until the budesonide was destroyed, which was monitored by LCMS. The reaction mixture was then diluted with DCM and washed with water. The organic solution was dried over sodium sulfate. After filtration, the solution was concentrated in vacuo and the residue (crude 3a) was used immediately for the next step without purification. (93 mg, yield 71%). ESI m/z: 572.2 (M+H)+.

Example 1c.

4- {2-[( 1 S,2S,4R,8S,9S, 11S, 12S, 13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}-4-oxobutanoic acid (1c)

See WO2015005459; WO2013074988; Research in Pharmaceutical Science, 2011, 6(2), 107-116; and International Journal of Pharmaceutics, 2009, 365(1-2), 69-76.

To a solution of budesonide (la, 0.10 g, 0.26 mmol) in DCM (1 ml) was added succinic anhydride (30 mg, 0.30 mmol), triethylamine (47 mg, 0.52 mmol) and DMAP (3 mg, catalyst, 0.02 mmol). The mixture was stirred at RT for 12 hours until the budesonide was destroyed, which was monitored by TLC and

- 110 045111

LCMS. The reaction mixture was then diluted with DCM and washed with water. The organic solution was dried over sodium sulfate. After filtration, the solution was concentrated in vacuo and the residue was purified by prep-HPLC to give the title compound 1c (22 mg, 18% yield) as a white solid. ESI m/z: 531.3 (M+H)+.

Ή NMR (500 MHz, CDCl ₃ ) δ7.48 (d J=10 Hz, 1H), 6.28-6.26 (m, 1H), 6.04-6.02 (m, 1H), 5.23-5.11 (m, 1H), 5.06 -4.99 (m, 1H), 4.83-4.68 (m, 1H), 4.45-4.43 (m, 1H), 2.76-2.63 (m, 5H), 2.41-2.39 (m, 1H), 2.26-2.12 (m, 2H), 1.99-1.66 (m, 6H), 1.57-1.38 (m, 6H), 1.32 (s, 1H), 1.16-0.94 (m, 8H) ppm.

Example 1d.

2-[( 1 S,2S,4R,8S,9S, 11 S,12S, 13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2, 9} .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-methyl-N-[2(methylamino)ethyl]carbamate (1d) hn— ° \ but . VI /AW ⁰ G Th JA

To a solution of crude intermediate 3a (63 mg, 0.11 mmol) in DCM (5 mL) was added N,N'dimethylethane-1,2-diamine (28 mg, 0.32 mmol) and triethylamine (38 mg, 0.38 mmol) at RT. The resulting mixture was stirred at RT for 2 hours until most of 3a was destroyed, which was monitored by LCMS. Volatiles were removed in vacuo and the residue was purified by prep-HPLC (Method B) to give 1d (4 mg, 4.5% yield) as a white solid. ESI m/z: 545.3 (M+H) ⁺ .

1H NMR (500 MHz, CDCl ₃ ) 57.32-7.30 (m, 1H), 6.28-6.25 (m, 1H), 6.02-6.01 (m, 1H), 4.42 (br s, 1H), 3.50-3.23 (m, 3H), 3.10-3.02 (m, 3H), 2.82-2.71 (m, 3H), 2.57-2.55 (m, 1H), 2.35-2.33 (m, 1H), 2.15-2.06 (m, 2H), 1.96- 1.79 (m, 12H), 1.60-1.56 (m, 3H), 1.46 (m, 3H), 1.37-1.33 (m, 2H), 1.23-1.04 (m, 4H), 0.92-0.86 (m, 3H) ppm .

Example 1e.

2-[(1S,2S,4R,8S,9S,11S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapenta cyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-[(hydrazinecarbonyl)methyl]carbamate (1e)

To a mixture of crude compound 3a (0.16 g, 0.28 mmol) in DMF (3 ml) was added Boc-2-(2aminoacetyl)hydrazine (80 mg, 0.42 mmol) and triethylamine (85 mg, 0.84 mmol). The reaction mixture was stirred at 25 °C for 16 h until the destruction of intermediate 3a was monitored by LCMS. The mixture was then cooled with water and extracted with ethyl acetate. The combined organic solution was dried over anhydrous sodium sulfate. After filtration, the solution was concentrated in vacuo. The residue was purified by silica gel column chromatography (30-50% ethyl acetate in petroleum ether) to give Boc-1e (70 mg) as a white solid (ESI m/z: 646 (M+H) ⁺ ), which was dissolved in DCM (2 ml). TFA (1 ml) was added dropwise to the solution at 0°C. The mixture was stirred at 25°C for 2 hours until the destruction of Boc-1e, which was monitored by LCMS. Volatiles were removed in vacuo and the residue was purified by prep-HPLC to give the title compound 1e (7 mg, 12% yield) as a white solid. ESI m/z: 546 (M+H) ⁺ .

Example 1g.

(1 S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-8-(2-{ [6-(hydroxymethyl)oxan-2-yl]oxy}acetyl)-9,13dimethyl- 6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^{2.9.0 4.8.0 13.18} ]icosa-14,17-dien-16-one (1g)

To a solution of budesonide (1a, 50 mg, 0.12 mmol) in anhydrous DCM (4 ml) was added (3,4-dihydro-2H- 111 045111 pyran-2-yl)methanol (0.11 g, 0.93 mmol) and p-toluenesulfonic acid (31 mg, 0.18 mmol) at 0°C.

The reaction mixture was stirred at RT for 4 days, which was monitored by LCMS.

Volatiles were removed in vacuo and the residue was dissolved in DMF and separated by prep-HPLC (Method B) to give the title compound 1g (13 mg, 20% yield) as a white solid.

ESI m/z: 545 (M+H)+.

Example 1h.

Methyl 2-[(6-{2-[( 1 S,2S,4R,8S,9S,11S,12S, 13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5, 7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}oxan-2-yl)methoxy]acetate (1h)

To a solution of (3,4-dihydro-2H-pyran-2-yl)methanol (2.0 g, 18 mmol) in THF (30 ml) was added sodium hydride (60% in mineral oil, 0.88 g, 22 mmol) portionwise at 0 °C in a protective nitrogen atmosphere. The suspension was stirred at 0°C until the evolution of hydrogen ceased. A solution of ethyl 2-bromoacetate (4.0 g, 26 mmol) in THF (18 ml) was then added to the resulting mixture. The mixture was stirred at RT overnight until almost complete destruction of the starting material according to LCMS. After cooling to 0°C, the reaction mixture was cooled with water under a protective nitrogen atmosphere and extracted with ethyl acetate. The combined organic solution was washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (0-8% ethyl acetate in petroleum ether) to give ethyl 2-((3,4-dihydro-2H-pyran-2-yl)methoxy)acetate (0.70 g, 21% yield) as colorless oil.

1H NMR (500 MHz, DMSo-d ₆ ) δ 6.36 (d, J=6.0 Hz, 1H), 4.67 (m, 1H), 4.16 (s, 2H), 3.92 (m, 1H), 3.65 (s, 3H ), 3.57 (d, J=5.5 Hz, 2H), 2.50-1.99 (m, 1H), 1.93-1.82 (m, 1H), 1.80 (m, 1H), 1.60 (m, 1H) ppm.

To a solution of budesonide (1a, 0.11 g, 0.25 mmol) in anhydrous DCM (8 ml) was added ethyl 2-((3,4dihydro-2H-pyran-2-yl)methoxy)acetate (0.35 g, 1.9 mmol), obtained by the method , described above, and ptoluenesulfonic acid (66 mg, 0.35 mmol) at 0°C. The reaction mixture was stirred at RT for 4 hours, which was monitored by LCMS. Volatiles were removed in vacuo and the residue was dissolved in DMF and separated by prep-HPLC (Method A) to give the title compound 1h (46 mg, 29% yield) as a white solid. ESI m/z: 617 (M+H) ⁺ .

Example 1i.

(1 S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-9,13-dimethyl-6-propyl-8-(2-{[(2R,3R,4S,5S,6R )-3,4,5trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}acetyl)-5,7-dioxapentacyclo[10.8.00 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa14.17 -dien-16-one (1i, with epimers)

To a mixture of budesonide (1a, 0.22 g, 0.50 mmol) and acetobromo-α-D-glucose (0.33 g, 0.80 mmol) in DCM (15 ml), 4 A molecular sieves (1.0 g) were added and the mixture was stirred at RT for half an hour , and then added silver trifluoromethanesulfonate (0.19 g, 0.75 mmol) at 0°C. The suspension was stirred in the dark at RT over the weekend (72 h) under a protective nitrogen atmosphere. The mixture was filtered through celite and concentrated in vacuo. The residue was purified by prep-HPLC (method B) to give Ac-1i (25 mg, 9.2% yield) as a white solid. ESI m/z: 761 (M+H) ⁺ .

1H NMR (DMSO-d ₆ , 400 MHz) (with epimers) δ 7.34-7.30 (m, 1H), 6.19-6.14 (m, 1H), 5.91 (s, 1H), 5.29 (t, J=9.2 Hz, 1H), 5.17-5.15 (m, 0.5H), 5.03-5.01 (m, 0.5H), 4.96-4.90 (m, 1H), 4.85-4.74 (m, 3H), 4.70-4.67 (m, 1H), 4.64-4.63 (m, 0.5H), 4.59-4.56 (m, 0.5H), 4.39-4.28 (m, 2H), 4.20-4.16 (m, 1H), 4.05-3.99 (m, 2H), 2.562.40 (m, 1H), 2.32-2.26 (m, 1H), 2.13-1.89 (m, 14H), 1.83-1.63 (m, 3H), 1.59-1.48 (m, 3H), 1.44-1.21 (m, 6H) , 1.15-0.91 (m, 2H), 0.86-0.81 (m, 6H) ppm.

To a solution of compound Ac-1i (30 mg, 39 µmol) obtained above in water (1 ml) and methanol (3 ml) was added lithium hydroxide monohydrate (17 mg, 0.40 mmol) at 0°C. After stirring at 0°C for one hour, the mixture was purified directly by prep-HPLC (method B) to give 1i (24 mg, 98% yield) as a white solid. ESI m/z: 593 (M+H) ⁺ .

^1H NMR (MeOD-d ₄ , 400 MHz) (with epimers) δ 7.46 (t, J=10.4 Hz, 1H), 6.26 (dt, J=10.0 and 2.0 Hz, 1H),

- 112 045111

6.02 (s, 1H), 5.21 and 4.64 (t, J=4.8 Hz, 1H), 5.16 (t, J=7.2 Hz, 0.5H), 4.94-4.80 (m, 2.5H), 4.57-4.47 (m, 1H),

4.44-4.41 (m, 1H), 4.34-4.31 (m, 1H), 3.90-3.87 (m, 1H), 3.70-3.64 (m, 1H), 3.39-3.24 (m, 4H), 2.70-2.62 (m ,

1H), 2.41-2.36 (m, 1H), 2.27-2.08 (m, 2H), 2.00-1.93 (m, 1H), 1.88-1.81 (m, 1H), 1.74-1.32 (m, 9H), 1.22- 0.90 (m, 8H) ppm.

Example 1j.

(2S,3S,4S,5R,6R)-3,4,5-trihydroxy-6-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9, 13-dimethyl16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.00 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy} oxane2-carboxylic acid (1j, with epimers)

Following similar procedures as 1i, except replacing acetobromo-α-D-glucuronic acid with acetobromo-α-D-glucose methyl ester, compound lj (24 mg, 9.2% yield in 2 steps) was obtained as a white solid. ESI m/z: 607 (M+H) ⁺ .

1H NMR (MeOD-d ₄ , 400 MHz) δ 7.48 (t, J=10.0 Hz, 1H), 6.28-6.24 (m, 1H), 6.02 (s, 1H), 5.22-5.15 (m, 1H), 4.98 -4.81 (m, 2.5H), 4.65-4.56 (m, 1.5H), 4.45-4.39 (m, 2H), 3.62-3.59 (m, 1H), 3.52-3.46 (m, 1H), 3.41 (t, J=8.8 Hz, 1H), 3.30-3.28 (m, 1H), 2.70-2.62 (m, 1H), 2.40-2.36 (m, 1H), 2.27-2.10 (m, 2H), 1.95-1.92 (m, 2H), 1.75-1.54 (m, 3H), 1.50 (s, 3H), 1.51-1.32 (m, 3H), 1.12-0.90 (m, 8H) ppm.

Example 1k.

(2, 2113ethoxy)({2 1H)S,2S,4R,SS,9S,11S,12S,13R)13okcu)({2 1H), 3.30-3.28 (m, 1H), 2.70-2.62 (m, 1H ), 2.40-2.36 (m, 1H) ^2.9 .0 ^4.8 .0 ^13.18 ]icos)({2 1H), 3.30-3.28 oxoethoxy})phosphinic acid (1k)

To a solution of triethylamine (0.67 g, 6.6 mmol) in chloroform (4 ml) was added phosphorus oxychloride (0.51 mg, 3.3 mmol) at 0°C, and then a solution of budesonide (1a, 1.3 g, 3.0 mmol) in chloroform (4 ml) was added ). After stirring at 20°C for 2 hours, the mixture was cooled to 0°C and a solution of Vosethanolamine (0.41 mg, 2.6 mmol) in chloroform (4 ml) and pyridine (3 ml) was added. After stirring at 20°C for one hour until the reaction was complete according to LCMS, the reaction mixture was cooled by adding water (2 ml) at 0°C. The mixture was stirred at 20°C overnight. Volatiles were removed in vacuo and the residue was purified by prep-HPLC (Method B) to give crude Boc-1k (330 mg, 17%) as a yellow film. ESI m/z: 676 (M+Na) ⁺ .

To a solution of Boc-1k (0.18 g, 0.28 mmol) in DCM (5 ml) was added trifluoroacetic acid (0.5 ml) at 0°C. The resulting mixture was stirred at 23°C for 2 hours until Boc was completely removed according to LCMS. Volatiles were removed in vacuo. The residue was purified by prep-HPLC (Method B) to give 1k as a white solid (90 mg, 59% yield). ESI m/z: 554 (M+H) ⁺ .

1H NMR (500 MHz, MeOD-d ₄ ) δ 7.49 (d, J=10.1 Hz, 1H), 6.28 (d,J=10.1 Hz, 1H), 6.03 (s, 1H), 5.24-5.15 (m, 1H ), 4.87-4.62 (m, 3H), 4.45 (d, J=6.1 Hz, 1H), 4.18-4.15 (m, 2H), 3.22-3.20 (m, 2H), 2.71-2.64 (m, 1H), 2.40 (d, J=13.4 Hz, 1H), 2.29-1.71 (m, 6H), 1.65-1.32 (m, 9H), 1.22-0.91 (m, 7H) ppm. Anal. HPLC: >99.9%, retention time: 3.90 min (method B).

Example 1l.

(2-{ [(9H-fluoren-9-ylmethoxy)carbonyl] amino} ethoxy)phosphonic acid.

See J. Am. Chem. Soc, 2016, 138(4), 1430-1445; WO2015153401; and Phosphorus, Sulfur and Silicon and the Related Elements, 2000, 165, 83-90.

O

FmocHN^^\^ >

To a mixture of Fmoc-ethanolamine (1.1 g, 3.9 mmol) in THF (16 ml) was added diphosphoryl chloride (2.2 g, 8.8 mmol) by syringe at -40°C. After stirring at -40°C for one hour until complete destruction of the starting material was monitored by LCMS, the reaction mixture was cooled with water (1 ml) at -40°C, exposed to saturated aqueous sodium bicarbonate (200 ml) and kept at 10-20°C overnight. The resulting mixture was acidified with conc. HCl to pH 2 and then extracted with ethyl acetate. The combined organic solution was dried over sodium sulfate and concentrated to give the crude title compound (1.6 g, crude) as a white solid, which was used for the next step without purification. ESI m/z: 364 (M+H) ⁺ .

(2-{ [(9H-fluoren-9-ylmethoxy)carbonyl] amino} ethoxy)( {[hydroxy({2- 113 045111

[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy})phosphoryl]oxy})phosphinic acid (Fmoc1l)

NHFmoc o, Ο, k° pO OH obH iThTx ⁰ h II th I h

Following the above procedure, except for the substitution of budesonide (1a, 0.86 g, 20 mmol) for Fmoc-ethanolamine, the budesonide phosphonic intermediate 1a-PO ₃ H ₂ (1.1 g, crude) was obtained as a white solid (ESI m/ z: 551 (M+H)+).

To a solution of crude (2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethoxy)phosphonic acid (0.29 g, 0.80 mmol) in DMF (5 ml) was added triethylamine (81 mg, 0.80 mmol) and 1, 1'-carbonyldiimidazole (CDI, 0.32 g, 2.0 mmol) at 10°C. The mixture was stirred at 10-20°C for 30 min and then the above phosphonic intermediate 1a-PO ₃ H ₂ (0.41 g, 0.80 mmol) and zinc chloride (0.87 g, 6.4 mmol) were added to the reaction mixture. The resulting mixture was stirred at 10-20°C overnight and the starting material was completely destroyed according to LCMS. The reaction mixture was then cooled with dilute water. HCl (1 N, 50 ml) and extracted with ethyl acetate. The combined organic solution was concentrated and the residue was purified by prep-HPLC (Method B) to give Fmoc-1l (0.32 g, 47% yield) as a white solid. ESI m/z: 856 (M+H) ⁺ .

(2-aminoethoxy)( {[hydroxy( {2-[( 1 S,2S,4R,8S,9S, 11S,12S, 13R)-11 -hydroxy-9,13-dimethyl-16-oxo-6propyl-5 ,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14D7-dien-8-yl]-2oxoethoxy})phosphoryl]oxy})phosphinic acid (1l)

See WO2015153401.

Piperidine (67 mg, 0.79 mmol) was added to a solution of Fmoc-11 (0.10 g, 0.12 mmol) in DCM (2 ml) at 10°C. The reaction mixture was stirred at 10-20°C for 16 hours. Compound Fmoc-1l was completely destroyed by LCMS. Volatiles were removed in vacuo and the residue was purified by prep-HPLC (method B) to give 1L (50 mg, 68% yield) as a white solid. ESI m/z: 634 (M+H) ⁺ .

1H NMR (500 MHz, MeOD-d4) δ 7.50 (d, J=10.0 Hz, 1H), 6.28 (d, J=9.9 Hz, 1H), 6.03 (s, 1H), 5.23-5.16 (m, 1H) , 5.02-4.97 (m, 1H), 4.88-4.67 (m, 1H), 4.45 (d, J=3.4 Hz, 1H), 4.23 (s, 2H), 3.24 (s, 2H), 2.67(dd,J =13.5, 8.3 Hz, 1H), 2.40 (d, J=11.3 Hz, 1H), 2.28-1.32 (m, 15H), 1.23-0.92 (m, 8H) ppm. Anal. HPLC: >99.9%, retention time: 2.74 min (method B).

In the table below. 1a shows steroids prepared using the methods described herein.

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Table la

Structure and chemical and physical properties of compounds

No. Structure HPLC Purity cLogP M.F. MB (Cal.) MS (M+H) 1a d-4 [τΥτ ι t O' o 95 2.73 C25H34O6 430.55 431.3 1s ^°Ъхх==\_ ° 7vXa==/ He O 98 3.00 C29H38O9 530.25 531.2 ID n n ^OTL ⁿ / 7\_/° \ O ' » ^he HN & 93 2.92 C30H44N2O7 544.69 545.3 1e ^ШК ° n^°daХХ=/° η,ν. A k! „ 1 -\ n Υ ° ⁿ o 100 1.44 C28H39N3O8 545.62 546.2 1g -X /l \| U-U n )=x G 1 oTl ⁿ LA /=° “Άα>·Υ4α 92 3.65 C31H44O8 544.68 545.3 1h about ΛΑΓΑΡΗ. 9 θΤΤνί/Τνν ^{0 о >3} Ън 98 3.19 C34H48O10 616.32 616.32 li _Q UуН ^Н /—\ ηο^ ^ο υ°^° ° ⁿ but'' γ 'he he 100 0.96 C31H44O11 592.67 593.4 ij t o, XX _o ° ILx ? Xq Qi 98 1.28 C31H42O12 606.66 607.3

- 115045111

Ik ο 4^0^0=° h ₂ n~ р ^v _oh he 100 1.18 C27H40NO9P 553.58 554.1 eleven __ РдУХ \=\ '—\ J—/ \-L /=o ~ 9 he r-P=o /— he h ₂ n 100 0.55 C27H41NO12P2 633.56 634.0 Im so N G~\ /7\-N /=\ ^N °77\2у7\=/ ° _{m +} O _h ° OH Na 'p-O Ό \\ Na ⁺ O 100 1.15 C2iH27Na ₂ O ₈ P 484.39 590.3 100 О Г “-Ζ Г ^{4 1} ijS: J Ζγς о χ-4—\\ О^/О / V о Ζ X 100 2.44 S ₂₅ Nz2r ₂ Ob 466.51 467 101a F Н Н ν οθΐΜ^ ΗΝ·^/ у Ън ¹ О >95 2.40 C29H40F2N2O7. C2HF3O2 663.66 567 101b F n n ΗΝ'^- ^Χ γ he ¹ O >95 2.63 C30H42F2N2O7. C2HF3O2 678.69 581

- 116 045111

101c HN^k °> ία F n n,—C F /=\ y_/ U=o ⁰ he 100 3.34 C32H46F2N2O7. C2HF3O2 722.74 609 lOld HN^ A°~ O N Drn ,.F , 1 ^H Y ^F X ⁰ i*Yl he Ch/Tse 100 2.46 C30H40F2N2O7 578.64 579 102c HS^ -\ /° g θ4 ABOUT -ln /—\ /7\ n 4 L n >-/ V ^z W/\ /=o '° bn >95 3.47 C28H39NO7S 533.68 531 102d hs^ —\_(° t o4 o Ά' ^Η /—\ *<-//\ 4° '° he >95 3.69 C29H41NO7S 547.71 548 102e H.S. —\(° 'T b4 ^t° o _a h _W ^F * V_//\ /=o '° he >95 3.17 C28H37F2NO7S 569.66 570 102f HS^ —\_(°G o4 O o V'” O >95 3.40 C29H39F2NO7S 583.69 584 —\ Ύ ⁿ /—\ 103a H h ₂ ny ⁿ -°— O gS/L /=o % AN ^and he 98 1.58 C28H40N2O7 516.64 517 103b —d Η ° Η ₂ Ν-γ ^Ν -°— Ο ..^H^ ^{F to} °Чн^ 98 1.29 C28H38F2N2O7 552.62 553 104a n ₂ D lY' Ρ° n G *7 /Y ^F >90 3.67 C3 ₅ H ₅ 0F ₂ N2O8 664.79 665 hXXS ^ΊΟ 104b Η ₂ Ρ .“. he -Hk ⁿ n D Υ^χ.,έ hX^S >95 2.19 C34H48F2N2O9 666.76 667

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Scheme 2

Synthesis of VC-PAB spacer budesonide

General Procedure A for the synthesis of MS spacer budesonide (2a and 2c)

R-NH ₂ (4 or 9)

DIPEA. DMF

O

O O CT, 4 p.m.

5a, X = CH, 2a,c

5b, X = (OSN ₂ CH ₂ ) ₃ O

To a solution of vcPAB-budesonide (4а А=СО, 1.0 eq.) or amine 9 (1.0 eq.) according to scheme 2 in DMF (ca. 1 ml per 10 mg of amine), activated NHS ester 5 (1.5-3.0 eq.) was added. in the table below and DIPEA (2.0 eq.) at CT. The reaction mixture was stirred at RT overnight until the NHS ester and most of the amine were destroyed according to LCMS spectra. After filtration, the reaction solution was directly purified by prep-HPLC or reverse phase flash chromatography to obtain the desired amide 2 (ca. 17% yield) as a white solid.

Amines Activated Ether 5 Base/reagents Solvent Time (h) Cleaning Product 2 4a ^c 50 mg, 56 µmol 5a 28 mg, 91 µmol DIPEA (23 mg, 0.18 mmol) DMF (3 ml) 16 Prep-HPLC (method A) 2a (10 mg, 17%) reaction solution 9 5b 8.8 mg, 20 µmol DIPEA (4.0 mg, 31 µmol) DMF (1 ml) 16 Prep-HPLC (method A) 2s (3.5 mg, 4%)

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With. TFA salt General procedure B for the synthesis of amides 2b, 2l, 2t-precursor from acid

To a solution of acid 5 (1.0-1.5 eq.) in DMF or DCM or THF (1 ml per 5 mg 5) added DIPEA (2.05.0 eq.) and HATU (1.4-2.2 eq.) at RT. The resulting mixture was stirred at this temperature for 0.5-1 h, and then vcPAB-budesonide (4, 1.0 eq.) was added. The reaction mixture was stirred at RT for 3-16 hours until complete destruction of the amine, which was monitored by LCMS. The reaction mixture was membrane filtered and the filtrate was then separated by prep-HPLC or reverse phase flash chromatography to provide amide 2 (21-54% yield) as a white solid. The table below provides additional information.

Amines acid Base/reagents Solvent Time (h) Cleaning Product 2 4b 10 mg, eleven µmol 5s 10 mg, 18 µmol DIPEA (6.2 mg, 48 µmol) HATU (9.0 mg, 24 µmol) DMF (1 ml) 16 Prep-HPLC (method B) 2b (3.0 mg, 21%)

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4s 58 mg, 61 µmol 5s 37 mg, 67 µmol DIPEA (15 mg, 0.12 mmol) HATU (34 mg, 89 µmol) DMF (5 ml) 3 Prep-HPLC (method B) 2i (21 mg, 24%) 4s 8.9 mg, 9.4 µmol 5d 6.3 mg, 14 µmol DIPEA (3.6 mg, 27 µmol) HATU (8.0 mg, 20 µmol) DMF (1 ml) 16 Prep-HPLC (method B) 21 (7 mg, 54%) 4s 20 mg, 21 µmol 5e 13 mg, 21 µmol DIPEA (8.0 mg, 62 µmol) HATU (12 mg, 31 µmol) THF (5 ml) 16 Prep-HPLC (method A) 2m represents precursor (5 mg)

General Procedure C for the synthesis of intermediates 7 and 10

To a solution of intermediate 6 (1.2-1.4 eq) in DMF, HATU (1.5-1.7 eq) was added at RT. The resulting solution was stirred at RT for an hour. To this suspension was added a solution of vcPAB-budesonide (4a, 1.0 eq.) in DMF (0.10 ml per mg of 4a) and then NMM (2.3-3.0 eq.). The reaction mixture was stirred at RT for 16 hours until it became clear. The reaction was monitored by LCMS until compound 4a was completely destroyed. Diethylamine (excess) was then added to the reaction mixture and the resulting mixture was stirred at RT for 1-3 hours until the Fmoc was removed by LCMS. Volatiles were removed in vacuo and the residue was purified by prepHPLC (Method A) to give compound 7 (18% yield from compound 4a), or purified by prepHPLC (Method B) to give compound 10 (5- % yield) as a white solid. The table below provides additional information.

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Amines Intermediate 6 Base/reagents Solvent Time (h) Cleaning Product 4a 60 mg, 64 µmol 6a 46 mg, 86 µmol NMM (15 mg, 0.15 mmol) HATU (41 mg, 0.11 mmol) then Et2NH (0.5 ml) DMF (3 ml) 16 hours then 3 hours PrepHPLC (method A) 7(13 mg, 18%) 4a 0.19 g, 0.20 mmol 6b 0.15 g, 0.24 mmol ΝΜΜ (61 mg, 0.60 mmol) HATU (0.11 g, 0.29 mmol) then Et2NH (1 ml) DMF (5 ml) 16 hours then 1 hour PrepHPLC (method B) 10(13 mg, 5%)

General Procedure D for the synthesis of carbamates 2j, 2k, 2r and 2s

JJ R ^1m

o* _o .

11a-c

NO ₂

R ^1m R ^2m NH (1c-e _T 1k4) DIPEA, (HOBt) * DMF, KT, 3-24 4.

R ^m

2j. 2k. 2r, 2s

The mixture was stirred at RT for 3-24 hours until the PNP activated ester was completely destroyed, which was monitored by LCMS. The reaction mixture was membrane filtered and the filtrate separated directly by prep-HPLC to give compound 2 (with or without diastereoisomers, 11-63% yield) as a white solid. The table below provides additional information.

Spacerbudesonide 21 Activated Ether 23 Base/reagents Solvent Time (h) Cleaning Product 2 1e 15 mg, 28 µmol Pa 15 mg, 20 µmol DIPEA (12 mg, 93 µmol) HOBt (4.0 mg, 30 µmol) DMF (1 ml) 12 PrepHPLC (method A) 2j (3.0 mg, 13%) ID 20 mg, 37 µmol 11a 22 mg, 30 µmol DIPEA (12 mg, 93 µmol) HOBt (6.0 mg, 44 µmol) DMF (1 ml) 12 PrepHPLC (method A) 2k-A (3.3 mg, 10%) 2k-B (4.1 mg, 12%) 1k 50 mg, 90 µmol 11s 30 mg, 95 µmol DIPEA (50 mg, 0.39 mmol) DMF (1 ml) 3 PrepHPLC (method B) 2g (40 mg, yield 61%)

Synthesis 2a-d.

Budesonide linkers 2a-d were prepared according to three approaches according to Scheme 2. The first approach was directly through coupling reactions of amides from vcPAB-budesonide (4a), which was obtained from the activated budesonide ester 3a with linkers 5. The second approach was through initial reactions coupling of amides from vcPAB budesonide (4a) to intermediate 6a, resulting in budesonide linkers 7, followed by a 3+2 cyclization with galactose azide (8a), resulting in intermediates 9, followed by secondary coupling reactions of amides with 5. The third approach was carried out through the first coupling reactions of amides 4 with intermediate 6b, followed by the formation of amides 5b and sequential deprotection of acetone, resulting in 2d.

- 121 045111

Intermediate 4a

In dry (1.4 mmol) DMF (10 ml) added Boc-vc-PAB (12a) [WO2008/34124 A2] (0.59 g, 1.2 mmol), DMAP (0.30 g, 2.4 mmol) and pyridine (0.29 g, 3.7 mmol ). The mixture was stirred at RT for 16 h until 3a was completely destroyed according to LCMS. The reaction mixture was directly purified by reverse phase flash chromatography (50-80% acetonitrile in water) to yield intermediate Boc-4a (0.74 g, 38% yield, ESI m/z: 936 (M+H)+ ) as a white solid, which was dissolved in DCM (40 ml). To 5 ml of DCM solution (containing 94 mg Boc-4a) TFA (0.5 ml) was added dropwise at 0°C. After stirring at RT for 1.5 hours until Boc4a was destroyed as monitored by LCMS, the resulting mixture was concentrated in vacuo to give crude title product 4a (83 mg, 34% yield from budesonide) as the TFA salt as a colorless oil that can be used without purification for the next stage synthesis. 20 mg of crude 4a was purified by prep-HPLC (Method B) to obtain pure 4a (8 mg) as free base for plasma stability testing. ESI m/z: 836 (M+H) ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 10.17 (s, 1H), 8.12 (br s, 1H), 7.62 (d, J=8.5 Hz, 2H), 7.31 (d, J=10.2 Hz, 3H) , 6.17 (d, J=10.1 Hz, 1H), 5.97 (t, J=5.7 Hz, 1H), 5.92 (s, 1H), 5.40 (s, 2H), 5.22-5.01 (m, 4H), 4.89- 4.62 (m, 3H), 4.53-4.40 (m, 1H), 4.30 (s, 1H), 3.09-2.87 (m, 3H), 2.36-2.22 (m, 1H), 2.14-1.87 (m, 4H), 1.81 (d, J=5.6 Hz, 2H), 1.75 (s, 2H), 1.62-1.52 (m, 4H), 1.51-1.41 (m, 2H), 1.40-1.19 (m, 8H), 1.18-0.92 ( m, 2H), 0.92-0.82 (m, 9H), 0.78 (d, J=6.8 Hz, 3H) ppm.

Example 2a.

N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({2-[(1S,2S,4R,8S,9S,11S,12S,13R )-11-hydroxy-9,13dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^{2.9.0 4.8.0 13.18} ]icosa-14,17-diene-8 -yl]-2oxoethoxy}carbonyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]-6-(2,5-dioxo2,5-dihydro-1H-pyrrol-1-yl)hexanamide (2a )

General Procedure A provided the title compound 2a (10 mg, 17% yield) as a white solid. ESI m/z: 1029 (M+H) ⁺ . Anal. HPLC: 92.5%, retention time: 7.66 min (method A).

Example 2b.

1-(4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4- oxobutanes[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-ido)-N-[(1S )-1-{[(1S)-4(carbamoylamino)-1-[(4-{[({2-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14, 17-dien-8-yl]-2oxoethoxy}carbonyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]-3,6,9,12tetraoxapentadecan-15-amide (2b)

- 122 045111

General Procedure B gave title compound 2b (3.0 mg, 21% yield) as a white solid. ESI m/z: 686 (M/2+H)+.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.05 (s, 1H), 8.15 (d, J=7.5 Hz, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.77 (t, J= 5.7 Hz, 1H), 7.71-7.66 (m, 1H), 7.66-7.59 (m, 3H), 7.52-7.27 (m, 9H), 6.17 (d, J=10.0 Hz, 1H), 6.03-5.95 (m , 1H), 5.92 (s, 1H), 5.79-5.74 (m, 1H), 5.43 (s, 2H), 5.22-4.99 (m, 6H), 4.88-4.84 (m, 1H), 4.84-4.60 (m , 2H), 4.42-4.34 (m, 1H), 4.33-4.26 (m, 1H), 4.26-4.20 (m, 1H), 3.65-3.55 (m, 3H), 3.48-3.44 (m, 12H), 3.32 -3.26 (m, 2H), 3.12-2.91 (m, 4H), 2.63-2.54 (m, 1H), 2.41-2.19 (m, 3H), 2.03-1.94 (m, 4H), 1.84-1.78 (m, 2H), 1.63-1.22 (m, 14H), 1.03-0.82 (m, 15H) ppm. Anal. HPLC: 96.9%, retention time: 8.10 min (method B).

Example 2c.

(2R)-2-amino-N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({2-[(1S,2S,4R,8S ,9S,11S,12S,13R)-11hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icos -14,17-dien-8yl]-2-oxoethoxy}carbonyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]-6-[2(cyclooct-2-yn-1-yloxy)acetamido ]hexanamide (7)

O YA ⁰ Y ⁰

Ύϋ

ESI m/z: 565 (M/2+H) ⁺ .

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 10.17-10.00 (m, 1H), 8.47-7.73 (m, 2H), 7.69-7.57 (m, 3H), 7.38-7.26 (m, 3H), 6.17 (d, J=10.0 Hz, 1H), 5.99 (s, 1H), 5.92 (s, 1H), 5.76 (s, 1H), 5.42 (s, 2H), 5.23-5.01 (m, 4H), 4.884. 61 (m, 3.4H), 4.43-4.16 (m, 4.6H), 3.85 (d, J=14.7 Hz, 1H), 3.73 (d, J=14.5 Hz, 1H), 3.23-3.18 (m, 1H) , 3.092.90 (m, 4H), 2.33-2.18 (m, 3H), 2.18-2.04 (m, 3H), 2.03-1.66 (m, 10H), 1.64-1.50 (m, 7H), 1.49-1.22 ( m, 13H), 1.17-0.90 (m, 4H), 0.90-0.79 (m, 12H) ppm.

(2R)-2-amino-N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({2-[(1S,2S,4R,8S ,9S,11S,12S,13R)-11hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icos -14,17-dien-8yl]-2-oxoethoxy}carbonyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]-6-[2-({1[(2R,3R,4S, 5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]-1H,4H,5H,6H,7H,8H,9Hcycloocta[d] [1,2,3]triazol- 4-yl}oxy)acetamido]hexanamide (9)

After filtration, the resulting solution 9 was used directly for the next step. ESI m/z: 667 (M/2+H) ⁺ .

N-[(1R)-1-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({2-[(1S,2S,4R,8S ,9S,11S,12S,13R)-11hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icos -14,17-dien-8yl]-2-oxoethoxy}carbonyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2methylpropyl]carbamoyl}-5-[2-({1-[(2R,3R,4S ,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol- 4-yl}oxy)acetamido]pentyl]-1-(2,5-dioxopyrrol-1yl)-3,6,9,12-tetraoxapentadecan-15-amide (Mc-PEG ₄ -N(caxap-COT)Lys- vc-PAB-budesonide) (2c)

- 123 045111

ESI m/z: 831 (M/2 + N) ⁺ .

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.73 (s, 1H), 8.21-8.07 (m, 3H), 7.94-7.82 (m, 1H), 7.71-7.59 (m, 3H), 7.39-7.26 (m, 4H), 7.02 (s, 1H), 6.17 (d, J=10.0 Hz, 1H), 6.09-5.98 (m, 1H), 5.92 (s, 1H), 5.56-5.28 (m, 4H), 5.25-5.01 (m, 4H),4.90-4.62 (m, 5H), 4.39-4.13 (m, 5H), 3.84-3.69 (m, 4H), 3.59-3.40 (m, 16H), 3.17-2.79 (m , 9H), 2.43-1.71 (m, 13H), 1.71-1.42 (m, 15H), 1.40-1.19 (m, 11H), 1.04-0.77 (m, 15H) ppm. Anal. HPLC: 97.6%, retention time: 7.63 min (method A).

Example 2d.

(2R)-2-amino-N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{[({2-[(1S,2S,4R,8S ,9S,11S,12S,13R)-11hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icos -14,17-dien-8yl] -2-oxoethoxy} carbonyl)oxy] methyl} phenyl)carbamoyl] butyl] carbamoyl} -2 -methylpropyl] -N'-[(2S)-2 [(4R,5R)- 5-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2,2-dimethyl-1,3-dioxolan-4-yl]-2hydroxyethyl]pentanediamide (10)

Following General Procedure C, compound 10 (13 mg, 5% yield) was obtained as a white solid. ESI m/z: 605 (M/2 + N) ⁺ .

^1H NMR (500 MHz, DMSO- _d6 ) δ 10.08 (s, 1H), 8.34-8.17 (m, 1H), 7.98 (s, 1H), 7.93-7.81 (m, 1H), 7.62 (d, J =8.4 Hz, 2H), 7.42-7.26 (m, 3H), 6.17 (d, J=10.1 Hz, 1H), 5.99 (s, 1H), 5.92 (s, 1H), 5.76 (s, 1H), 5.42 (s, 2H), 5.23-5.01 (m, 4H), 4.91-4.61 (m, 4H), 4.43-4.20 (m, 3H), 4.10-3.72 (m, 6H), 3.60-3.54 (m, 1H) , 3.24-3.09 (m, 2H), 3.07-2.91 (m, 2H), 2.32-2.25 (m, 1H), 2.22-2.10 (m, 2H), 2.04-1.93 (m, 2H), 1.88-1.67 ( m, 5H), 1.641.22 (m, 27H), 1.17-0.93 (m, 3H), 0.91-0.81 (m, 12H) ppm.

(2R)-2-amino-N-[( 1S)-1-{[(1 S)-4-(carbamoylamino)-1 -[(4- {[({2-[( 1 S,2S,4R ,8S,9S, 11S,12S,13R)-11hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8yl] -2-oxoethoxy} carbonyl)oxy] methyl} phenyl)carbamoyl] butyl] carbamoyl} -2 -methylpropyl] -N'[(2S,3R,4R,5R)-2 ,3,4,5,6-pentahydroxyhexyl]pentanediamide (10A)

To a mixture of compound 10 (13 mg, 11 µmol) in DCM (1 ml) was added TFA (1 ml) dropwise. The mixture was stirred at RT for an hour, which was monitored by LCMS. Volatiles were removed in vacuo to obtain the crude deprotected product 10A (13 mg) as a light yellow oil, which was used for the next step without further purification. ESI m/z: 565 (M/2+H)+.

(2R)-N-[(1S)-1-{[(1S)-4-(carbamoylαamino)-1-[(4-{[({2-[(1S,2S,4R,8S,9S,11S ,12S,13R)-11-hydroxy9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14.17 -dien-8-yl]-2oxoethoxy} carbonyl)oxy] methyl} phenyl)carbamoyl] butyl] carbamoyl} -2 -methylpropyl] -2-[1-(2,5dioxopyrrol-1-yl)-3,6,9 ,12-tetraoxapentadecane-15-amido]-N'-[(2S,3R,4R,5R)-2,3,4,5,6pentahydroxyhexyl]pentanediamide (2d)

- 124 045111

General Procedure A provided title compound 2d (3.5 mg, 1% overall yield from 4a) as a white solid. ESI m/z: 829.7 (M/2+H)+.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 10.10 (s, 0.25H), 9.79 (s, 0.75H), 8.22 (d, J=7.1 Hz, 1H), 8.12 (d, J=7.5 Hz, 1H), 8.07 (d, J=7.8 Hz, 1H),7.79-7.71 (m, 1H), 7.69-7.60 (m, 2H), 7.38-7.27 (m, 4H), 7.02 (d, J=1.4 Hz , 2H), 6.17 (dt, J=7.6, 1.6 Hz, 1H), 6.03-5.97 (m, 1H), 5.92 (s, 1H), 5.46-5.40 (m, 2H), 5.35-5.02 (m, 4H ), 4.89-4.61 (m, 4H), 4.50-4.45 (m, 1H), 4.40-4.16 (m, 7H), 3.65-3.37 (m, 21H), 3.07-2.90 (m, 3H), 2.44-2.23 (m, 4H), 2.171.93 (m, 6H), 1.90-1.71 (m, 4H), 1.62-1.20 (m, 15H), 1.06-0.76 (m, 15H) ppm. Anal. HPLC: >99%, retention time: 6.40 min (method A).

Scheme 3

Synthesis 2e

Example 2e.

2-[( 1 S,2S,4R,8S,9S, 11 S,12S, 13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2, 9} .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-[(4aminophenyl)methyl]carbamate (13)

To a solution of 4-aminobenzylamine (9.7 mg, 79 µmol) and DIPEA (12 mg, 93 µmol) in THF (10 ml) was added a solution of 3a (24 mg, 44 µmol) in THF (5.0 ml) dropwise at RT. The mixture was stirred at RT for 3 hours until 3a was completely destroyed, which was monitored by LCMS. Volatiles were removed in vacuo and the residue was purified by prep-HPLC (Method B) to give compound 13 (5.6 mg, 22% yield) as a white solid. ESI m/z: 579.2 (M+H) ⁺ .

^1H NMR (400 MHz, MeOD-d ₄ ) δ 7.48-7.45 (m, 3H), 7.31-7.28 (m, 2H), 6.27-6.24 (m, 1H), 6.02 (s, 1H), 5.22 (m , 1H), 5.12-5.11 (m, 1H), 4.96 (m, 1H), 4.86-4.81 (m, 2H), 4.66 (m, 1H), 4.44-4.35 (m, 3H), 2.66-2.65 ( m, 1H), 2.40-2.36 (m, 1H), 2.15-2.10 (m, 2H), 1.97-1.60 (m, 6H), 1.53-1.36 (m, 6H), 1.09-0.92 (m, 7H) ppm .

2-[( 1 S,2S,4R,8S,9S, 11 S,12S, 13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2, 9} .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-({4-[(2S)-5(carbamoylamino)-2-[(2S) -2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]-3methylbutanamido]pentanamido]phenyl}methyl)carbamate (2e)

- 125 045111

To a mixture of MC-VC-OH 5f (WO2014/191578 A1) (5.7 mg, 12 µmol) in dry DMF (1.5 ml) was added HATU (4.6 mg, 12 µmol) and NMM (4.0 mg, 40 µmol) at RT. The mixture was stirred at RT for 10 min, after which compound 13 (4.7 mg, 8.1 μmol) was added to the reaction mixture. The resulting mixture was stirred at RT overnight. Compound 13 was completely destroyed and the desired product was detected as the main product by LCMS. The mixture was purified directly by prep-HPLC to give 2e (2.9 mg, 35% yield) as a white solid. ES m/z: 1029.1 (M+H)+, 514.7 (M/2+H) ⁺ . Anal. HPLC: >99%, retention time: 7.54 min (method A).

Scheme 4

Synthesis of linker-spacer-budesonide (via ether) 2f u 2g

Example 2f.

1-{4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido ]3-methylbutanamido]phenyl}methyl 4-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo6-propyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl}butanedioate (2f)

o^nh ₂

MC-VCPAB (12b, 0.11 g, 0.19 mmol), TBTU (63 mg, 0.19 mmol), HOBt (26 mg, 0.19 mmol) and DIPEA (41) were added to solution 1c (20 mg, 38 µmol) bDMF (2 ml). mg, 0.19 mmol) at CT. The resulting mixture was stirred at 70°C for 12 hours. No more product 2f was formed, which was monitored by LCMS. The reaction mixture was purified directly by prep-HPLC to give the title compound 2f (2.6 mg, 6% yield) as a white solid. ESI m/z: 1085.3 (M+H) ⁺ .

^1H NMR (400 MHz, CDCl ₃ ) δ7.55-7.52 (m, 1H), 7.34-7.30 (m, 1H), 6.67 (br s, 1H), 6.30-6.27 (m, 1H), 6.03 (br s, 1H), 5.16-5.11 (m, 2H), 5.09-4.87 (m, 2H), 4.80-4.61 (m, 1H), 4.60-4.30 (m, 2H), 3.47-3.44 (m, 1H), 3.21-3.10 (m, 1H), 3.03-2.96 (m, 2H), 2.79-2.55 (m, 4H), 2.35-1.73 (m, 28H), 1.71-1.32 (m, 15H), 1.29-1.07 (m , 4H), 0.99-0.85 (m, 9H) ppm.

Example 2g.

- {4-[(2S)-2-[(2S)-2-[1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5, 7,13,15-hexaen-10-yn-2yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecan-15-amido]-3-methylbutanamido]-5-

- 126 045111 (carbamoylamino)pentanamido]phenyl}methyl 4-{2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13dimethyl-16-oxo-6-propyl -5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethyl} butanedioate (2g)

nh ₂

Following the procedures of Example 2f, except using DIBAC-suc-PEG ₄ -vcPAB (12c) instead of 12b, compound 2g (15 mg, 19% yield) was obtained as a white solid. ESI m/z: 714 (M/2+H)+.

¹ H NMR (500 MHz, MeOD-d ₄ ) δ 7.67-7.60 (m, 4H), 7.48-7.46 (m, 4H), 7.38-7.32 (m, 4H), 7.26 (d, J=6.8 Hz, 1H ), 6.27 (d, J=10.0 Hz, 1H), 6.03 (s, 1H), 5.24-5.10 (m, 4H), 5.02-4.97 (m, 1H), 4.85-4.66 (m, 2H), 4.61 ( s, 1H), 4.53 (dd, J=9.1,4.9 Hz, 1H), 4.45-4.43 (m, 1H), 4.22 (dd, J=6.7, 4.7 Hz, 1H), 3.78-3.69 (m, 3H) , 3.60-3.55 (m, 11H), 3.47-3.40 (m, 2H), 3.26-3.10 (m, 4H), 2.78-2.54 (m, 8H), 2.41-2.35 (m, 2H), 2.25-2.11 ( m, 4H), 2.021.31 (m, 17H), 1.21-0.92 (m, 14H) ppm. Anal. HPLC: 99.5%, retention time: 7.86 min (method B).

Scheme 5

Synthesis of linker-spacer-budesonide 2j-2n (via amide or carbamate)

DIBAC-suc-PEG4-Val-Cit-OH (5d)

HATU, NMM, DMFKT, 2.5 h I.SP ^x -Bud HN

R ⁴ mc-vc-PAB-PNP (11a)

MnMDlBAC-suc-PEG4-v(

DIPEA. HOBt. DMF. KT. 12KT, at

1d (R ⁴ is Sc-alkyl; SP ^X is

-CH ₂ CH ₂ N(CH ₃ )-C(O)-)

1e (R ^q is H; SP ^X is

NHC(O)CH2NHC(O)-)

1) Fmoc-vc-PAB-PNP (11 d) DIPEA, DMAP, DMF,

KT, 3 h

2) piperidine, KT, 1 h

ABOUT

SP ^x -Bud (5a) (5c) (5d)

4b (vcPAB-1f)

4c (vcPAB-1d) then Et ₂ NH or mc-dLys(suc-DIBAC)-OH (5e), then CD-N ₃ (8b)

In scheme 5, Bud means budesonide.

Most budesonide linkers (2) having a VC-PAB moiety were synthesized using three amide or carbamate approaches (Scheme 5). Compounds 1d, 1e were separately conjugated to linkers via amide coupling, and the amide or carbamate was obtained (Scheme 5). The table below provides additional information.

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No. connect Research Institute R ^ql in 2j, 2k, 21, 2m, 2n n in 2j-n R ^C| -NHspacerbudesonide 2j O 1 le 2k ,Ο o 1 ID 21 Η H O ^h 1 ID 2m AD xbVV Du 3¾ _HO XWW° HO \ OH 1 ID 2n O h ₂ ny^ _OH 1 ID

Intermediate connection 4c.

2-( 1 S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)-2-[(2S)-2amino -3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl](methyl)amino}ethyl)-N-methylcarbamate (4c)

o^nh ₂

Fmoc-vcPAB-PNP (11d, 60 mg, 78 µmol), DMAP (9.0 mg, 74 µmol) and DIPEA (20 mg, 0.16 mmol) were added to a solution of compound 1d (40 mg, 73 µmol) in DMF (3 ml). at room temperature. The mixture was stirred at room temperature for 3 hours until most of the starting material was destroyed, which was monitored by LCMS. Piperidine (1 ml) was then added to the reaction mixture. After stirring at room temperature for an hour until the de-Fmoc reaction was complete as monitored by LCMS, the reaction mixture was directly purified by prep-HPLC (Method B) to give compound 4c (9.0 mg, 13% yield, ^2nd peak in LC ) as a white solid. ESI m/z: 951 (M+H)+, 973 (M+Na) ⁺ .

Example 2j.

2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-({N'-[({4-[(2S)-5(carbamoylamino)-2- [(2 S)-2-[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanamido]-3 methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]hydrazinecarbonyl}methyl ) carbamate (2j)

- 128 045111

ESI m/z: 1144.3 (M+H)+.

1H NMR (400 MHz, CDCl ₃ ) δ10.02 (s, 1H), 9.77 (s, 1H), 9.18 (s, 1H), 8.12(d, J=7.2 Hz, 1H), 7.83 (d,J= 8.4 Hz, 1H), 7.73-7.70 (m, 1H), 7.61-7.59 (m, 2H), 7.32-7.29 (m, 3H), 7.01 (s, 2H), 6.19-6.16 (m, 1H), 6.00 -5.92 (m, 2H), 5.05-5.00 (m, 2H), 4.95-4.85 (m, 2H), 4.71-4.62 (m, 2H), 4.40-4.17 (m, 3H), 3.71-3.58 (m, 2H), 3.453.39 (m, 2H), 3.06-2.89 (m, 3H), 2.33-2.28 (m, 2H), 2.20-2.07 (m, 3H), 2.02-1.91 (m, 3H), 1.81- 1.75 (m, 2H), 1.75-1.62 (m, 2H), 1.60-1.44 (m, 11H), 1.42-1.20 (m, 6H), 1.19-1.12 (m, 4H), 1.00-0.92 (m, 2H ), 0.88-0.81 (m, 10H) ppm.

Example 2k.

2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9, 13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl

N-(2-{[({4-[(2S)-5(carbamoylamino)-2-[(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-nirrol- 1-yl)hexanamido]-3methylbutanamido]pentanamido]phenyl}methoxy)carbonyl](methyl)amino}ethyl)-N-methylcarbamate (2G-A and

2k-V)

Following General Procedure D, the title compounds 2k-A (3.3 mg, 10% yield, second LC peak) and 2k-E (4.1 mg, 12% yield, first LC peak) were obtained as diastereoisomers as white solids . 2k-A: ESI m/z: 1143.4 (M+H) ⁺ , retention time 1.70 min (method A). 2k-b: ESI m/z: 1143.4 (M+H) ⁺ , retention time: 1.65 min (method A).

Example 2l.

Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-[(4- {[( {2-[( {2[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8. 0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl)oxy]methyl} phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamoyl}3,6,9,12-tetraoxatetradecane-1-yl)carbamate (2l)

o ^z nh ₂

ESI m/z: 687.5 (M/2+H) ⁺ , 1396.8 (M+Na) ⁺ (50%),

1H NMR (500 MHz, MeOD-d ₄ ) δ 7.66 (d, J=7.5 Hz, 2H), 7.50 (d, J=10.0 Hz, 1H), 7.36 (d, J=7.0 Hz, 2H), 6.28 ( d, J=10.0 Hz, 1H), 6.04 (s, 1H), 5.15-5.05 (m, 2H), 4.87-4.62 (m, 4H), 4.53(s, 1H), 4.46(s, 1H), 4.21 (d, J=6.5 Hz, 1H), 4.16 (d, J=8.0 Hz, 2H), 3.83-3.72 (m, 2H), 3.66-3.60 (m, 12H), 3.56-3.52 (m, 3H), 3.49-3.42 (m, 1H), 3.30 (s, 3H), 3.25-3.09 (m, 3H), 3.02-2.96 (m, 4H), 2.87 (dd, J=16.6, 4.8 Hz, 2H), 2.72- 2.64 (m, 1H), 2.58 (t, J=6.0 Hz, 2H), 2.40 (d, J=13.1 Hz, 1H), 2.31-2.10 (m, 9H), 2.01-1.91 (m, 3H), 1.81 -1.70 (m, 2H), 1.63 (s, 7H), 1.51 (s, 3H), 1.48-1.30 (m, 4H), 1.11 (s, 1H), 1.02-0.91 (m, 15H) ppm. Anal. HPLC: >99.9%, retention time: 9.40 min (method A).

Example 2m.

2-[( 1 S,2S,4R,8S,9S, 11 S,12S, 13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2, 9} .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)-2-[(2S) -2-[(2R)6-(4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaene-10-yne- 2-yl}-4-oxobutanamido)-2- 129 045111

[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]hexanamido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl](methyl) amino }ethyl)-N-methylcarbamate (2mprecursor)

According to General Procedure B, crude compound 2m-precursor (2m precursor) (5 mg) was obtained as a light yellow oil, which was directly used for the next step. ESI m/z: 780 (M/2+H)+.

2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)-5(carbamoylamino)-2-[ (2S)-2-[(2R)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]-6-[4-(3{[31 ,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20,25,30-pentakis(hydroxymethyl)2,4,7,9,12,14 ,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 3,6.2 ⁸ , ^{11.2 13} , ^{16.2 18} , ^{21.2 23 , 26} ]dotetracontan-5yl]methyl}-3,4 ,5,13-tetraazatetracyclo[13.4.0.0 ²⁶ .0 ⁷ d ² ]nonadeca-1(15),2(6),4,7(12),8,10.16,18-octaen-13yl)-4-oxobutanamido ]hexanamido]-3-methylbutanamido]pentanamido]phenyl}methoxy)carbonyl](methyl)amino} ethyl)-N-methylcarbamate (2m)

To a solution of crude product 2t (5 mg) in DMF (1 ml) was added CD-N3 (8b, 63 mg, 63 µmol). The resulting mixture was stirred at RT for 72 hours, which was monitored by LCMS. The resulting mixture was immediately purified by prep-HPLC (Method A) to give 2m (2 mg, 4% yield from 4c) as a white solid. ESI m/z: 1278.8 (M/2+H) ⁺ . Anal. HPLC: 97.9%, retention time: 6.49 min (method A).

Example 2n.

2-[(1S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)-2-[(2S)-2 -[(4R)4-amino-4-[(2-azidoethyl)carbamoyl]butanamido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl](methyl)amino}ethyl)-K-methylcarbamate (2n)

The reaction mixture was then stirred at RT overnight. Connection 4c was completely destroyed according to LCMS. The mixture was separated by prep-HPLC (method A) and after lyophilization by

- 130 045111 compound Fmoc-2n (20 mg) was treated, which was then dissolved in DMF (2 ml). Diethylamine (4 drops, see 0.08 ml) was added to the DMF solution. The reaction mixture was stirred at RT for 2 h until the de-Fmoc reaction was complete, as monitored by LCMS. The mixture was filtered through a filtration membrane and the solution was purified by prep-HPLC (Method A) to give the title compound 2n (10 mg, 8.4% yield) as a white solid. ESI m/z: 1147 (M+H) ⁺ . Anal. HPLC: 99.6%, retention time: 5.67 min (method A); 7.72 min (method B).

Scheme 6

Synthesis of linker budesonide 2q (THP analogue)

Example 2q.

(2S)-(3,4-dihydro-2H-pyran-2-yl)methyl ureidopentanoate (15) o

O

1) Budesonide (1a), pTsOH, DCM Ar, 30 °C, 12 h.

2) Et ₂ NH, DCM, cold, 1 h

PHP _g aunt, Et< DMF

2q

2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-5 To a mixture of Fmoc-Cit-OH (14, 0.29 g, 0.73 mmol) in DMF (5 ml) was added DHP (0.10 mg, 0.88 mmol), TBTU (0.70 g, 2.2 mmol) and triethylamine (0.37 g, 3.7 mmol) at RT. The resulting mixture was stirred at RT for 24 hours. Detection of 15% of the required mass was carried out by LCMS. The reaction mixture was cooled with water (30 ml) and extracted with ethyl acetate (10 ml x 3). The combined organic solution was washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by reverse phase flash chromatography (0-100% acetonitrile in water) to give compound 15 (110 mg, 30% yield) as a colorless oil. ESI m/z: 494 (M+H)+.

- 131 045111 (6- {2-[( 1 S,2S,4R,8S,9S, 11S,12S, 13R)-11 -hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}oxan-2-yl)methyl (2S)-2amino-5- (carbamoylamino)pentanoate (16)

To a solution of compound 15 (80 mg, 0.16 mmol) in anhydrous DCM (3 ml) was added a solution of budesonide (1a, 70 mg, 0.16 mmol) and p-toluenesulfonic acid (42 mg, 0.24 mmol) in anhydrous DCM (2 ml) by syringe in a protective atmosphere of argon at RT. The reaction mixture was stirred at 30°C under a protective argon atmosphere for 12 hours. Most of compound 15 was destroyed by LCMS. The reaction mixture was cooled to RT and diethylamine (1 ml) was added to the reaction mixture. The reaction mixture was stirred at RT for 1 h until the de-Fmoc reaction was complete, as monitored by LCMS. Volatiles were removed in vacuo and the residue was purified by prep-HPLC (Method B) to give the title compound 16 (17 mg, 31% yield) as a white solid. ESI m/z: 702 (M+H)+.

(S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanoic acid (5i)

To a solution of L-valine (0.12 g, 1.0 mmol) in anhydrous DMF (2 ml) was added compound 5a (0.31 g, 1.0 mmol) and triethylamine (0.51 g, 5.0 mmol) at RT. The mixture was stirred at RT for 8 h until compound 5a was completely destroyed, which was monitored by LCMS. The reaction mixture was filtered through a filtration membrane and the filtrate was immediately purified by prep-HPLC (Method A) to give the title compound 5i (0.14 g, 46% yield) as a colorless oil. ESI m/z: 311 (M+H) ⁺ .

6- {2-[( 1 S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}oxan-2-yl)methyl (2S)-5-(carbamoylamino)-2- [(2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamudo]-3methylbutanamido]pentanoate (2q)

To a solution of compound 5i (5.8 mg, 19 µmol) in DMF (1 ml) was added HATU (10 mg, 26 µmol) and DIPEA (6.6 mg, 51 µmol) at RT. The mixture was stirred at RT for 15 min and then compound 16 (12 mg, 17 μmol) was added to the solution at RT. The resulting mixture was stirred at RT for 4 h until compound 5i was completely destroyed, which was monitored by LCMS. The reaction mixture was then filtered through a filter membrane and the filtrate was purified directly by prep-HPLC (Method A) twice to give the title compound 2q (2 mg, 12% yield) as a white solid. ESI m/z: 995.3 (M+H) ⁺ . Anal. HPLC: 83.5%, retention time: 10.36 min (method B).

Scheme 7. Synthesis of linker-phosphate-budesonide 2r and 2s

Phosphate budesonides were coupled with BCN-PNP (11c) to obtain BCN phosphate budesonides (compound 2r and 2s) (Scheme 7).

- 132 045111

Example 2r.

{2-[({bicyclo[6.1.0]non-4-yn-9-ylmethoxy}carbonyl)amino]ethoxy}({2[(1S,2S,4R,8S,9S,11S,12S,13R)- 11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl ]-2-oxoethoxy})phosphinic acid (2r)

Following General Procedure D, 2r (40 mg, 61% yield) was obtained as a white solid. ESI m/z: 730 (M+H) ⁺ .

^1H NMR (500 MHz, MeOD-d ₄ ) δ 7.49 (d, J=10.1 Hz, 1H), 6.29-6.26 (m, 1H), 6.03 (s, 1H), 5.23-5.16 (m, 1H), 4.88-4.64 (m, 3H), 4.45 (dd, J=8.0, 3.1 Hz, 1H), 4.23-4.13 (m, 2H), 3.97 (dd, J=11.9, 5.7 Hz, 2H), 3.36 (t, J=4.7 Hz, 2H), 2.67 (td, J=13.4, 5.3 Hz, 1H), 2.40 (d, J=9.6 Hz, 1H), 2.30-1.34 (m, 23H), 1.21-0.92 (m, 10H ) ppm. Anal. HPLC: >99.9%, retention time: 5.26 min (method B).

Example 2s.

{2-[({bicyclo[6.1.0]non-4-yn-9-ylmethoxy}carbonyl)amino]ethoxy}({[hydroxy({2[(1S,2S,4R,8S,9S,11S,12S ,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-diene -8-yl]-2 oxoethoxy})phosphoryl]oxy})phosphinic acid (2s)

ESI m/z: 810 (M+H)+.

1H NMR (500 MHz, MeOD-d ₄ ) δ 7.51 (d, J=10.1 Hz, 1H), 6.28 (d, J=10.0 Hz, 1H), 6.03 (s, 1H), 5.22-5.16 (m, 1H ), 4.98-4.66 (m, 3H), 4.46 (s, 1H), 4.12 (dt, J=45.0, 13.0 Hz, 4H), 3.39 (t,J=11.9 Hz, 2H), 2.67 (dd,J= 13.3, 8.0 Hz, 1H), 2.40 (d, J=11.2 Hz, 1H), 2.30-1.32 (m, 23H), 1.22-0.92 (m, 10H) ppm.

Anal. HPLC: >99.9%, retention time: 4.03 min (method B).

Experimental procedures for intermediates

Table 4

Key intermediates and starting materials

Structures Intermediate connection Links or synthesis AAA/ ο ΧΙ J?H 1 But% 1a (budesonide) Available for sale (51333-22-3) O AL ° J >° y o '—< >=o m T 4a (vcPABbudesonide) See diagram 2

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n Η Well--y ^°ΥΡνΓ>° Υ Η 9 Υθ^θ Ън '^ΝΗ Ο^ΝΗ ₂ 4c (vcPAB-ld) See diagram 5 Υυ ⁰ Υζ ο Ο Ο=^ Ο / γ-Ζ γΥο 5a (mc-NHS) Available for sale (55750-63-5) γΥ° Υζ Ο ο '1 ο ο ο ,ο ^Ζ Ύ Ο=\Υ 5b (MALPEG4-NHS) Available for sale (1325208-25-0) ο if / ^{0 0} W ⁰ 5c (DIBAC-sukPEG4acid) Available for sale (1537170-85-6) „ Ο ο ⁿ К у \V/ ^H 5d (BCNPEG4 acid) Available for sale (1421932-54-8) o Q _r ° θ λ Ο^ΝΗ υΥ · o he 5e Scheme 8

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SY ⁿ { o ⁰ / H GS O^NH ₂ 5f (mc-Val-CitOH) WO2014/19157 8 Al O 1( / U Ν Y n 9 Υγ^^θ^ο^θ^ο'^ΝΥ'Απ ^{0 Η} θ NGCH ο^νη ₂ 5g (DIBACsuc-PEG4-ValCit-OH) Scheme 9 ο FmocHN l^k _he N ₃ ^^Aq 5h Scheme 10 ο^α ζ-Α / Ο )=Ο ΙΖ / °Υ ' Ο τ 5i See diagram 6 0 FmocHN^X j UM ΗΝ^Ο / \ όΑ^/ 6a Scheme 11 o <υΗγ FmocHN. .ν\ A _kl /\ Al/A' ⁰ IN IV /V OH ^u no^o 6b J. Org. Chem. 2010, 75, 3685- 3691 ^N3 '·^OH ΗΟ^^Ύ'ΌΗ OH 8a Available for sale (35899-89-9)

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he oAt---n--^9 L AZS° ^0H \V /OH W O IT. he^^ ^N o C O1^L-on o o \on he/ CHL οη/Ά' /A A°b°~AL~O OH ^0>/ \ he 8b Synth. Commun., 2002, 32(21), 33673372 J. Am. Chem. Soc., 2012, 134(46), 1910819117 J. Med. Chem., 1997, 40(17), 2755-2761 J. Am. Chem. Soc., 1993, 115(12), 50355040 see about A o o=< o b ZI IZ '--i Z A ° n / ZX o = / ° h / V—Z AA° Pa (MC-VCPAB-PNP) Available on sale (159857-81-5) WO2014/19157 8 Al SOp z-vV 1 ζζ O O O U ΖΖ / °А °А ^g h. _$o ΖΖ h o >° o 0 Ζ o 11b (DIBACsuc-PEG4-vcPAB-PNP) Scheme 12 o Z ABOUT 11c (BCNPNP) WO2013/18169 7A1 Angew. Chem. Int. Ed., 2010, 49 (49), 94229425 o iV° ² AAJ o A ooo^ 1st A AJ FmocHN y G fl o k An oT _N h ₂ lid (Fmoc-vcPAB-PNP) Available for sale 863971-53-3 Y N n PG ^0N LxA AJ* BocHN T Ά N 0 A ⁿ ΗΙΨ O^An ₂ 12a (Boc-vcPAB) Available for sale (870487-09-5) WO2008/34124 A2 IZ / У1 о=о Z \--< ZI “ ^Х А IZ 7 О I 12b (MC-VCPAB) Available for sale 159857-80-4 O/O ? and γ n? ps° ⁿ O ⁰ o Ζ ΗΝ^ cTnh ₂ 12c (DIBACsuc-PEG4-vcPAB) Scheme 12

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Scheme 8

Synthesis 5e

5а 18 5е (2R)-6-(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4,6,8,13,15-hexaen-10-yn-2-yl } -4oxobutanamido)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]hexanoic acid (5 e)

To a mixture of H-D-Lys(Boc)-OH (17, 0.25 g, 1.0 mmol) in DMF (10 ml) was added 6-maleimidocaproic acid-NHS (5a, 0.31 g, 1.0 mmol) and diisopropylethylamine (DIPEA, 0.26 g 2.0 mmol ) with CT. After this, the reaction mixture was stirred at RT overnight, compound 5a was completely destroyed. The mixture was directly separated by reverse phase flash chromatography (0-100% acetonitrile in water (0.05% TFA)) to obtain intermediate Boc-18 (ESI m/z: 440 (M+H)+) as a colorless oil. which was dissolved in DCM (5 ml). TFA (0.5 ml) was added dropwise to the solution at 0°C and the mixture was stirred at RT for an hour. The reaction was monitored by LCMS and intermediate Boc-18 was completely destroyed. Volatiles were removed in vacuo to obtain crude product 18 (ESI m/z: 340 (M/2+H)+), which was used for the next step without further purification. To a mixture of crude 18 (0.21 g, 0.62 mmol) in DMF (5 ml) was added activated ester 19 (0.20 g, 0.50 mmol) and DIPEA (50 mg, 0.39 mmol) at RT. The reaction mixture was then stirred at RT overnight and compound 19 was completely destroyed as monitored by LCMS. The reaction mixture was then directly separated by reverse phase flash chromatography (0-100% acetonitrile in water) to obtain the title compound 5e (0.10 g, 20% yield from 3 steps of 5a) as a white solid. ESI m/z: 627 (M+H)+.

¹ H NMR (DMSO-d ₆ , 400 MHz): δ 7.89 (d, J=13.2 Hz, 1H), 7.69-7.62 (m, 3H), 7.48-7.47 (m, 3H), 7.367.28 (m, 3H), 6.99 (s, 2H), 5.03 (d, J=13.6 Hz, 1H), 4.09-4.04 (m, 1H), 3.60 (d, J=13.6 Hz, 1H), 3.38-3.34 (m, 1H ), 2.90-2.87 (m, 2H), 2.61-2.55 (m, 1H), 2.25-2.17 (m, 1H), 2.08-1.51 (m, 5H), 1.46-1.15 (m, 12H) ppm.

Scheme 9

Synthesis 5 g

(2S)-2-[(2S)-2-[ 1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15 -hexaen-10-in-2-yl}-4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbuanamido]-5(carbamoylamino)pentanoic acid (5g)

- 137 045111

HATU (0.31 g, 0.81 mmol) and DIPEA (0.14 g, 1.1 mmol) were added to a solution of compound 5c (0.30 g, 0.54 mmol) in DMF (10 mL) at RT. The mixture was stirred at RT for 15 minutes. Val-Cit-OH (20 (CAS No. 159858-33-0), 0.21 g, 0.76 mmol) was added to the reaction mixture at RT and the resulting mixture was stirred at RT for 3 h until most of the materials were destroyed, as monitored by LCMS . The reaction mixture was filtered through a filter membrane and the filtrate was directly purified by reverse flash chromatography (0-100% acetonitrile in water (with 10 mmol/L ammonium bicarbonate)) to give the title intermediate 5g (0.25 g, 57% yield) in as a white solid. ESI m/z: 809.5 (M+H) ⁺ .

Scheme 10

Synthesis of intermediates 5h

(4R)-4-[(2-azidoethyl)carbamoyl]-4-{[(9H-fluorene-9tert-butyl ylmethoxy)carbonyl]amino}butanoate (22)

To a mixture of Fmoc-£>D-Glu(OTBU)-OH (21, (104091-08-9), 0.30 g, 0.71 mmol) and 2-azidoethanamine (87156-40-9, 73 mg, 0.85 mmol) in DCM (50 ml) HATU (0.41 g, 1.1 mmol) and triethylamine (0.3 ml) were added at RT. The reaction mixture was stirred at RT overnight. Most of compound 21 was destroyed according to TLC and LCMS. The reaction mixture was then cooled with water (50 ml) at RT, the organic solution was washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by prep-TLC (silica gel, eluting with petroleum ether/ethyl acetate (v/v=1)) to give the title compound 22 (0.30 g, 76% yield) as a yellow viscous oil. ESI m/z: 494 (M+H)+.

^1H NMR (400 MHz, DMSO-d6): δ 8.09 (m, 1H), 7.84-7.90 (m, 4H), 7.33-7.44 (m, 4H), 6.28 (s, 2H), 3.35 (m, 6H ), 3.26 (m, 1H), 2.50 (m, 2H), 2.25 (m, 1H), 1.80 (m, 1H), 1.39 (s, 9H) ppm.

(4R)-4-[(2-azidoethyl)carbamoyl]-4-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}butanoic acid (5h)

The resulting mixture was stirred at RT for 2 hours until compound 22 was completely destroyed, which was monitored by TLC and LCMS. Volatiles were removed in vacuo to give crude product 5h (17 mg, 96% yield) as a yellow oil, which was used for the next step without further purification. ESI m/z: 438 (M+H) ⁺ .

Scheme 11

Synthesis of intermediates 6a

O

OSu

NHFmoc Fmoc-(D)-Lys-OH

Et ₃ N, DMF

KT, 30 min.

O 6a (2R)-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}hexanoic acid (6a)

- 138 045111

To a mixture of commercial compound 23 (65 mg, 0.23 mmol, CAS: 1425803-45-7) in DMF (2 ml) was added Fmoc-D-Lys-OH (85 mg, 0.23 mmol) and triethylamine (52 mg, 0.51 mmol) . The reaction mixture was stirred at RT for 30 min. The mixture was directly separated by reverse phase flash chromatography (0-100% acetonitrile in water (0.05% TFA)) to obtain intermediate 6a (85 mg, 70% yield) as a white solid. ESI m/z: 533 (M+H)+.

Ή NMR (MeOD-d4, 500 MHz): δ 7.70 (d, J=7.5 Hz, 2H), 7.59 (t, J=8.0 Hz, 2H), 7.30 (t, J=7.5 Hz, 2H), 7.22 ( t, J=7.4 Hz, 2H), 4.35-4.22 (m, 2H), 4.22-4.09 (m, 2H), 4.09-3.99 (m, 1H), 3.94-3.81 (m, 1H), 3.79-3.67 ( m, 1H), 3.15 (t, J=6.9 Hz, 2H), 2.17-1.96 (m, 3H), 1.96-1.86 (m, 1H), 1.85-1.66 (m, 4H), 1.66-1.41 (m, 5H), 1.41-1.25 (m, 3H) ppm.

Scheme 12

Synthesis of intermediates 11b and 12c

-(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-in-2-yl} -4oxobutanamido)- N-[( 1S)-1-{[(1 S)-4-(carbamoylamino)-1 - {[4-(hydroxymethyl)phenyl]carbamoyl}butyl]carbamoyl}-2-methylpropyl] -3,6,9 ,12-tetraoxapentadecane-15-amide (12c)

The mixture was stirred at RT for 15 minutes. vc-PAB (24 (159857-79-1), 0.21 g, 0.54 mmol) was added to the reaction solution at RT, and the resulting mixture was stirred at RT for 3 h until most of the materials were destroyed, as monitored by LCMS. The reaction mixture was filtered through a filter membrane and the filtrate was directly purified by reverse phase flash chromatography (0-100% acetonitrile in water (with 10 mmol/L ammonium bicarbonate)) to obtain the title intermediate 12c (0.30 g, 60% yield) as white solid matter. ESI m/z: 617 (M+H)+.

{4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7 ,13,15-hexaen-10-yn-2yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecanp-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methyl 4- nitrophenyl carbonate (11b)

To a solution of compound 12c (0.15 g, 0.16 mmol) in DMF (10 ml) was added bis(4-nitrophenyl) carbonate (0.15 g, 0.49 mmol) and DIPEA (63 mg, 0.49 mmol) at 0°C. The mixture was then stirred at RT for 3 hours until compound 12c was almost completely destroyed, as monitored by LCMS. The reaction mixture was filtered through a filter membrane and the filtrate was directly purified by reverse phase flash chromatography (0-100% acetonitrile in water (with 10 mmol/L ammonium bicarbonate)) to give the title intermediate 11b (50 mg, 28% yield) in as a white solid. ESI m/z: 1079 (M+H)+.

In the table below. 5 shows linker loads prepared using the methods described herein.

Table 5

Examples of linker loads

- 140 045111

2e la MC-VC-PABA H O 1 >C n IU _avd aa - ^! - V Al ΉΗ ° c> nh ₂ 2f 1c MC-VC-PAB ⁷ VZ £ zi 5 / ^Z IZ ZI o y— z 2g 1c DIBAC-suc-vcPAB ^H *X /—\ О °^ИИ° О fl fl Th fl <у>А^о-!Х _о he ΉΗ CT nh ₂ 2j le MC-VC-PAB I O ^H O A ⁿ L-<N> 1 ^NO 7L Chsh O^NHj ° 2k ID MC-VC-PAB • О )-0/==° Z— — Z о У\О '--Ч 2=0 ZI IZ ^z 1

- 141 045111

- 142 045111

Table 6

Characteristic data for linker loads

LP (LN) cLogP M.F. M.B. HPLC Purity (%) HPLC KT (min) MC (m/z) 100% Highest peak m/z 2a 4.18 C ₅₄ H ₇₂ N ₆ Oi4 1029.2 92 7.6(A) 1029 (M+H) 1029 (M+N) 2b 5.42 C74H95N7O18 1370.6 97 8.1(B) 685.8 (M/2+H) 685.8 (M/2+N) 2s 1.03 C81H117N11O26 1660.9 97 7.0(A) 830.8 (M/2+H) 830.8 (M/2+N) 2d -1.87 C70H ₁₀₂ N ₈ O25 1455.6 100 6.48(A) 728 (M/2+H) 1456 (M+N) (10%) 2e 3.45 C54H73N7O13 1028.2 100 7.54(A) 1028.3 (M+H) 1028.3 (M+N) 2f 3.54 C ₅ 7H ₇ 6N ₆ O1 ₅ 1085.2 98 1.71 (LCMS) 1085.3 (M+H) 1085.3 (M+N) 2g 4.77 C77H99N7O19 1426.7 >99 7.86(B) 714.0 (M/2+H) 1427 (M+N) (15%) 2j 2.16 C57H77N9O16 1144.3 98 1.57 (LCMS) 1144.3 (M+H) 1144.3 (M+N) 2k 3.54 C59H82N8O15 1143.3 98 1.70 (LCMS) 1143.4 (M+H) 1143.4 (M+N) 21 4.66 C71H104N8O19 1373.6 100 9.40 (A) 687.5 (M/2+H) 1396.8 (M+Na) (50%) 2m -4.89 C120H166N14O47 2556.7 100 6.49(A) 1278.8 (M/2+H) 1278.8 (M/2+N) 2n 1.68 C ₅₆ H ₈ 2N12O14 1147.3 100 7.72(B) 1147.5 (M+H) 1147.5 (M+N) 2q 3.52 C52H75N5O14 994.2 79 10.36 (B) 995.3 (M+H) 995.3 (M+H) 2r 4.88 C ₃ 8H ₅₂ NO11P 729.8 100 5.26(B) 686.2 (weak, May) 1459.1 (2M+N) 2s 0.55 C38H ₅₃ NO1 ₄ P2 809.8 100 4.03 (B) 766.1 (M-44) 810.1 (M+H) (70%)

- 143 045111

Synthesis of linker loads LP1-4.

Scheme 101

Synthesis of load 100, its spacer loads 101a-d and linker loads LP1-4

Fluocinolone acetonide

Q C ₃ H ₇ CHO, 70% NSJ ₄

CT scan, on

R ¹² Μ

R ¹³

101-2a-d

1) 101-2a-d, Et ₃ N, DMAP, DCM

2) TFA

101-2a, R ¹¹ = H, R ¹² = Me, R ¹³ = Boc

101-2b, R ¹¹ = R ¹² = Me, R ¹³ = H

101-2c, R ¹¹ = R ¹² = Et, R ¹³ = BOC

101-2d, Boc-piperazine

O

1) 11d, NOVT, DIPEA, DMF, K.T.

2) piperidine, b.t.

ABOUT

101a-d

101a, R ¹¹ = N, R ¹² = Me

101b, R ¹¹ = R ¹² = Me

101c, R ¹¹ = R ¹² = Et

101d, R ¹¹ , R ¹² together = -CH ₂ CH ₂ - (formation of piperazine)

DIBAC-suc-PEG ₄ -vcPAB-PNP (11b)

Et ₃ N, DMAP, DMF

101-3a,b,d = H, R ¹² = N = R ¹² = Me d, R ¹¹ , R ¹² together

LP1, R ¹¹ = H, R ¹² = Me

LP2, R ¹¹ = R ¹² = Me

LP3, R ¹¹ = R ¹² = Et

LP4, R ¹¹ ,R ¹² BMecTe = -CH ₂ CH ₂ - (formation of piperazine) (1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy- 8-(2-hydroxyacetyl)-9,13dimethylamine-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (100)

To a mixture of fluocinolone acetonide (10 g, 22 mmol) and silica gel (100 g) in heptanes (900 ml) was added butyraldehyde (3.0 ml, 33 mmol) below 10°C and the suspension was stirred at 10-20°C for 30 minutes. Perchloric acid (70%, 7.6 ml, 91 mmol) was added dropwise to the mixture at 0°C. The reaction mixture was then stirred at RT overnight. Most of the fluocinolone acetonide was destroyed by TLC and LCMS. The reaction mixture was diluted with petroleum ether and cooled down. water sodium carbonate. The suspension was filtered and the solid was washed with DCM/methanol (v/v=1:1). The combined filtrate was concentrated in vacuo. The residue was purified by flash chromatography (0-1.5% methanol in DCM) to give compound 100 (8.0 g, yield: 78%) as a white solid. ESI m/z: 467.1 (M+H)+.

- 144 045111

1-[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl- 5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}carbonyl)oxy] pyrrolidine-2,5-dione ( 101-1a)

To a solution of compound 100 (0.47 g, 1.0 mmol) in DCM (20 ml) was added bis(2,5-dioxopyrrolidin1-yl) carbonate (0.28 g, 1.1 mmol), triethylamine (0.40 g, 4.0 mmol) and DMAP (3.0 mg , cat.). The reaction mixture was stirred at RT for 4 hours until the destruction of compound 100, which was monitored by LCMS. The reaction mixture was then diluted with DCM and washed with water. The organic solution was dried over sodium sulfate. After filtration, the solution was concentrated in vacuo and the residue (crude product 101-1a) was used directly for the next step without purification. ESI m/z: 608.2 (M+H) ⁺ .

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-8-{2-[(4nitrophenoxycarbonyl)oxy]acetyl}- 6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17dien-16-one (101-1b)

A mixture of compound 100 (0.80 g, 1.7 mmol), bis(4-nitrophenyl) carbonate (1.6 g, 5.2 mmol) and DIPEA (1.1 g, 8.6 mmol) in THF (20 ml) was stirred at RT for 4 h, which was monitored via LCMS. The mixture was concentrated in vacuo. The crude product was purified by silica gel column chromatography (50% ethyl acetate in petroleum ether) to give 101-1b (0.75 g, 69% yield) as a colorless oil. ESI m/z: 632 (M+H) ⁺ .

General procedure for connection 101.

(formation of piperazine)

To a solution of compound 101-1a or 101-1b (crude product calculated to be 1.0 mmol) in DCM (20 ml) was added diamine 101-2(a, b, c or d) (1.1 mmol) and DMAP (0.1 mmol ). The reaction mixture was stirred at RT for 4 hours and TFA (1 ml) was added. The mixture was stirred at RT for half an hour (for R ¹³ = Boc, the reaction mixture was stirred until Boc was completely removed, which was monitored by LCMS). The resulting mixture was concentrated in vacuo and the residue was purified by reverse phase flash chromatography (50-80% acetonitrile in aq. TFA (0.5%)) to give compound 101(a, b, c or d) as a colorless oil.

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-[2(methylamino)ethyl]carbamate (101a)

F

Following the general procedure and using compound 101-1a with Boc-diamine 1012a, compound 101a (0.38 g, 57% in 2 steps, TFA salt) was obtained as a colorless oil. ESI m/z: 567 (M+H) ⁺ .

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-methyl-N-[2- 145 045111 (methylamino)ethyl] carbamate (101b)

Following the general procedure and using compound 101-1a with diamine 101-2b, compound 101b (0.45 g, 66% yield in 2 steps, TFA salt) was obtained as a colorless oil. ESI m/z: 581 (M+H)+.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ² ' ⁹ .0 ⁴ ' 8.0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2-oxoethyl N-ethyl-N-[2(ethylamino)ethyl]carbamate (101c)

Following the general procedure and using compound 101-1b with diamine 101-2c, compound 101c (24 mg, 18% yield in 2 steps, TFA salt) was obtained as a colorless oil. ESI m/z: 609 (M+H) ⁺ .

1H NMR (500 MHz, DMSO-d ₆ ) δ 8.38 (s, 2H), 7.30 (d, J=10.5 Hz, 1H), 6.24 (dd, J=10.5 Hz, 2.0 Hz, 1H), 6.11 (s, 1H), 5.70-5.54 (m, 2H), 5.10-4.91 (m, 1H), 4.85-4.65 (m, 3H), 4.25-4.16 (m, 1H), 3.60-3.40 (m, 2H), 3.33- 3.20 (m, 4H), 3.20-2.90 (m, 4H), 2.67-2.53 (m, 1H), 2.30-2.20 (m, 1H), 2.10-2.00 (m, 2H), 1.80-1.70 (m, 1H ), 1.64-1.51 (m, 4H), 1.49 (s, 3H), 1.40-1.30 (m, 2H), 1.20-1.10 (m, 4H), 1.10-1.00 (m, 1H), 0.90-0.80 (m , 6H) ppm.

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ² ' ⁹ .0 ⁴ ' 8.0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2-oxoethylpiperazine-1carboxylate (101d)

Following the general procedure, using compound 101-1b with piperazine (1012d) without exposure to TFA and purification by silica gel column chromatography (0-10% methanol in DCM) gave compound 101d (0.21 g, 49% yield in 2 steps , free base) as a colorless oil. ESI m/z: 579 (M+H) ⁺ .

General procedure for connection 101-3

Fmoc-VC-PAB-PNP 11d was added to a solution of compound 101a, b or d (0.20 mmol) in DMF (3 ml)

- 146 045111 (0.17 g, 0.22 mmol), NOBT (41 mg, 0.30 mmol) and DIPEA (77 mg, 0.60 mmol). The mixture was stirred at RT for 3 hours, which was monitored by LCMS. Piperidine (0.3 ml) was then added to the mixture. The reaction mixture was stirred at RT for an hour until complete removal of Fmoc according to LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (50-80% acetonitrile in aq. NH4HCO3 (10 mM)) to give compound 101-3a, b or d as a white solid.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)- 2[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl](methyl)amino}ethyl)carbamate (101-3a)

Following the general procedure and using compound 101a, compound 1013a (0.11 g, 57% yield) was obtained as a white solid. ESI m/z: 972 (M+H)+.

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6N-methyl-N- [2propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ] icosa-14,17-dien-8-yl]-2-oxoethyl (methylamino)ethyl]carbamate (101 -3b)

Following the general procedure and using compound 101b, compound 1013b (0.13 g, 65% yield) was obtained as a white solid. ESI m/z: 986 (M+H) ⁺ .

1-{4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl 4{2-[( 1 S,2S,4R,SS ,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl5,7-dioxapentacyclo[10.8.0.0 ² ' ⁹ .0 ⁴ ' ⁸ .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2-oxoethyl}piperazine-1,4dicarboxylate (101-3d)

Following the general procedure and using compound 101d (58 mg, 0.10 mmol), compound 101-3d (52 mg, 53% yield) was obtained as a white solid. ESI m/z: 984 (M+H)+.

1H NMR (500 MHz, DMSO-d ₆ ) δ 10.0 (s, 1H), 8.14 (d, J=7.5 Hz, 1H), 7.60 (d, J=8.5 Hz, 2H), 7.31 (d, J=8.5 Hz, 2H), 7.26 (d, J=8.5 Hz, 1H), 6.30 (dd, J=1.5 Hz, 10.5 Hz, 1H), 6.11 (s, 1H), 6.02-5.88 (m, 1H), 5.705. 50 (m, 2H), 5.42 (s, 2H), 5.20-4.92 (m, 3H), 4.80-4.62 (m, 3H), 4.50-4.45 (m, 1H), 4.25-4.15 (m, 1H), 3.503.35 (m, 8H), 3.05-2.90 (m, 3H), 2.70-2.54 (m, 2H), 2.32-2.20 (m, 1H), 2.10-1.50 (m, 10H), 1.46 (s, 3H ), 1.451.30 (m, 5H), 0.95 (d, J=7.5 Hz, 1H), 0.90-0.80 (m, 9H), 0.77 (d, J=7.0 Hz, 3H) ppm.

- 147 045111

General procedure for connections LP1, LP2 and LP4

HATU (27 mg, 71 µmol) and DIPEA (20 mg, 0.15 mmol) were added to a solution of compound 5c (34 mg, 60 µmol) in DMF (1 ml). The mixture was stirred at RT for 5 min and compound 101-3a, b or d (50 µmol) was added to the mixture. The mixture was stirred at RT for 2 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (Method B) to obtain compound LP1, 2 or 4 as a white solid.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)- 2[^)-2-[1-(4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaene-10-yne -2-yl}-4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl](methyl)amino}ethyl)carbamate ( LP1)

Following the general procedure and using compound 101-3a, linker load LP1 (20 mg, 26% yield) was obtained as a white solid. ESI m/z: 754 (M/2+H)+.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S)- 2[(2S)-2-[1-(4-{2-azatriciclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaene-10- in-2-yl}-4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl](methyl)amino}ethyl)- N-methylcarbamate (LP2)

Following the general procedure and using compound 101-3b, linker load LP2 (20 mg, 26% yield) was obtained as a white solid. ESI m/z: 761 (M/2+H) ⁺ .

^1H NMR (500 MHz, DMSO-d ₆ ) δ 9.98 (s, 1H), 8.12 (d, J=7.4 Hz, 1H), 7.87 (d, J=8.6 Hz, 1H), 7.76 (t, J= 5.4 Hz, 1H), 7.69-7.66 (m, 1H), 7.64-7.57 (m, 3H), 7.51-7.44 (m, 3H), 7.40-7.33 (m, 2H), 7.32-7.25 (m, 4H) , 6.33-6.27 (m, 1H), 6.11 (s, 1H), 5.98 (t, J=5.5 Hz, 1H), 5.70-5.55 (m, 2H), 5.41 (s, 2H), 5.08-4.91 (m , 4H), 4.794.66 (m, 2H), 4.41-4.35 m, 1H), 4.25-4.17 (m, 2H), 3.64-3.56 (m, 3H), 3.49-3.41 (m, 15H), 3.30- 3.27 (m, 2H), 3.13-3.00 (m, 3H), 2.98-2.75 (m, 8H), 2.65-2.54 (m, 2H), 2.48-2.43 (m, 1H), 2.41-2.34 (m, 1H ), 2.28-2.19 (m, 2H), 2.07-1.94 (m, 4H), 1.81-1.67 (m, 3H), 1.63-1.53 (m, 5H), 1.48 (s, 3H), 1.45-1.31 (m , 5H), 0.88-0.82 (m, 13H) ppm.

1-{4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyl[10.4.0.0 ^4.9 ]hexadeca-1(12),4(9),5 ,7,13,15-hexaen-10-in-2yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5-148 045111 (carbamoylamino)pentanamido] phenyl}methyl 4-{2-[(1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11hydroxy-9,13-dimethylamine-16-oxo-6- propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icos-14,17dien-8-yl]-2-oxoethyl}piperazine-1,4-dicarboxylate (LP4)

F

St nh ₂

Following the general procedure and using compound 101-3d, linker load LP4 (35 mg, 44% yield) was obtained as a white solid. ESI m/z: 760 (M/2+H)+.

1H NMR (500 MHz, DMSO-d6) δ 10.0 (s, 1H), 8.14 (d, J=7.5 Hz, 1H), 7.88 (d, J=8.5 Hz, 1H), 7.77 (t, J=5.0 Hz , 1H), 7.70-7.66 (m, 1H), 7.63-7.55 (m, 3H), 7.52-7.43 (m, 3H), 7.40-7.27 (m, 6H), 6.30 (d, J=10.5 Hz, 1H ), 6.11 (s, 1H), 6.02-5.88 (m, 1H), 5.70-5.50 (m, 2H), 5.42 (s, 2H), 5.20-4.92 (m, 4H), 4.80-4.62 (m, 3H ), 4.40-4.35 (m, 1H), 4.26-4.20 (m, 2H), 3.64-3.55 (m, 3H), 3.50-3.35 (m, 17H), 3.30-3.25 (m, 3H), 3.15-2.90 (m, 4H), 2.64-2.54 (m, 2H), 2.50-2.45 (m, 1H), 2.41-2.34 (m, 1H), 2.32-2.20 (m, 2H), 2.10-1.92 (m, 4H) , 1.80-1.68 (m, 3H), 1.62-1.50 (m, 5H), 1.46 (s, 3H), 1.45-1.40 (m, 2H), 1.40-1.30 (m, 3H), 1.22-1.20 (m, 1H), 0.90-0.80 (m, 13H) ppm.

{4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 ⁴ , ⁹ ]hexadeca-1(12),4(9),5,7 ,13,15-hexaen-10-yn-2yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methyl N- ethyl-N-[2-(ethylamino)ethyl]carbamate (101-4)

cr nh ₂

N,N'diethylethylenediam (58 mg, 0.50 mmol) and DMAP (1 mg, 0.01 mmol) were added to a solution of DIBAC-suc-PEG ₄ -vcPAB-PNP 11b (0.11 g, 0.10 mmol) in DMF (5 ml). The reaction mixture was stirred at RT for 4 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (Method B) to give compound 101-4 as a colorless oil. ESI m/z: 1056.6 (M+H) ⁺ , 529 (M/2+H) ⁺ .

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2-{[({4-[(2S) -2[(2S)-2-[1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1 (12),4(9),5,7,13,15-hexaene-10 -in-2-yl}-4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamidoo]phenyl}methoxy)carbonyl](ethyl)amino}ethyl) -N-ethylcarbamate (LP3)

A mixture of compound 101-4 (16 mg, 15 μmol), 101-1b (11 mg, 17 μmol) hDIPEA (4.0 mg, 31 μmol) in DMF (5 ml) was stirred at RT for 4 h, which was monitored by LCMS. The reaction mixture was purified directly by prep-HPLC (Method B) to obtain linker load LP3 (3 mg, 13% yield) as a white solid. ESI m/z: 775 (M/2+H) ⁺ .

^1H NMR (500 MHz, DMSCve) δ 10.0 (s, 1H), 8.14 (d, J=7.5 Hz, 1H), 7.88 (d, J=8.5 Hz, 1H), 7.77 (t, J=5.0 Hz, 1H), 1.10-1.66 (m, 1H), 7.63-7.55 (m, 3H), 7.52-7.43 (m, 3H), 7.40-7.27 (m, 7H), 6.30 (d, J=10.5 Hz, 1H) , 6.11 (s, 1H), 6.02-5.88 (m, 1H), 5.70-5.50 (m, 2H), 5.42 (s, 2H), 5.20-4.92 (m, 4H), 4.80-4.62 (m, 3H) , 4.404.35 (m, 1H), 4.26-4.20 (m, 2H), 3.64-3.55 (m, 4H), 3.50-3.35 (m, 18H), 3.12-2.90 (m, 7H), 2.64-2.54 ( m, 3H), 2.41-2.34 (m, 2H), 2.32-2.20 (m, 3H), 2.10-1.92 (m, 5H), 1.88-1.68 (m, 3H), 1.62-1.50 (m, 6H), 1.50-1.40 (m, 6H), 1.40-1.30 (m, 4H), 1.22-1.20 (m, 1H), 1.02-0.90 (m, 4H), 0.90-0.78 (m, 6H) ppm.

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Synthesis of cysteamine prodrugs: linker loads LP5, 6, 7 and LP8

2-(pyridin-2-yldisulfanyl)ethanamine hydrochloride (102-1e) was commercially available under CAS 83578-21-6. tert-butyl (2-mercaptoethyl)(methyl)carbamate (102-1f) was synthesized according to Eur. J. Med. Chem., 2015, 95, 483-491.

Endo-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}ethyl)carbamate (5f) was synthesized according to WO2016168769.

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-[2-(pyridin-2yldisulfanyl)ethyl] carbamate (102 -3 e)

DIPEA (0.22 g, 1.7 mmol), 102-1e (0.19 g, 0.86 mmol) and DMAP (10 mg, 86 μmol) were added to a solution of compound 101-1b (0.54 g, 0.86 mmol) in acetonitrile (10 ml). The reaction mixture was stirred at RT for 48 hours until 101-1b was completely destroyed according to LCMS. The mixture was then filtered and the filtrate was concentrated in vacuo. The residual oil was purified by silica gel column chromatography (20-50% ethyl acetate in hexane) to give 102-3e (0.47 g, 80% yield) as a white solid. ESI m/z: 679.2 (M+H)+.

2-[( 1 S,2S,4R,8S,9S,11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-methyl-N-[2(pyridin-2-yldisulfanyl) ethyl]carbamate (102-3f)

Following the procedure for 102-3e, except substituting 102-1f for 102-1e, afforded 102-3f (0.45 g, 75% yield) as a white solid. ESI m/z: 693.2 (M+H)+.

2-[( 1 S,2S,4R,8S,9S,11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-(2sulfanylethyl)carbamate (102e)

- 150 045111

To a solution of compound 102-3e (1.4 g, 2.1 mmol) in chloroform (30 ml) was added triethylamine (0.82 ml, 5.9 mmol) and 1,4-dithiothreitol (Cleland's reagent, DTT) (1.2 g, 7.8 mmol). The reaction mixture was stirred at RT under nitrogen atmosphere for 4 hours, which was monitored by LCMS. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (50-70% ethyl acetate in hexane) to give 102e (0.95 g, 83% yield) as a white solid. ESI m/z: 570.2 (M+H)+.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl N-methyl-N-(2sulfanylethyl)carbamate (102f)

Following the procedure for 102e, except substituting 102-3f for 102-3e, afforded 102f (0.97 g, 83% yield) as a white solid. ESI m/z: 584.3 (M+H)+.

3-({2-[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16oxo- 6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}carbonyl)amino]ethyl}disulfanyl) -3-methylbutanoic acid (102-5e)

To a solution of compound 102-3e (0.68 g, 1.0 mmol) in methanol (5 ml) was added compound 102-4 (0.13 g, 1.0 mmol). The reaction mixture was stirred at RT for 18 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (Method B) to give compound 102-5e (0.39 g, 55% yield) as a white solid. ESI m/z: 702.2 (M+H)+.

3-({2-[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16oxo- 6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}carbonyl)(methyl)amino]ethyl }disulfanyl)-3-methylbutanoic acid (102-5f)

Following the procedure for 102-5e, except substituting 102-3f for 102-3e, afforded 102-5f (0.29 g, 40% yield) as a white solid. ESI m/z: 716.3 (M+H)+.

2,5-dioxopyrrolidin-1-yl 3-({2-[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11hydroxy-9 ,13-dimethylamine-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17dien-8-yl]-2-oxoethoxy }carbonyl)amino]ethyl}disulfanyl)-3-methylbutanoate (LP5)

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To a solution of compound 102-5e (0.20 g, 0.29 mmol) in DCM (10 ml), HOSu (73 mg, 0.64 mmol) and EDCI (0.12 g, 0.64 mmol) were added and the mixture was stirred at RT for 24 h, which was monitored by LCMS. The resulting mixture was diluted with DCM (50 ml) and the organic solution was washed with water and brine, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was purified by prep-HPLC (Method A) to give LP5 (85 mg, 37% yield) as a colorless oil. ESI m/z: 799.3 (M+H)+.

2,5-dioxopyrrolidin-1-yl-3-({2-[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11hydroxy- 9,13-dimethylamine-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17dien-8-yl]-2- oxoethoxy}carbonyl)(methyl)amino]ethyl}disulfanyl)-3-methylbutanoate (LP6)

Following the procedure for LP5 except substituting 102-5f for 102-5e, LP6 (86 mg, 36% yield) was obtained as a colorless oil. ESI m/z: 813.3 (M+H)+.

endo-bicyclo[6.1.0]non-4-yn-9-ylmethyl N- {2-[2-(2- {2-[3-( {2-[( {2[(1S,2S,4R, 8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ⁴ ' ⁸ .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2oxoethoxy}carbonyl)amino]ethyl}disulfanyl)-3methylbutanamido]ethoxy}ethoxy)ethoxy]ethyl}carbamate (LP7)

HATU (0.11 g, 0.29 mmol) and DIPEA (75 mg, 0.58 mmol) were added to a solution of compound 102-5e (0.20 g, 0.29 mmol) in DMF (10 ml). The reaction mixture was stirred at RT for 10 min and then compound 5f (0.11 g, 0.29 mmol) was added. The reaction mixture was stirred at RT for 18 hours, which was monitored by LCMS. The resulting mixture was directly purified by prep-HPLC (Method B) to give LP7 (0.16 g, 55% yield) as a white solid. ESI m/z: 526.7 (M/2+H)+.

Ethoxyendo-bicyclo[6.1.0]non-4-yn-9-ylmethyl N- {2-[2-(2- {2-[3-( {2-[( {2[(1 S,2S,4R ,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,72.9 /\4_8 /^13.18-1 dioxapentacyclo [10.8.0.0 ' .0 ' .0 ' ]icosa-14,17-dien-8-yl]-2-oxoethoxy}carbonyl)(methyl)amino]ethyl}disulfanyl)-3-methylbutanamido]ethoxy}ethoxy)ethoxy ]ethyl}carbamate (LP8)

Following the procedure for LP7 except substituting 102-5f for 102-5e, LP8 (0.13 g, 41% yield) was obtained as a white solid. ESI m/z: 533.8 (M/2+H)+.

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Synthesis of AMO linker loads LP9-11.

Scheme 103A

Synthesis of loads 103a,b and linker loads LP9-10

(2-(((9H-fluoren-9-yl)methoxy)carbonylamino)acetamido)methyl acetate (103-1)

To a mixture of Fmoc-Gly-Gly-OH (4.3 g, 12 mmol) in THF (0.12 L) and toluene (40 ml) was added pyridine (1.2 ml, 15 mmol) and lead tetraacetate (6.8 g, 15 mmol). The reaction mixture was heated at reflux for 5 hours, which was monitored by LCMS. After cooling to RT, the reaction mixture was filtered through celite to remove insoluble material and the filtrate was concentrated in vacuo. The residue was dissolved in ethyl acetate and the solution was washed with water and brine. The organic solution was dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (10% ethyl acetate in petroleum ether) to give 103-1 (3.0 g, 67% yield) as a colorless solid. ESI m/z: 391 (M+Na) ⁺ .

9H-fluoren-9-ylmethyl N-{[( {2-[( 1 S,2S,4R,8S,9S, 11S,12S,13R)-11 -hydroxy-9,13-dimethylamine-162.9 r,4_8 Zl13 .18l oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^, .0 ^, .0 ^, ]icosa-14,17-dien-8-yl]-2oxoethoxy} methyl)carbamoyl]methyl}carbamate (103- 2a)

Potassium tert-butoxide (0.22 g, 2.0 mmol) was added to a solution of compound 103-1 (0.37 g, 1.0 mmol) and budesonide 1a (1.3 g, 3.0 mmol) in THF (4 ml) at 0°C. The reaction mixture was stirred at RT for 15 min, which was monitored by TLC. Ethyl acetate and water were added to the reaction solution at 0°C, and it was extracted using ethyl acetate and chloroform. The combined organic solution was dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (30-35% ethyl acetate in petroleum ether) to give 103-2a (0.19 g, 40% yield) as a white solid. ESI m/z: 739 (M+H)+.

9H-fluoren-9-ylmethyl N-{[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-

2.9 /\4.8 /\13.18l dimethylamine-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^, .0 ^, .0 ^, ]icosa-14,17-dien-8-yl]-2oxoethoxy} methyl)carbamoyl]methyl}carbamate (103-2b)

A solution of compound 103-1 (0.30 g, 0.81 mmol) and compound 100 (0.38

- 153 045111 g, 0.81 mmol) in DCM (4 ml) and pyridinium p-toluenesulfonate (21 mg, 81 µmol) at RT. The reaction vessel was sealed and shaken at 50°C for 24 h, which was monitored by LCMS. After cooling, the reaction mixture was concentrated in vacuo and the residue was purified by prep-HPLC (Method A) to give compound 103-2b (0.23 g, 37% yield) as a white solid. ESI m/z:

775(M+H)+.

2-amino-N-( {2-[(1S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo [10.8.0.0 ² ' ⁹ .0 ⁴ ' ⁸ .0 ^13.18 ] icosa-14,17-dien-8-yl]-2-oxoethoxy} methyl)acetamide (103 a)

Piperidine (35 mg, 0.41 mmol) was added to a solution of compound 103-2a (0.10 g, 0.14 mmol) in DMF (2 ml). The reaction mixture was stirred at RT for 2 h until complete removal of Fmoc was monitored by LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (0-100% acetonitrile in aqueous TFA (0.05%)) to give compound 103a (50 mg, 70% yield) as a white solid. ESI m/z: 517.6 (M+H)+.

2-amino-N-( {2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine16-oxo- 6-propyl-5,7-dioxapentacyclo[10.8.0.0 ² ' ⁹ .0 ^{4 '} 8 .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)acetamide (103b)

Following the procedure for 103a, except substituting 103-2b for 103-2a, afforded 103b (0.26 g, 65% yield) as a white solid. ESI m/z: 553.2 (M/2+H) ⁺ .

NHFmoc

Compound 105(b) was prepared according to the above procedure.

{4-[(2S)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.O ^{4 9} ]hexadekα-1(12),4(9),5, 7,13,15-hexaen-1O-in-2yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methyl N -{[({2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13dimethylamine-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8. 0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)carbamoyl]methyl}carbamate (LP9)

- 154 045111

To a solution of compound 103a (15 mg, 29 µmol) in DMF (1.0 ml) was added compound 11b (30 mg, 28 µmol), HOBT (2.0 mg, 15 µmol) and DIPEA (7.7 mg, 60 µmol). The reaction mixture was stirred at RT for 2 hours, which was monitored by LCMS. The reaction mixture was purified directly by prep-HPLC (Method B) to obtain linker load LP9 (5.0 mg, 11% yield) as a white solid. ESI m/z: 729 (M/2+H)+.

{4-[(2S)-2-[(2S)-2-[1 -(4- {2-zatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7 ,13,15-hexaen-10-in-2-yl} 4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5 (carbamoylamino)pentanamido]phenyl}methyl N -{[({2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl -5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17dien-8-yl]-2-oxoethoxy} ethyl)carbamoyl]methyl}carbamate (LP10)

Following the procedure for LP9, except substituting 103b for 103a, the linker load LP10 (23 mg, 58% yield) was obtained as a white solid. ESI m/z: 747.0 (M/2+H) ⁺ ; 513.8 (cleaved by LCMS, cleaved by NHCH2-Q).

1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.80-8.72 (m, 1H), 8.15 (d, J=6.8 Hz, 1H), 7.89 (d, J=8.8 Hz, 1H) , 7.80-7.75 (m, 1H), 7.68 (d, J=7.6 Hz, 1H), 7.64-7.58 (m, 3H), 7.54-7.22 (m, 9H), 6.29 (d, J=10.0 Hz, 1H ), 6.10 (s, 1H), 6.04-5.96 (m, 1H), 5.78-4.10 (m, 15H), 3.66-3.54 (m, 5H), 3.54-3.42 (m, 13H), 3.32-3.26 (m , 2H), 3.20-2.50 (m, 7H), 2.42-2.20 (m, 4H), 2.05-1.90 (m, 4H), 1.85-1.25 (m, 15H), 0.90-0.80 (m, 13H) ppm.

Scheme 103B

Synthesis of linker load LP11

HOSu, EDCL DCM

H-Gly-Gly-Phe-OH, DIPEA, DMF

(2S)-2-(2- {2-[1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15- hexaen-10-in-2-yl}-4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]acetamido}acetamido)-3-phenylpropanoic acid (103-3)

HOSu (73 mg, 0.64 mmol) and EDCI (0.12 g, 0.64 mmol) were added to a solution of compound 5c (0.16 g, 0.29 mmol) in DCM (20 ml). The mixture was stirred at RT for 24 h until compound 5 was completely destroyed according to LCMS. The resulting mixture was diluted with DCM (50 ml) and the organic solution was washed with water (50 ml) and brine (50 ml), dried with anhydrous sodium sulfate and concentrated in vac

- 155 045111 uo and obtained OSu active ester (0.16 g, 84% yield) as a colorless oil, which was used directly for the next step.

To a solution of H-Gly-Gly-Phe-OH (10 mg, 36 µmol) in DMF (0.5 ml) was added OSu active ester (23 mg, 36 µmol), prepared as described above, and DIPEA (9.0 mg, 72 µmol). The reaction mixture was stirred at RT overnight. The mixture was directly purified by prep-HPLC (Method B) to give 103-3 (15 mg, 51% yield) as a white solid. ESI m/z: 408.2 (M/2+H)+.

-(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-in-2-yl}-4oxobutanamido)- N -{[({[(1S)-1-({[({2-[(19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)carbamoyl]methyl}carbamoyl)-2-phenylethyl]carbamoyl} methyl)carbamoyl]methyl} 3,6,9,12-tetraoxapentadecane-15-amide (LP11)

HATU (14 mg, 36 µmol) and DIPEA (9.3 mg, 72 µmol) were added to a solution of compound 103-3 (15 mg, 18 µmol) in DMF (0.5 ml). The reaction mixture was stirred at RT for 10 min and then compound 103b (10 mg, 18 µmol) was added to the mixture. The reaction mixture was then stirred at RT overnight. The resulting mixture was purified directly by prep-HPLC (Method B) to obtain linker load LP11 (5.0 mg, 20% yield) as a white solid. ESI m/z: 420.2 (M/3+N) ⁺ .

Dipeptide prodrug synthesis: linker-load LP12

Scheme 104A

Load synthesis 104a

Alpha-azidoisobutyric acid 104-1 was synthesized according to Org. Lett, 2001, 3(5), 781-783.

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-azido-2methylpropanoate (104-2a)

To a solution of compound 100 (0.10 g, 0.21 mmol) in DCM (30 ml), compound 104-1 (52 mg, 0.40 mmol), DCC (90 mg, 0.44 mmol) and DMAP (10 mg) was added and the mixture was stirred at RT for throughout the night, which was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (Method A) to give compound 104-2a (80 mg, 70% yield) as a white solid. ESI m/z: 578.3 (M+H) ⁺ .

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6 propyl-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl methylpropanoate (104-3 a)

2-amino-2-

To a solution of compound 104-2a (75 mg, 0.15 mmol) in THF (5 ml) was added PPh ₃ (0.15 g, 0.57 mmol), water (5 ml) and conc. HCl (1 drop) and the reaction mixture was stirred at RT overnight.

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The mixture was concentrated in vacuo and diluted with DMF. The precipitate was removed by filtration. The filtrate was purified by prep-HPLC (Method A) to give compound 104-3a (45 mg, 54% yield) as a white solid. ESI m/z: 552.3 (M+H)+.

2-[( 1 S,2S,4R,8S,9S,11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-[(2S)-2-{[(tert-butoxy)carbonyl ] amino} -4-methylpentanamido]-2-methylpropanoate (104-4a)

AhU o \/ /=о ⁰

DIPEA (30 mg, 0.23 mmol), DMAP (2 mg) and compound 104-3a ( 30 mg, 54 µmol). The mixture was stirred at RT for an hour, which was monitored by LCMS. The mixture was directly purified by prep-HPLC (Method A) to give compound 104-4a (25 mg, 55% yield) as a white solid. ESI m/z: 787.4 (M+Na) ⁺ .

2-[( 1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-[(28)-2-amino-4methylpentanamido]-2 -methylpropanoate (104a)

To a mixture of compound 104-4a (25 mg, 33 μmol) in DCM (1.5 ml) was added TFA (0.15 ml) and the mixture was stirred at RT for an hour until complete removal of Boc by LCMS. The reaction mixture was concentrated in vacuo and the residue was purified by prep-HPLC (Method A) to give compound 104a (13 mg, 61% yield) as a white solid. ESI m/z: 665.4 (M+H) ⁺ .

1H NMR (400 MHz, DMSO-06) δ 8.39 (s, 1H), 8.26 (s, 1H), 7.27 (d,J=10.0 Hz, 1H), 6.31 (d, J=10.1 Hz, 1H), 6.11 (s, 1H), 5.63 (t, J=24.2 Hz, 2H), 5.30-5.05 (m, 1H), 4.96 (d, J=17.7 Hz, 1H), 4.81-4.65 (m, 2H), 4.20 ( s, 1H), 2.69-2.54 (m, 1H), 2.36-2.15 (m, 1H), 2.12-1.94 (m, 2H), 1.85-1.63 (m, 2H), 1.63-1.53 (m, 3H), 1.521.41 (m, 11H), 1.41-1.10 (m, 6H), 0.97-0.76 (m, 12H) ppm.

Circuit 104B

Synthesis of linker load LP12

- 157 045111 tert-butyl (2S)-2- {[({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2- {[(9H-fluoren-9ylmethoxy)carbonyl]amino }-3-methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]amino}-4methylpropanoate (104-5)

DIPEA (0.26 g, 2.0 mmol) was added to a solution of compound 11b (0.30 g, 0.39 mmol) and H-Leu-OTBU-OH (0.14 g, 0.63 mmol) in DMF (3 mL), and the reaction mixture was stirred at RT for 2 hours. The resulting mixture was directly purified by prep-HPLC (Method A) to give compound 104-5 (0.11 g, 35% yield) as a white solid. ESI m/z: 815.4 (M+H)+.

(2S)-2-{[({4-[(2S)-5-(carbamoylamino)-2-[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3methylbutanamido] pentanamido]phenyl}methoxy)carbonyl]amino}-4-methylpentanoic acid (104-6)

To a solution of compound 104-5 (25 mg, 31 µmol) in DCM (4 ml) was added TFA (0.8 ml) and the mixture was stirred at RT for 2 hours. The resulting mixture was concentrated in vacuo and the residue was purified by prep-HPLC (method A ) to obtain 104-6 (15 mg, 67% yield) as a white solid. ESI m/z: 759.3 (M+H) ⁺ .

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-[(2S)-2-{[({4[( 2S)-5-(carbamoylamino)-2-[(2S)-2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-3methylbutanamido]pentanamido]phenyl}methoxy)carbonyl]amino}-4-methylpentanamido ]-2methylpropanoate (104-7 a)

To a solution of compound 104-5 (20 mg, 26 µmol) and compound

104-3 (17 mg, 32 µmol) in DMF (2 ml), HATU (20 mg, 52 µmol), DIPEA (13 mg, 0.10 mmol) and DMAP (1 mg) were added and the reaction mixture was stirred at RT for an hour, which was monitored via LCMS. The resulting mixture was directly purified by prep-HPLC (Method A) to give compound 104-7a (30 mg, 89% yield) as a white solid. ESI m/z: 1293 (M+H) ⁺ .

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-[(2S)-2-{[({4[( 2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl]amino}-4-methylpentanamido]-2-methylpropanoate (104-8a)

To a solution of compound 104-7a (25 mg, 19 µmol) in DMF (2 ml) was added piperidine (0.2 ml) and

- 158 045111 the mixture was stirred at RT for half an hour until complete removal of Fmoc according to LCMS. The resulting mixture was directly purified by reverse phase flash chromatography (0-50% acetonitrile in aq. TFA (0.03%)) to give compound 104-8a (20 mg, 95% yield) as a white solid. ESI m/z: 1070.5 (M+H)+.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-[(2S)-2-{[({4[( 2S)-2-[(2S)-2-[1-({[endo-bicyclo[6.1.0]non-4-yn-9-ylmethoxy]carbonyl}amino)-3,6,9,12tetraoxapentadecane-15 -amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl]amino}-4-methylpentanamido]-2methylpropanoate (LP12)

HATU (22 mg, 58 µmol), DIPEA (15 mg, 0.12 mmol) and DMAP ( 1 mg) and the reaction mixture was stirred at RT for 2 hours, which was monitored by LCMS. The resulting mixture was purified directly by prep-HPLC (Method A) to obtain linker load LP12 (10 mg, 21% yield) as a white solid. ESI m/z: 747.6 (M/2+H) ⁺ .

¹ H NMR (400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.35-8.25 (m, 1H), 8.11 (d, J=7.4 Hz, 1H), 7.87 (d, J=8.6 Hz, 1H ), 7.58 (d, J=8.2 Hz, 2H), 7.34-7.18 (m, 4H), 7.17-7.04 (m, 1H), 6.30 (d, J=10.1 Hz, 1H), 6.11 (s, 1H) , 6.03-5.90 (s, 1H), 5.72-5.50 (m, 2H), 5.41 (s, 2H), 5.34-3.91 (m, 12H), 3.66-3.38 (m, 15H), 3.15-2.88 (m, 4H), 2.73-2.53 (m, 1H), 2.50-2.45 (m, 1H), 2.41-2.31 (m, 1H), 2.30-1.90 (m, 9H), 1.83-1.62 (m, 2H), 1.63- 1.17 (m, 27H), 0.92-0.77 (m, 20H) ppm.

Scheme 104C

Synthesis of load 104b and its linker load LP13

O ^Z NH ₂ 104-8b

LP13 ^{0 NH} 2

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6- 159 045111 propyl-5 ,7-dioxapentacyclo[10.8.0.0 ² ' ⁹ .0 ⁴ ' ⁸ .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2-oxoethyl fluoren-9-ylmethoxy)carbonyl] amino} -4 -methyl pentanoate (104-2b) (2S)-2-{[(9H-

EDCI (0.23 g, 1.2 mmol) and DMAP (12 mg, 0.10 mmol) were added to a solution of compound 100 (0.47 g, 1.0 mmol) and Fmoc-Leu-OH (0.39 g, 1.1 mmol) in DCM (10 ml). The mixture was stirred at RT for 16 hours, which was monitored by LCMS. The resulting mixture was diluted with water (50 ml) and extracted with DCM (50 ml x 2). The combined organic solution was dried over sodium sulfate and concentrated in vacuo. The residue was purified by silica gel column chromatography (10-50% ethyl acetate in petroleum ether) to give 104-2b (0.52 g, 65% yield) as a white solid. ESI m/z: 802.4 (M+H) ⁺ .

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl (2S)-2-amino-4methylpentonoate (104-3b)

Piperidine (1 mL) was added to a solution of compound 104-2b (0.50 g, 0.62 mmol) in DCM (9 mL) and the reaction mixture was stirred at RT for 30 min until complete removal of Fmoc by LCMS. The resulting mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography (5-10% methanol in DCM) to give 104-3b (0.32 g, 90% yield) as a white solid. ESI m/z: 580.3 (M+H) ⁺ .

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl (2S)-2-[(2S)-2{[(tert -butosy)carbonyl]amino}-3-hydroxypropanamido]-4-methylpentanoate (104-4b)

HATU (0.25 g, 0.66 mmol) and DIPEA (0.21 g, 1.6 mmol) were added to a mixture of compound 104-3b (0.32 g, 0.55 mmol) and Boc-Ser-OH (0.14 g, 0.66 mmol) in DMF (3 mL). with CT. The mixture was stirred at RT for 6 hours, which was monitored by LCMS. The resulting mixture was concentrated in vacuo and the residue was purified by silica gel column chromatography (10-50% ethyl acetate in petroleum ether) to give compound 104-4b (0.34 mg, 80% yield) as a white solid. ESI m/z: 767.4 (M+H)+.

- 160 045111

2-[( 1 S,2S,4R,8S,9S,11S,12R, 13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl (2S)-2-[(2S)-2amino-3- hydroxypropanamido]-4-methylpropanoate trifluoroacetic acid salt (104b)

To a solution of compound 104-4b (0.34 g, 0.44 mmol) in DCM (5 ml) was added TFA (1 ml). The reaction mixture was stirred at RT for 2 h until Boc was completely removed according to LCMS. The resulting mixture was concentrated and the residue (0.33 g, 95% yield) was sufficiently pure for the next step. 100 mg of crude product was further purified by prep-HPLC (Method A) to give compound 104b (80 mg, 80% yield) as a yellow solid. ESI m/z: 667.3 (M+H)+.

2-[( 1 S,2S,4R,8S,9S,11S,12R, 13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl (2S)-2-[(2S)-2-{[ ({4[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methoxy)carbonyl] amino} -3-hydroxypropanamido]-4-methylpropanoate (104 -8b)

To a solution of fresh crude compound 104b (0.10 g, 0.13 mmol) prepared as described above in DMF (3 ml) was added Fmoc-vcPAB-PNP (11d) (0.12 g, 0.16 mmol), DMAP (16 mg, 0.13 mmol) and DIPEA (50 mg, 0.39 mmol) at CT. The mixture was stirred at RT for 3 hours until most of the starting materials were destroyed, which was monitored by LCMS. Piperidine (1 ml) was then added to the reaction mixture. The mixture was then stirred at room temperature for an hour until complete removal of Fmoc according to LCMS. The resulting mixture was purified directly by prepHPLC (Method B) to give 104-8b (28 mg, 20% yield) as a white solid. ESI m/z: 536.8 (M/2+H) ⁺ .

2-[( 1 S,2S,4R,8S,9S,11S,12R, 13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl(2S)-2-[(2S)-2-{[ ({4[(2S)-2-[(2S)-2-[1 -({[endo-bicyclo[6.1.0]non-4-in-9-ylmethoxy]carbonyl}amino)-3,6, 9,12tetraoxapentadecane-15-amido]-3-methylbutanamido]-5 (carbamoylamino)pentanamido]phenyl}methoxy)carbonyl]amino}-3-hydroxypropanamido]-4methylpropanoate (LP13)

To a solution of compound 5d (10 mg, 22 µmol) in DMF (1 ml) HATU (9 mg, 22 µmol) and DIPEA (8.0 mg, 62 µmol) were added sequentially at RT. The mixture was stirred at RT for half an hour and a solution of compound 104-8b (20 mg, 19 µmol) in DMF (1 ml) was added. The resulting mixture was stirred at RT for 2 hours until the destruction of compound 104-8b, which was monitored by LCMS. After membrane filtration, the filtrate was directly purified by prep-HPLC (Method B) to obtain linker load LP13 (10 mg, 35% yield) as a white solid. ESI m/z: 748.4 (M/2+H) ⁺ .

- 161 045111

Scheme 105

Synthesis of LP18-21

- 162 045111

2,5-dioxopyrrolidin-1-yl 1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaene10-in- 2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-oate (5-1c)

To a solution of compound 5c (160 mg, 0.290 mmol) in DCM (20 mL), HOSu (73.3 mg, 0.637 mmol) and EDCI (122 mg, 0.637 mmol) were added and the mixture was stirred at RT for 24 h, which was monitored by LCMS. The reaction mixture was diluted with DCM (50 ml) and the organic layer was washed with water (50 ml) and brine, dried with anhydrous Na ₂ SO ₄ and concentrated in vacuo to give compound 51c (159 mg, 84% yield) as a colorless oil, which was used directly for the next stage. ESI m/z: 650 (M+H)+.

2,5-dioxopyrrolidin-1-yl 1-[({endo-bicyclo[6.1.0]non-4-yn-9-ylmethoxy}carbonyl)amino]3,6,9,12-tetraoxapentadecane-15-oate ( 5-1d)

Following the procedure for 5-1c, except substituting 5d for 5c, afforded 5-1d (150 mg, 54% yield) as a colorless oil, which was used directly for the next step without further purification. ESI m/z: 539 (M+H) ⁺ .

{4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N{[({2-[(1S,2S,4R,8S ,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^{4, 8} .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)carbamoyl]methyl}carbamate (103-8b)

h ₂ n^o

Following the procedure for 104-8b (see Scheme 104C), except substituting 103b for 104b, afforded 103-8b (28 mg, 20% yield) as a white solid. ESI m/z: 480 (M/2+H) ⁺ .

(4S)-4-amino-4- {[(1S)-1-{[(1 S)-4-(carbamoylamino)-1 -[(4- {[({[({2[(1S,2S ,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl]k arbamoyl}-2methylpropyl] carbamoyl}butanoic acid (103-9a)

HATU (0.19 g, 0.50 mmol) and DIPEA (0.13 g, 1.0 mmol) were added to a solution of the compound Boc-L-Glu(OTBU)-OH (0.15 g, 0.50 mmol) in DMF (5 ml). The reaction mixture was stirred at RT for 10 min, then compound 103-8b (0.48 g, 0.50 mmol) was added to the reaction mixture. The reaction mixture was then stirred at RT for 3 hours until 103-8b was completely destroyed according to LCMS. The mixture was extracted with EtOAc and the combined organic solution was washed with water, dried over anhydrous sodium sulfate and concentrated in vacuo. The residue was dissolved in DCM (10 ml). TFA (2 ml) was added to the solution and the reaction mixture was stirred at RT for 3 hours, which was monitored by LCMS. The reaction mixture was concentrated and the residue was directly purified by prep-HPLC (Method B) to give compound 103-9a (0.41 g, 75% yield) as a white solid. ESI m/z: 536.8 (M/2+H) ⁺ .

(4R)-4-amino-4- {[(1S)-1-{[(1 S)-4-(carbamoylamino)-1 -[(4- {[({[({2- 163 045111

[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo [10.8 .0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2 oxoethoxy}methyl)carbamoyl]methyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl] carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (103-9b)

Following the procedure for 103-9a, except substituting Boc-Z)-Glu(OTBU)-OH for BocL-Glu(OTBU)-OH, provided 103-9b (0.40 g, 74% yield) as a white solid . ESI m/z: 536.8 (M/2+H)+.

(4S)-4-amino-4-{[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-({4-[({[(1S)-1-{[( 2S)-1-{2[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6- propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}-4-methyl-1-oxopentan-2yl ]carbamoyl}-2-hydroxyethyl]carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (104-9a)

Following the procedure for 103-9a, except substituting 104-8b for 103-8b, afforded 104-9a (0.39 g, 65% yield) as a white solid. ESI m/z: 601.3 (M/2+H) ⁺ .

(4R)-4-amino-4-{[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-({4-[({[(1S)-1-{[( 2S)-1-{2[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6- propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}-4-methyl-1-oxopentan-2yl ]carbamoyl}-2-hydroxyethyl]carbamoyl}oxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (104-9b)

Following the procedure for 103-9a, except for replacing 104-8b instead of 103-8b, Boc-DGlu(OTBU)-OH instead of Boc-L-Glu(OTBU)-OH, we obtained compound 104-9b (0.39 g, 65% yield) as a white solid. ESI m/z: 601.3 (M/2+N) ⁺ .

(4S)-4-[1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1 (12),4(9),5,7,13,15-hexaene-10-yne- 2-yl}-4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-4-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[( 4{[({[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo -6propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)carbamoyl]methyl}carbamoyl)oxy ]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (LP18)

- 164 045111

To a solution of compound 103-9a (57 mg, 53 µmol) in DMF (1 ml) was added compound 5-1c (36 mg, 56 µmol) and DIPEA (27 mg, 0.21 mmol). The reaction mixture was stirred at RT for 4 hours, which was monitored by LCMS. The resulting mixture was then directly purified by prep-HPLC (Method B) to obtain LP18 (12 mg, 15% yield) as a white solid. ESI m/z: 811.4 (M/2+H)+.

(4R)-4-[1 -(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1 (12),4(9),5,7,13,15-hexaene-10-yne- 2-yl} -4oxobutanamido)-3,6,9,12-teraoxapentadecane-15-amido]-4- {[(1S)-1-{[(1 S)-4-(carbamoylamino)-1 -[( 4{[({[({2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo -6propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2oxoethoxy}methyl)carbamoyl]methyl}carbamoyl)oxy ]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (LP19)

Following the procedure for LP18, except substituting 103-9b for 103-9a, LP19 (14 mg, 17% yield) was obtained as a white solid. ESI m/z: 811.4 (M/2+H) ⁺ .

(4S)-4-{1-[( {endo-bicyclo[6.1.0]non-4-yn-9-ylmethoxy} carbonyl)amino]-3,6,9,12tetraoxapentadecane-15-amido}-4- {[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-({4-[({[(1S)-1-{[(2S)-1-{2[(1S ,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2 .9} .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}-4-methyl-1-oxopentan-2yl]carbamoyl}-2-hydroxyethyl]carbamoyl} hydroxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (LP20)

To a solution of compound 104-9a (64 mg, 53 µmol) in DMF (1 ml) was added compound 5-1d (30 mg, 56 µmol) and DIPEA (27 mg, 0.21 mmol). The reaction mixture was stirred at RT for 4 hours, which was monitored by LCMS. The resulting mixture was then directly purified by prep-HPLC (Method B) to obtain LP20 (17 mg, 20% yield) as a white solid. ESI m/z: 813.0 (M/2+H) ⁺ .

(4R)-4-{1-[( {endo-bicyclo[6.1.0]non-4-yn-9-ylmethoxy} carbonyl)amino]-3,6,9,12tetraoxapentadecane-15-amido}-4- {[(1S)-1-{[(1 S)-4-(carbamoylamino)-1-({4-[({[(1S)-1-{[(2S)-1-{2[(1S ,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difloro-11-hydroxy-9,13-dimethylamine-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2 .9} .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}-4-methyl-1-oxopentan-2yl]carbamoyl}-2-hydroxyethyl]carbamoyl} hydroxy)methyl]phenyl}carbamoyl)butyl]carbamoyl}-2methylpropyl]carbamoyl}butanoic acid (LP21)

- 165 045111

Following the procedure for LP20, except substituting 104-9b for 104-9a, LP21 (14 mg, 17% yield) was obtained as a white solid. ESI m/z: 813.0 (M/2+H)+.

In the table below. Figure 5a shows linker loads prepared using the methods described here.

Table 5 a

Examples of linker loads

LP (LN) Load Linker name Structures LP1 101a DIBAC-sucPEG4vcPABMEDA-100 F H Η nA O 5 hAHam /O I ! Yh ϊ ρρο^Ν^ ^Ν γ ^ο ^° ^on O on ⁿ o ί ⁿ Sih cYnh ₂ LP2 101b DIBAC-sucPEG4vcPABDMEDA-100 F n N Ngd /O 9 ; Υ n; γ \n 0 S ^{n n} ί 1 ⁿ %n cYnh ₂ LP3 101s DIBAC-sucPEG4vcPABDEEDA-100 О ьШК ιί ) о j ^^ofOtCr ⁰ C ^N i° ^{0 he} 0 1 ⁿ %n cY _NH2 LP4 101d DIBAC-sucPEG4vcP AB-Pip100 O O A o and γ n i? |Сс° ^l о °7p>% ^F 1 x ° Ν ΝΗ ₂ LP5 102e NHSdisulfideSEA-100 Ш /° St I ° лЛ Υ°7= ^ο / о ZI ω W 5 с>р LP6 102f NHS disulfideSEMA-100 /О Оо Н Λύ ⁰ \ / о I <^ ₀ Αχ ₈ _Λ _ο γΜ ⁰ О γ / / κΥγγ

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LP7 102e BCN-PEG3disulfideSEA-100 Η 7 ^H Ο ⁰ \ A _s - ^S \/ Loh o T ΛΑ ^x°>< ^H W HO b ⁿ n r^A-'' ^F Ό LP8 102f BCN-PEG3disulfideSEMA-100 € Η U 0 '°Al^ Ο K L^o H ο 1 У-. XX Λ θΧ ' ¹ ° HO G ⁿ n Y^x.aF O LP9 103a DIBAC-sucPEG4vcPAB-GlyAMO-Bud Η H ^N NH; °>=° °L (7 •'xYrg ⁰ o—< yu θΑ? ^zen o LP10 103b DIBAC-sucPEG4vcPAB-GlyAMO-100 О tL W ⁰ ABOUT Η X/ X—0^ XN NH ₂ Ο-Ά ^H A F ⁴ aLL^v ⁰ ΤΓνΑί ¹ D' OH Ό LP11 103b DIBAC-suc- PEG4-GGFG- AMO-100 -ο Ο ABOUT χ. Η Ο A. /· γ·Α'.Α o o S ⁱ n 7 /A' ^{1F H} ° ^F < A LP12 104a BCN-PEG4vcPAB-LeuAib-100 Ά' ''LA X/ ''/'L I &X LP13 104b BCN-PEG4vcPAB-SerLeu-100 /^^'''Α^Ν-^χχ-Οχ^^θχχ^Ο^^θ/χ Α?γ^ 0^ MH ^x nh ₂ V°v ^H 4aD ⁿ n LP18 103b DIBAC-sucPEG4-EVCPAB-GlyAMO-100 /=\o Η x-HA ο ο > ο ΗΟ^Ο Η A η ₂ ν^4 ill 1 h ABOUT U n H'A'A'·' F AaLLL ⁰ VtvAf ' ⁰ A ¹ ΌΗ [ ⁰ o F LP19 103b DIBAC-sucPEG4-dEVCPAB-GlyAMO-100 47 ⁴ N o 3 ⁰ Η Ν^θ^Οχ nSAo nA η ₂ ν^ο // vA-vA 7 ⁿ S VtvA? ¹ to ¹ he is about

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LP20 104b BCN-PEG4EVC-PABSer-Leu-100 O ^H h »Y ° nsОо Sih oA _N h ₂ LP21 104b BCN-PEG4dEVC-PABSer-Leu-100 o U ^0H o T°X hoOd Sih oAh ₂

Table 6a Characterization data for linker loads

LP (LN) cLogP M.F. M.B. HPLC Purity (%) MC (m/z) 100% Highest peak m/z LP1 4.26 C78H101F2N9O19 1506.71 95 753.9 (m/2+H) 753.9 (m/2+H) LP2 4.48 C79H103F2N9O19 1520.71 97 761.0 (m/2+H) 761.0 (m/2+H) LP3 5.20 C81H107F2N9O19 1548.76 95 775.0 (m/2+H) 775.0 (m/2+H) LP4 4.31 C79H101F2N9O19 1518.69 99 760.0 (m/2+H) 760.0 (m/2+H) LP5 3.40 C37H48F2N2O11S2 798.91 99 799.3 (m+H) 799.3 (m+H) LP6 3.62 C38H50F2N2O11S2 812.94 97 813.3 (m+H) 813.3 (m+H) LP7 5.20 C52H75F2N3O13S2 1052.29 97 526.7 (m/2+H) 526.7 (m/2+H) LP8 5.42 C ₅ 3H77F ₂ N3O13S2 1066.32 98 533.8 (m/2+H) 533.8 (m/2+H) LP9 3.65 C77H101N9O19 1456.70 96 729.0 (m/2+H) 729.0 (m/2+H) LP10 3.35 C77H99F2N9O19 1492.68 95 513.8 (fragment) 747.0 (m/2+H) LP11 1.96 C71H87F2N7O17 1348.51 95 450.2 (m/3+H) 675.2 (m/2+H) LP12 5.61 C76H110F2N8O20 1493.75 90 747.6 (m/2+H) 747.6 (m/2+H) LP13 4.13 C75H108F2N8O21 1495.70 95 748.4 (m/2+H) 748.4 (m/2+H) LP18 2.46 C82H106F2N10O22 1621.77 95 811 (m/2+H) 811 (m/2+H) LP19 2.46 C82H106F2N10O22 1621.77 95 811 (m/2+H) 811 (m/2+H) LP20 3.25 C80H115F2N9O24 1624.81 95 813 (m/2+H) 813 (m/2+H) LP21 3.25 C80H115F2N9O24 1624.81 95 813 (m/2+H) 813 (m/2+H)

Example 2.

This example demonstrates specific procedures for site-specific conjugation of an alkyne linker load to an antibody.

In this example, site-specific conjugates are created in two steps. The first step is the MTG-based enzymatic coupling of a small molecule, such as an azide-PEG ₃ -amine (supra), to an antibody having a Q-tag (eg, WO2017/147542) (hereinafter referred to as MTG-based conjugation). An antibody having an N297Q mutation or an N297D mutation has Q295/Q297 or Q295 that is induced to attach to MTG for enzyme-based site-specific conjugations, where Q295 and/or Q297 have been defined as a Q tag. At the second stage, the linker load is attached to the azido-functionalized antibody through [2+3] cycloaddition, at

- 168 045111 example, 1,3-bipolar cycloaddition between azides and cyclooctynes (also called click chemistry without copper compounds). See documents Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C R. PNAS 2007, 104. (43), 16793-7, the contents of which are incorporated herein in their entirety by reference, where the reactive group (RG) is a DIBAC moiety, conjugation is performed with an azido-functionalized antibody via [2+ 3] cycloaddition. This process produces site-specific and stoichiometric conjugates.

ADC conjugation via [2+3] click reaction.

Step 1. Preparation of azido-functionalized antibody.

IgG aglycosylated human antibody (IgG1, IgG4, etc.) or human IgG1 isotype with N297Q mutation (EU nomenclature), in PBS (pH 6.5-8.0) mixed with >200 molar equivalents of azido-dPEG ₃ -amine (MW = 218.26 g/mol). The resulting solution is mixed with MTG (EC 2.3.2.13 from Zedira, Darmstadt, Germany, or Modernist Pantry [L# 210115A] - ACTIVA TI contains maltodextrin from Ajinomoto, Japan) (25 U/ml; 5 U MTG per mg antibody), resulting which results in a final antibody concentration of 0.5-5 mg/ml, and then the solution is incubated at 37°C for 4-24 hours with gentle shaking. The reaction is monitored by ESI-MS. Once the reaction is complete, excess amine and MTG are removed by SEC or protein A column chromatography to yield an azido-functionalized antibody. This product is characterized by SDSPAGE and ESI-MS. Azido-dPEG ₃ -amine is added to two antibody sites, resulting in an increase of 204 Da for the 2DAR antibody-PEG ₃ -azide conjugate.

Step 2. Preparation of site-specific drug-antibody conjugates using click chemistry.

Site-specific antibody-drug conjugates with human IgG (IgG1, IgG4, etc.) in table. 7 is prepared via a [2+3] click reaction between azido-functionalized antibodies and an alkyne-containing linker load. The following describes the detailed conjugation procedure. Site-specific linker-loaded (LP) antibody conjugate is prepared by incubating mAb-PEG3-N3 (1-3 mg/ml) in an aqueous medium (eg, PBS, PBS with 5% glycerol, HBS) with >6 molar. equivalents of LP dissolved in a suitable organic solvent such as DMSO, DMF or DMA (i.e. the reaction mixture contains 5-20% organic solvent, v/v) from 24°C to 37°C for more than 6 hours The reaction progress is monitored by ESI-MS and the absence of mAb-PEG ₃ -N ₃ indicates completion of conjugation. Excess LP and organic solvent are removed by SEC via PBS elution or by Protein A column chromatography via acid buffer elution followed by neutralization with Tris (pH8.0).

The final product is concentrated by ultra-centrifugation and characterized by UV, SEC, SDS-PAGE and ESI-MS.

Example 3.

This example demonstrates a method for performing non-site-specific conjugation of a drug to an antibody using a thiol-maleimide reaction.

Conjugation through antibody cysteines is performed in two steps using methods similar to those described in Mol Pharm. 2015 Jun 1; 12(6): 1863-71.

Monoclonal antibody (mAb, 10 mg/ml in 50 mM HEPES, 150 mM NaCl) pH 7.5 is reconstituted with 1 mM dithiothreitol (0.006 mg per mg antibody) or TCEP (2.5 molar antibody equivalent) at 37°C for 30 min. Antibody concentration was calculated based on the absorbance at 280 nm on a Nanodrop (ThermoFisher Scientific) and the extinction coefficient of the antibody. After gel filtration (G-25, pH 4.5 sodium acetate), compound LP in DMSO (10 mg/ml) was added to the reconstituted antibody and the pH of the mixture was adjusted to pH 7.0 using 1 M HEPES (pH 7.4). The mixture was allowed to react for 314 hours. The resulting conjugate was purified by SEC. DAR values are determined using measured ncADC absorbance and antibody and compound LP extinction coefficients.

Example 4.

ADC conjugation.

In one example, site-specific conjugates are prepared by two-step conjugation of an antibody with the N297Q or N297D mutation based on a microbial transglutaminase (MTG) (MTG EU 2.3.2.13, Zedira, Darmstadt, Germany) (here MTG-based). In the first step, an antibody with the N297Q mutation is functionalized with an azido-PEG ₃ -amine through an MTG-based enzyme reaction. See, for example, PCT International Patent Application No. PCT/US17/19537, filed February 24, 2017, the contents of which are incorporated herein by reference in their entirety for all purposes. In the second step, the alkyne-functionalized linker load is added to the azido-functionalized antibody via a [2+3] 1, 3-bipolar cycloaddition reaction with conjugation to the azido-functionalized antibody obtained via [2+3] cyclization). This process produces site-specific and stoichiometric conjugates.

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Example 5.

The following example describes a method for preparing the azido-functionalized antibody-drug conjugate shown in Table 1. 7.

N297Q mutated AB Azido-functionalized antibody

ABOUT

Ab-DIBACT-PEG ₄ -VC-PAB- _H arpy3Ka

Aglycosylated antibody with the human IgG1 isotope in BupH™ (pH 7.6-7.8) was mixed with >200 molar. equivalents of azido-dPEG ₃ -amine (MB 218.26 g/mol). The resulting solution was mixed with transglutaminase (25 units/ml; 5 units of MTG per mg of antibody), resulting in a final antibody concentration of 0.5-3 mg/ml, and then the solution was incubated at 37°C for 4-24 hours with gentle shaking. The reaction was monitored by SDS-PAGE or ESI-MS. Upon completion of the reaction, excess amine and MTG were removed by size exclusion chromatography (SEC) to obtain the azido-functionalized antibody. This product was analyzed by SDS-PAGE and ESI-MS. Azido-dPEG ₃ -amine was added to two sites - Q295 and Q297 - of the antibody, which gave an increase of 804 Da for the 4DAR aglycosylated antibody-PEG ₃ -azide conjugate.

The following example demonstrates a method for performing site-specific drug-antibody conjugation using click chemistry reactions.

Site-specific aglycosylated antibody-drug conjugates with human IgG1 harboring the N297Q mutation were prepared via a [2+3] click reaction between azido-functionalized antibodies with an alkyne-containing linker load. Specifically, anti PRLR AbPEG3-N3 was conjugated to 2b, 2g, 2l, 2r, or 2s; and anti-HER2 Ab-PEG ₃ -N ₃ conjugated to 2b, 2g, 2l, 2r, or 2s. These anti-HER2 antibody conjugates were synthesized to act as isotype controls in the assays described below. In the following, for convenience, anti-HER2 antibodies are referred to as isotopic antibodies.

The detailed conjugation procedure is described below. Site-specific linker-loaded antibody conjugate (LP or 2) was prepared by incubating mAb-PEG ₃ -N ₃ (1-3 mg/ml) in an aqueous medium (eg, PBS, PBS with 5% glycerol, HBS) with >6 molar equivalents of LP dissolved in a suitable organic solvent such as DMSO, DMF or DMA (i.e. reaction mixture contains 5-20% organic solvent, v/v) at 24°C to 37°C for more than 6 hours. The reaction was monitored by ESI-MS, and the absence of mAb-PEG3N3 indicated completion of conjugation. Excess LP and organic solvent were removed by SEC via PBS elution or by Protein A column chromatography via acid buffer elution followed by neutralization with Tris (pH8.0). Purified conjugates were analyzed by SEC, SDS-PAGE and ESI-MS. In table 7 provides a list of steroidal antibody conjugates from the corresponding LPs, their molecular weights and ESIDAR values.

In a specific example, an azido-functionalized antibody (1 mg) in 0.800 ml PBSg (PBS, 5% glycerol, pH 7.4) was exposed to 6-molar. equivalents of 2b (conc. 10 mg/ml in DMSO) for 6–12 h at RT and excess linker load (LP) was removed by size exclusion chromatography (SEC, Superdex 200 HR, GE Healthcare). The final product was concentrated by ultra-centrifugation and characterized by UV, SEC, SDS-PAGE and ESI-MS.

Example 6.

Characterization of ADCs.

SDS-PAGE is used to analyze the integrity and purity of ADCs.

In one method, SDS-PAGE conditions include unreduced and reduced samples (2-4 μg) along with a prestained protein marker reference set (Invitrogen, cat. no. 10748-010; lot no. 1671922.) placed in lane at (1.0 mmx10 holes) Novex 4 - 170 045111

20% Tris-glycine gel and the process is performed at 180 V, 300 mA for 80 min. The unreduced sample was prepared using Novex Tris-glycine SDS buffer (2x) (Invitrogen, cat.

No. LC2676), and the reconstituted sample was prepared using SDS sample buffer (2x) containing 10% 2-mecaptoethanol.

The molecular weight of antibodies and ncADCs by SDS-PAGE was determined for non-reducing and reducing conditions. Mass shifts may not be obvious under non-reducing conditions due to the relatively small percentage of mass changes. However, the heavy chain masses increase from naked antibodies to azido-functionalized antibodies and further to the ncADC conjugate. Cross-linked material may or may not be detected.

ADCs are analyzed for purity by size exclusion chromatography (SEC).

Size exclusion chromatography is performed to determine the purity of antibody-drug conjugates. SEC analytical experiments are performed using a Waters 600 instrument, on a Superdex 200 HR column (1.0x30cm), flow rate 0.80 ml/min using PBS pH 7.4, and monitored at λ280 nm using a Waters 2998 PDA. The test sample consists of 200 µl PBS (pH 7.4) with 30-100 µl of test sample. Preparative SEC purifications are performed using an ACTA instrument from GE Healthcare, on a Superdex 200 PG column (2.6x60 cm), at a flow rate of 2 ml/min, eluting with PBSg at pH 7.4 and monitored at λ280 nm.

The antibody and ADC are analyzed by mass analysis of the parent material using LCESI-MS, which is described below.

Precursor mass measurements of ADC samples by LC-ESI-MS were performed to determine the drug-loading distribution profile and calculate the average DAR ratio. Each test sample (20-50 ng, 5 µl) was placed in an Acquity UPLC Protein BEH C4 column (10K psi, 300 A, 1.7 µm, 75 µmx100 mm; cat. no. 186003810). After 3 min of desalting, the protein was eluted and mass spectra were obtained using a Waters Synapt G2-SL mass spectrometer.

In table 7 provides a list of budesonide-spacer-linker-antibody conjugates from the corresponding LPs (2b, 2g, 2l, 2r, and 2s), their molecular weights of naked antibodies, azido-functionalized antibodies, LPs and steroidal ADCs, and ESI-DAR values. In table Ab means antibody, Ab-N ₃ means azido-functionalized antibody, and Ab-steroid conjugates mean noncytotoxic steroid antibody conjugates with the corresponding LPs. As indicated in table. 7, most site-specific ADCs have 4DAR.

Table 7

LP (LN) Ab, Ab-N3, or Ab-steroid conjugates EX MS m/z Name MB (Yes) DAR Anti-PRLR Ab 144602 - PEG3-N3 218.3 Anti-PRLR Ab-N3 145385 4 2b 1369.7 Anti-PRLR Ab-2b 150894 4 2g 1426.7 Anti-PRLR Ab-2g 151097 4 21 1373.7 Anti-PRLR Ab-21 150884 4 2g 729.8 Anti-PRLR Ab-2r 148330 4 2s 809.8 Anti-PRLR Ab-2s 148623 4 Ahth-HER2 (isotype) Ab 145126 - PEG3-N3 218.3 Isotype Ab -N3 145930 4 2b 1369.7 Isotype Ab-2b 151439 3.8 2g 1426.7 Isotype Ab-2g 151652 4 21 1373.7 Isotype Ab-21 151445 3.9 2r 729.8 Isotypic Ab -2r 148876 4 2s 809.8 Isotype Ab-2s 149169 4

Example 7.

To analyze the time-dependent activation of GR by budesonide and the steroids described here, a cell-based glucocorticoid receptor (GR) co-activator luciferase reporter gene assay was performed.

To create a cell line for analysis, the HEK293 cell line was constructed for

- 171 045111 expression of human full-length PRLR. This stable cell line was then further transfected with pBIND-GR vector (Cat. No. E1581, Promega) and pGL4.35 [Luc2P/9xGAL4UAS/Hygro] vector (Cat. No. E1370, Promega) so that the luciferase gene was transcribed upon steroid binding on GR. The resulting stable cell line is called here HEK293/PRLR/GRE-Luc.

The activity of steroids in HEK293/PRLR/GRE-Luc cells was studied after 24, 48 and 72 hours of incubation after the addition of the test reagents. For this assay, 20,000 cells were plated in 96-well plates in DMEM supplemented with 10% FBS and penicillin/streptomycin (complete medium) and cultured overnight at 37°C in 5% CO ₂ . To generate dose response curves for free steroids and/or their ncADCs, serially diluted reagents from 100 nM to 5.1 pM were added to the cells and incubated for 24, 48, and 72 h at 37°C. Luciferase activity was determined by adding 100 μl of One-Glo™ reagent (Promega, cat. no. E6130), incubating at room temperature for 5 min, and relative light units (RLUs) were measured on a Victor luminometer (Perkin Elmer).

In the first step of the analysis of anti-PRLR Ab ncADC (Anti-PRLR Ab-2r, Anti-PRLR Ab-2s and Anti-PRLR Ab-2b), isotype. Ab ncADC (isotype Ab-2r, isotype Ab-2s and isotype Ab-2b) and budesonide free load were analyzed in the HEK293/PRLR/GRE-Luc cell assay using the protocol described above with incubation for 24, 48 or 72 h during one analysis step. In the second step of the analysis of anti-PRLR Ab ncADC (anti-PRLR Ab-2b, anti-PRLR Ab-2l, and anti-PRLR Ab-2g), isotype. Ab ncADC (isotype Ab-2b, isotype Ab-2l and isotype Ab-2g), antibodies only (anti-PRLR Ab and isotype Ab) and budesonide free load were analyzed in the HEK293/PRLR/GRE-Luc cell assay using the 72 hour incubation protocol described above. EC ₅₀ values were determined using a four-parameter 10-point logistic equation using GraphPad Prism. Steroid delivery caused activation of the Luc reporter in HEK293/PRLR/GRE-Luc cells.

Fig. 1A-C and table. 8 illustrates the results of the first stage of analysis. As shown in FIG. 1A-C, the anti-PRLR Ab-2b conjugate induced activation of HEK293/PRLR/GRE-Luc cells at 24, 48 and 72 h, with an EC ₅₀ value of less than <10 nM at all three time points and showing >15- multiple selectivity relative to the control isotype-AL-2b. The anti-PRLR Ab-2s conjugate induced activation in HEK293/PRLR/GRE-Luc cells at 24, 48 and 72 h, however, it did not show selectivity for the isotype control-2s. The anti-PRLR Ab-2r conjugate induced activation in 293/PRLR/GRE-Luc cells with <5-fold selectivity at 24 and 48 h, but increased activation only at 72 h with an EC ₅₀ of 13.4 nM.

At the second stage of the analysis, which is shown in Fig. 2 and in table. 9, the anti-PRLR Ab-2b conjugate induced activation in HEK293/PRLR/GRE-Luc cells, with an EC ₅₀ of 0.48 nM and showed approximately 14-fold selectivity over the control isotype-Ab-2b. The anti-PRLR Ab-2g conjugate induced activation in HEK293/PRLR/GRE-Luc cells with an EC ₅₀ of 1.9 nM with approximately 3.7-fold selectivity over the isotype control-Ab-2g. The anti-PRLR conjugate Ab-2l induced activation in HEK293/PRLR/GRE-Luc cells with an EC ₅₀ of 8.98 nM, while isotype-AB-21 did not demonstrate activation in this assay.

Table 8

Molecule HEK293/PRLR/GRE-Luc cells ECso (24 h) ECso (48 h) ECso (72 h) Anti-PRLR Ab-2s 0.9078 0.4239 0.4823 Anti-PRLR Ab-2r 34.98 18.33 13.35 Anti-PRLR Ab-2b 1.225 0.4674 0.4291 Isotype Ab-2s 1.461 0.6594 0.4904 Isotype Ab-2r 109.2 64.01 310.9 Isotype Ab-2b 15.04 8.201 5.243 la 0.5732 0.5679 1.043

Table 9

Steroid ADCs HEK293/PRLR/GRE-Luc cells ECso (nM) after 72 hours Anti-PRLR AB-2b 0.29 Anti-PRLR Ab-21 8.98

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Example 8.

This example describes a mouse model of LPS-induced cytokine release.

The purpose of the present study was to evaluate the test compounds for inhibition of LPS-induced cytokine release in mice. Test compounds were administered 48 hours, 24 hours and 2 hours before the LPS assay, and levels of cytokines in blood samples, including TNF-α and IL6, were measured 2 hours and 4 hours after the LPS assay.

Materials and reagents.

Lipopolysaccharide (LPS) derived from E. Coli K12 can be purchased from Invivogen (San Diego, CA, USA, cat. no. Tlrl-eklps), Dexamethasone can be purchased from ADAMAS (Emeryville, CA, USA, cat. no. 50-02-2). The mouse TNF-α ELISA kit is available from ebioscience (ThermoFisher Scientific, cat. #88-7324). The mouse IL6 ELISA kit is available from ebioscience (ThermoFisher Scientific, cat. #88-7064).

Experimental methods.

Animals.

A total of 18 drug-naïve C57BL/6j mice were used in this study. These animals were males, weighing 18-20 g at the start of the study. Animals were purchased from Shanghai Laboratory Animal Center, CAS (SLAC), and housed in the ChemPartner vivarium in a pathogen-free environment. Upon arrival, the animals were examined for health status, including coat, limbs, orifices, and abnormal signs of posture or movement, and were acclimatized to the environment for more than 7 days.

The animals were placed 3 mice per polycarbonate cage with individual ventilation in an environment free from pathogenic flora; The environmental controls in the cells were set to maintain a temperature of 20-26°C, humidity of 40-70%, and a 12-hour light/12-hour dark cycle.

Standard food (SLAC-M01, from Shanghai Laboratories Animal Center) and purified water (filtered municipal drinking water quality) were provided ad libitum throughout the study period.

Experimental procedures.

Group allocation: animals were randomly assigned to 6 groups (A-F) before the start of the study. Each group included 3 mice. Group A served as a naïve control group; Group B received dexamethasone and served as a positive control; Group C received the test compound, and Groups D-F received the test compounds.

Experimental procedure.

All mice received LPS dissolved in PBS at a dose of 0.5 mg/kg via intraperitoneal injection. Mice in group A received PBS, mice in group B received Dex (5 mg/kg), and mice in group C received 4b (5 mg/kg) by intraperitoneal injection, 2 hours before the LPS challenge; mice in groups D, E, and F received a dose of 5 mg/kg via intraperitoneal injection 2 h, 24 h, and 48 h before LPS challenge, respectively.

Blood samples were taken 2 hours and 4 hours after LPS challenge, in tubes with heparin. Blood samples were centrifuged, plasma samples were collected and stored at -80°C until analysis.

Plasma TNFa levels were measured using ELISA kits according to standard procedures. Various PK parameters were measured (eg, _Tmax , _Cmax , limit t1 _/2 , AUC _last and AUCINF).

Blood samples were collected 2 h after LPS challenge; measured plasma TNF-α levels. Data were expressed as mean ±8EM (mean error), *p<0.05, **p<0.01 vs Group A, by one-way analysis of variance (ANOVA).

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Example 9.

Mouse dendritic cells.

To determine the effect of the test compound on ex-vivo LPS-induced inflammatory immune responses, CD11c ⁺ dendritic cells (DCs) were isolated from the spleens of wild-type C57B1/6 mice (Jackon Labs, Protocol #426.0). DCs from spleens were isolated using Collagenase D digestion (400 U/ml collagenase D (Roche cat. no. 11088858001), 20 μg/ml DNase I (Roche cat. no. 10104159001), 2% FCS in HEPES-buffered RPMI-1640) and incubated at 37°C for 25 minutes. After incubation, spleen tissue was washed with RPMI-1640 and filtered through a 70 μm filter, then erythrocyte lysis was performed using ASA lysis buffer (Gibco cat. no. A1049201) for 1 min. The cell suspension was subsequently washed twice using RPMI-1640. Classical DCs were isolated from mononuclear cell suspensions using CD11c magnetic microbeads (Milteny Biotec cat. no. 130108338). To summarize the process, the cell suspension was washed twice using autoMACS running buffer (Milteny Biotec cat. no. 130091221) prior to 30 min incubation at 4°C with CD11c ⁺ microbeads, according to validated Milteny Biotec protocols. CD11c ⁺ cells were isolated by positive selection, washed, suspended in complete RPMI medium [RPMI-1640 (ThermoFisher Scientific, cat. no. 15140122) containing 10% FBS (ThermoFisher Scientific, cat. no. 10082147) and 1% penicillin-streptomycin (ThermoFisher Scientific, Cat. No. 11875093)] and performed quantitative analysis before culturing 2x105 cells per well. Control complete RPMI medium, complete RPMI medium with compound of Formula (III) (at 10 nM and 100 nM) or complete RPMI medium with dexamethasone (Sigma, cat. no. D4902-25 mg) (at 10 nM and 100 nM) were added to the cells into a 96-well culture dish. DC/control, cells exposed to test compound or dexamethasone were incubated for 24 hours at 37°C before stimulation with 10 ng/ml LPS for 24 hours.

Human dendritic cells.

To determine the effect of the test compound on ex-vivo LPS-induced inflammatory immune responses in human innate immune system cells, CD14+ monocytes (Lonza cat. no. 2W-400C) were isolated and cultured in the presence of complete RPMI medium [RPMI-1640 (ThermoFisher Scientific, cat. 15140122) containing 10% FBS (ThermoFisher Scientific, cat. no. 10082147) and 1% penicillin-streptomycin (ThermoFisher Scientific, cat. no. 11875093)] supplemented with human IL4 (50ng/ml) (Milteny Biotec, cat. no. 130-093-922) and human GM-CSF (100 ng/ml) (Milteny Biotec, cat. no. 130093866) for 7 days. Complete RPMI medium with IL4 and GM-CSF was changed every three days. Two specific cultivation conditions are created:

condition 1: incubation of CD14+ monocytes with RPMI complete medium control, RMPI complete medium with test compound (with 10 nM and 100 nM) or RMPI complete medium with dexamethasone (Sigma) (with 10 nM and 100 nM) for 7 days of culture, or condition 2: incubate CD14+ monocytes with control complete RPMI medium for 5 days before incubation with control complete RPMI medium, complete RPMI medium with test compound (with 10 nM and 100 nM) or complete RPMI medium with dexamethasone (Sigma, cat. no. D4902-25 mg) (with 10 nM and 100 nM) until day 7. On day 7, different experimental groups were stimulated with 10 ng/ml LPS for 24 hours.

Measurement of cytokines in supernatants 24 h after LPS exposure ex vivo.

Supernatants were collected into 96-well round-bottom tissue culture plates 24 h after LPS challenge and stored at −20°C until further analysis. Cytokine concentrations in supernatants were measured using the Proinflammatory Panel 1 (mouse) multiplex immunoassay kit (MesoScale Discovery, cat. no. K15048D) according to the manufacturer's instructions or the Pro-inflammatory Panel 1 (human) multiplex immunoassay kit. (MesoScale Discovery, cat. no. K15049D). Briefly, 50 μL/well of calibration markers and samples (diluted in Diluent 1:2) are added to precoated antibody plates and incubated at room temperature with shaking at 700 rpm for 2 hours. The plates are then washed. 3 times using 1xPBS containing 0.05% (w/v) Tween-20, followed by the addition of 25 μl of antibody detection solution diluted in Diluent 45. After 2 hours of incubation at room temperature with shaking, the plates are washed 3 times and each 150 µl of 2x reading buffer is added to the well. Electrochemiluminescence is then immediately determined on an MSD Spector® instrument. Data analysis is performed using GraphPad Prism™ software. Statistical significance within groups was determined by one-way Anova with Turkey's post hoc multiple comparison test and standard error of the mean (SEM±) calculated. The production of IL2p70, IL1β, IL6, KC-GRO and TNF-α by CD11c ⁺ DCs was measured. The expression of IL2p70, IL1β, IL6, and TNF-α was measured by DCs derived from human monocytes.

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Example 10.

Stability in plasma 103a.

Plasma stability can be measured according to the following protocol.

Preheated 0.05 M sodium phosphate and 0.07 M NaCl buffer, pH 7.4: 14.505 g/L Na ₂ HPO ₄ -12H2O, 1.483 g/L NaH ₂ PO ₄ -2H2O and 4.095 g/L NaCl were dissolved in deionized water. The stock solution was then titrated with phosphoric acid to pH 7.40 and stored refrigerated for up to 7 days. The pH was checked on the day of the experiment and adjusted if the external characteristics were 7.4 ± 1.

Plasma preparation: Frozen plasma was thawed at 37°C. The plasma was centrifuged at 3000 rpm for 8 min to remove clots, pipetted, and the supernatant was collected as plasma to be used in the experiment. Plasma pH was checked and recorded, and plasma was used only in the pH range of 7.4 to 8. If the pH level exceeded 8, the plasma was discarded. The initial plasma pH was not adjusted to pH 7.4 with acid or CO2 sparging. When using an incubator of 5% CO2 and PBS buffer, pH of 7.4 reached equilibrium dialysis after 4 hours. Plasma was placed on ice until used.

Additive solution of test compounds and reference compounds. 0.5 mM Test Compound Addition Solution A: 10 μL of a 10 mM test compound stock solution was added to 190 μL of DMSO. 0.02 mM Additive B Solution: 40 μL Additive A Solution was added to 960 μL of 0.05 mM Sodium Phosphate Buffer with 0.5% BSA.

The plasma and solution of additive B were preheated at 37°C for 5 minutes. 10 μl of prewarmed solution of additive B was added to the wells selected for all time points (0, 0.25, 1, 8, 24 h). For 0-min, 400 μl of IS-containing ACN was added to the wells of the 0-min plate and then 90 μl of plasma was added. 90 μl of prewarmed plasma was added to the wells assigned to the time points (0.25, 1, 8, 24 h) and the timing was started. After 0.25, 1, 8, 24 h, 400 μL of IS-containing ACN was added to the wells of the corresponding plates, respectively, to stop the reaction. After cooling, the plates were shaken on a shaker (PSA, MTS 2/4) for 10 min (600 rpm) and then centrifuged at 5594 g for 15 min (Thermo Multifugex3R). 50 μL of supernatant from each well was transferred to a 96-well sample plate containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for LC/MS analysis.

Stability data in human plasma for compound 103a are shown in the table below.

O

Test sample Remaining percentage (%) T1/2 (h) 0h 0.25h 1h 8h 24h Procaine 100.00 NPKO NPKO NPKO NPKO <0.25 103a 100.00 107.04 96.48 78.87 38.03 16.79 Area ratio Budesonide released from 103a 0.054 0.052 0.042 0.047 0.049

LLOQ: below the limit of quantitative detection.

Example 11.

pH stability of linker load.

Because the solubility of load 103a,b and their linker loads LP9,10 are low (<0.02 mg/mL aq DMSO (20%)), the pH stability of these compounds was not analyzed. LP9 was cooled with CD-N3 to obtain the following compound QLP9, which was water soluble and used for the pH stability test.

He

Synthesis of compound QLP9: To a solution of LP9 (5 mg, 3.4 µmol) in DMF (1.0 ml) compound was added

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CD-N ₃ (7.0 mg, 6.8 µmol). The reaction mixture was stirred at RT overnight, which was monitored by LCMS. The reaction mixture was purified directly by prep-HPLC to give QLP9 (5 mg, 60% yield) as a white solid. ESI m/z: 819.0 (M/3 + N) ⁺ .

pH stability in buffers: The stability of QLP9 in DMSO, pH 5.0, 7.4 and 8.5 PBS buffers (containing 20% DMSO) was analyzed and the compound was found to be stable in such solutions after 7 days.

Example 12.

Cellular analysis of the glucocorticoid receptor (GR) luciferase reporter gene.

A cell-based GR luciferase reporter gene assay was used to analyze the GR agonist activity of the compounds described herein. The following materials were used in this analysis.

Vector information (if applicable) HEK293 cells ATSS, cat. No. CRL-1573 HEK293/UAS-Luc/pBIND GR Culture media: DME high glucose, 10% FBS, 100 U/ml penicillin, 100 mg/ml, streptomycin, 53 µg/ml glutamine, 100 µg/ml hygromycin, 500 µg/ml G418 DME high glucose, 500 ml Irvine Scientific #9033 Fetal bovine serum Saradigm# 1500-500 L-glutamine solution of penicillin-streptomycin 100X Gibco #10378-016 Vector pGL4.35[luc2p/9XGAL 4UAS/rHrpo] Promega, cat. No. El37A Vector pBIND-GR, selectable marker is neomycin Promega, cat. No. El58A Hygromycin-B 100 mg/ml InvivoGen#ant-hg-5 G418 100 mg/ml GIBCO #11811-098 Nunclon delta 96-well immunoassay plate, sterile, white, flat bottom Thermo Scientific #13610 Analysis medium: Same as culture medium DMSO dimethylamine sulfoxide, for cell culture ATCC. #4-x. ONE Gio Promega, cat. No. E6130 Device Tablet reader Envision, Perkin Elmer Breeding plates Axygen Scientific, cat. No. P-DW-ll-C-S Trypsin-EDT A Millipore cat. No.SM-2004-C PBS IX without calcium and magnesium salts Irvine Scientific #9240 Test compound 1a Sigma/1278201/RO36WO Test compound 100 Test compound 105a Test compound 103a Test compound 103b Test compound Dexamethasone Sigma/D 1756-25MG/BCBV3214

To obtain a cell line for analysis, HEK293 cells were co-transfected with the vectors pBindGR (Promega, cat. no. E158A) and pGL4.35 [Luc2P/9xGAL4UAS/hygro] (Promega, cat. no. B137A). The pBind-GR vector expresses a fusion protein consisting of a yeast Gal4DNA-binding domain and a GR ligand-binding domain, which can bind to the Gal4 activating sequence (AS) in the luciferase expression vector and induce luciferase expression upon binding to a GR agonist. Cell selection was performed for a minimum of 2 weeks in G418+ hygromycin. The resulting cell line is called HEK293/UASLuc/pBIND-GR.

Cells were seeded in 96-well plates (20,000 cells/well) in assay medium (DME with high

Claims (38)

high glucose supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 53 µg/ml glutamine, 500 µg/ml G418 and 100 µg/ml hygromycin) one day before analysis. Three-fold serial dilutions of test compounds were prepared in 100% DMSO, transferred to fresh assay medium, and added to cells. The final concentration of DMSO was kept constant at 0.2%, and the final concentrations of the test compounds varied from 100 nM to 0.015 nM. The last well in the plate served as a blank control containing only assay medium and 0.2% DMSO (untreated well) and was plotted as a continuation of the 3-fold serial dilution. Forty-eight hours later, luciferase activity was determined after the addition of OneGlo™ reagent (Promega, cat. no. E6130). Relative light units (RLUs) were measured on an Envision luminometer (PerkinElmer), and EC ₅₀ values were determined using a four-parameter logistic equation from a ten-point dose response curve (GraphPad Prism). The signal to noise ratio (S/N) was determined using the ratio max. OSE on the dose response curve to OSE in untreated wells. As shown in the table below, EC ₅₀ values ranged from 51.8 pM to 3.24 nM, and S/N values ranged from 12.4 to 17.3. The test compound 100 was 5.6 times more effective compared to 1a (budesonide) and 5.9 times more effective than Dexamethasone. Spacer addition to 1a to produce 103a gave EC ₅₀ values comparable to 1a. Similarly, spacer addition to 100 to produce 103b gave _EC50 values comparable to 100. Spacer addition to 100 to produce 105a gave _EC50 values that were 2.7 times less efficient than 100. S/N values were comparable for of all tested compounds. GR reporter activity under the influence of GR agonists. ___________ Test compound ECso (M) S/N Dexamethasone 3.24E-09 17.3 1a 3.06E-10 15.3 103a 4.84E-10 14.6 100 5.47E-11 14.5 103b 5.18E-11 16.6 105b 1.49E-10 12.4 The embodiments and examples described above are for illustrative purposes only and are non-limiting. Those skilled in the art are familiar with methods for performing standard experimental work, many equivalents of specific compounds, materials and procedures. All such equivalents are deemed to come within the scope of the appended claims. CLAIM 1. Compound of formula (III) (P1); or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ⁴ represents C1- _C6 alkyl; both R ^x are hydrogen or both R ^x are fluorine; And SP is -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₁₀ -alkylene)-NR ^50a -, where the terminal NR ^50a is attached to L; R ⁵⁰ and R ^50a are independently hydrogen or C1-C ₆ alkyl; z is an integer selected from 1 to 30 inclusive; L is a linker of the formula -L1-L ² -(L ³ ) _0-1 -; where L ¹ is connected to BA and L ³ is connected to SP; L ¹ represents a reactive group residue selected from or him - 177 045111 regioisomer; or a regioisomer thereof; or where S refers to the sulfur atom on the cysteine residue through which the reactive group residue is added to BA, and O L, where N refers to the nitrogen atom on the lysine residue, through which the residue of the reactive group is connected to BA; L ² is a binding linker; L ³ , if present, is a self-cleaving linker; And BA is an antibody, an antibody fragment, or an antigen-binding fragment thereof. 2. Compound of formula (II) (P); or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ⁴ represents C1-C6 alkyl; both R ^x are hydrogen or both R ^x are fluorine; SP represents -(C ₁ -C ₁₀ -alkylene)-NR ⁵⁰ C(O)-(C ₁ -C ₁ o-alkylene)-NR ^50a -, where terminal NR ^50a joins (L ³ ) _0-1 ; R ⁵⁰ and R ^50a are independently hydrogen or C1-C6 alkyl; RG is a reactive group selected from L ² represents a connecting linker; And L ³ , if present, is a self-cleaving linker. 3. A compound according to claim 1 or 2, where R ⁴ represents a linear or branched C1-C6 alkyl. 4. The connection according to any of claims 1-3, where R ⁴ is in the (^-configuration. 5. A compound according to any one of claims 1 to 4, where SP is -(linear C ₁ -C ₃ -alkylene)NR ⁵⁰ C(O)-(linear C ₁ -C ₃ -alkylene)-NR ^50a -. 6. A compound according to any one of claims 1 to 5, wherein R ⁵⁰ and R ^50a are independently hydrogen or methyl. 7. A compound according to any one of claims 1 to 5, wherein R ⁵⁰ and R ^50a in each case represents hydrogen. 8. A compound according to any one of claims 1 to 5, wherein R ⁵⁰ and R ^50a is in each case methyl. 9. A compound according to any one of claims 1 to 8, wherein both R ^x represent hydrogen. 10. A compound according to any one of claims 1 to 8, wherein both R ^x represent fluorine. 11. A compound of formula (III) according to any one of claims 1 and 3-10, where z is selected from 1 to 10 inclusive. 12. A compound of formula (III) according to any of claims 1 and 3-11, where z is selected from 2 to 4 inclusive. 13. A compound of formula (III) according to any one of claims 1 and 3-12, where z is equal to 4. 14. A compound of formula (III) according to any one of claims 1 and 3-12, where z is equal to 2. 15. The compound of formula (III) according to any one of claims 1 and 3-14, where BA contains an antibody or antigen-binding fragment. 16. A compound of formula (III) according to any one of claims 1 and 3-15, where BA contains an antibody having the following the following structure ^z , where Ab is a monoclonal antibody, a polyclonal antibody, an antibody fragment or a bispecific antibody; R represents C _2-4 alkylene, n represents an integer from 1 to 10 inclusive, and z represents an integer from 1 to 10 inclusive. 17. A compound of formula (III) according to any one of claims 1 and 3-16, where BA contains an antibody having the following - 178 045111 blowing structure An Ab is a monoclonal antibody, polyclonal antibody, antibody fragment, or bispecific antibody; R represents C _2-4 alkylene; and n is an integer selected from 2 to 4 inclusive. 18. A compound of formula (III) according to any one of claims 1 and 3 to 17, wherein the BA contains an antibody or an antigen binding fragment thereof that binds macrophage scavenger receptor 1 (MSR1). 19. A compound of formula (III) according to any one of claims 1 and 3-18, where L represents -L1-L ² , where L ¹ is attached to BA and L ² is attached to SP; and where L ¹ represents a reactive group residue and L ² represents a connecting linker. 20. A compound of formula (III) according to any one of claims 1 and 3 to 18, where L represents -LYE ³ -. 21. A compound of formula (III) according to claim 19 or 20, where L ¹ is selected from or regioisomer; or regioisomer; and where S denotes the sulfur atom on the cysteine residue through which the reactive group residue is added to BA. 22. A compound of formula (II) according to any one of claims 2 to 10, where RG is selected from 23. A compound of formula (II) according to any one of claims 2 to 10, where RG represents 24. A compound of formula (II) according to any one of claims 2 to 10, where RG represents 25. A compound of formula (II) according to any one of claims 2 to 10, where RG represents 26. A compound according to any one of claims 1 to 21, where for a compound of formula (III) L represents -L ¹ - ABOUT L ² -(L ³ )0_i-; and where L ³ in a compound of formula (II) or (III) is ' where -NH group is attached to L ² when SP is -(Cl-Cl0-alkylene)-NR ⁵⁰ C(O)-(Cl-Cl ₀ -alkylene)NR ^50a -; or L ³ is absent in the compound of formula (II) or (III). 27. A compound according to any one of claims 1 to 26, where for a compound of formula (III) L represents -L ¹ L ² -(L ³ )0-i-; and wherein L ² in the compound of formula (II) or (III) contains a PEG, a dipeptide residue, a tripeptide residue, a tetrapeptide residue, a pentapeptide residue, -C(O)CH2CH ₂ C(O)-, a cyclodextrin (CD) residue, or -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ C(O) - or any combination thereof. 28. A compound according to any one of claims 1 to 26, where for a compound of formula (III) L represents -L ¹ L ² -(L ³ ) _0- i-; and where L ² in the compound of formula (II) or (III) contains - 179 045111 or a cyclodextrin residue (CD); or any combination thereof. 29. A compound according to any one of claims 1 to 26, where for a compound of formula (III) L represents -L ¹ L ² -(L ³ ) _0-1 -; and where L ² in the compound of formula (II) or (III) contains or a cyclodextrin (CD) residue, or any combination thereof. 30. The connection according to any one of claims 27-29, where the CD is selected from the group consisting of - 180 045111 31. A compound according to any one of claims 1 to 25, where for a compound of formula (III) L is -L ¹ L2-(L ³ )0-1-; and where -L2-(L ³ )0-1- in a compound of formula (II) or (III) contains - 181 045111 - 182 045111 - 183 045111 32. Compound of formula (I) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R ⁴ represents -C1-C ₆ -alkyl; a) both R ^x are hydrogen; And R ³ represents -(C1-C10-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁₀ -alkylene)-NR ^50a R ^50b ; or b) both R ^x are fluorine; And R ³ represents -(C1-C1o-alkylene)-NR ⁵⁰ C(O)-(C1-C ₁ o-alkylene)-NR ^50a R ^50b ; and in each case ^R50 , ^R50a and ^R50b are independently hydrogen or C1- _C6 alkyl. 33. The compound according to claim 32, where R ⁴ represents C1-C ₆ -alkyl. 34. The connection according to claim 32 or 33, where R ⁴ is in the ^-configuration. 35. The compound according to any one of claims 33, 34, where both R ^x represent hydrogen. 36. The compound according to any one of claims 33, 34, where both R ^x represent fluorine. 37. Compound one of the following formulas: or 38. A method of treating a disease, disorder or condition associated with glucocorticoid receptor signaling, comprising administering to a patient with said disease, disorder or a mixture thereof; - 184 -