EA045579B1 - STEROIDS AND THEIR CONJUGATES WITH PROTEINS - Google Patents

Description

Technical field of the invention

Presented herein are novel steroids, their protein conjugates, and methods of treating diseases, disorders, and conditions involving administration of the steroids and conjugates.

State of the art

Antibody-drug conjugates (ALCs) 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 efficacy of small molecule drugs. The therapeutic utility of antibody-drug conjugates has been confirmed in the treatment of cancer and is a major ongoing goal of research. ADCETRIS® (bentruximab vedotin) and KADCYLA® (adotrastuzumab emtansine) are antibody-drug conjugates approved for the treatment of certain types of cancer, and there are at least forty antibody-drug conjugates currently in clinical development.

Glucocorticoids (GCs) are low molecular weight steroids that bind to glucocorticoid receptors (GCRs) and are used in anti-inflammatory and immunosuppressive therapy. However, due to the universal expression of glucocorticoid receptors in many cell types, glucocorticoid treatment has been questioned due to toxicity to most organ systems. Thus, there is a need for both new glucocorticoids and new treatments that minimize the side effects encountered when glucocorticoids are administered, especially the effects resulting from activation of glucocorticoid receptors in non-target cells. The present invention provides solutions to meet the above needs as well as other needs in the field to which it relates. Included in the present invention are antibody-drug conjugates containing glucocorticoid payloads.

The essence of the invention

Provided herein are compounds and methods useful for treating various diseases, disorders or conditions. In certain aspects, a compound with the structure of formula (A) is provided:

R ³ (A);

or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof,

R ⁴ o^o where R ¹ and R ² together form ' where R ⁴ represents -C _1-20 alkyl;

R ³ represents -NR ^a R ^b ,

R ⁵ in each instance is independently -OH, halo, -C _1-20 alkyl or -C _6-20 arylC _1-20 alkyl and each R ⁵ is located on any ring atom;

R ^a and R ^b are each independently -H, -C _1-20 alkyl or -C _6-20 aryl, where -C _6-20 aryl is unsubstituted or substituted with an amino group; or

R ^a and R ^b cyclize to form heterocycloalkyl with three to six ring atoms, including one heteroatom, which is N, to which they are attached; and n is an integer from 0 to 19.

In certain aspects, the compounds are protein-drug conjugates, such as antibody-drug conjugates containing an antigen-binding protein, such as an antibody and a compound of Formula (A).

In some aspects, the compounds are protein-drug conjugates, for example, antibody-drug conjugates containing an antigen-binding protein, eg, an antibody, a compound of Formula (A), and a cyclodextrin group.

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Brief description of illustrations

In fig. 1 shows the sequence of synthesis of certain steroids described herein.

In fig. Figure 2 shows the sequence for changing the position of budesonide on the primary alcohol.

In fig. Figure 3 shows the sequence for changing the primary alcohol position of flumethasone.

In fig. Figure 4 shows the sequence for changing the position of dexamethasone on the primary alcohol.

In fig. Figure 5 shows a two-dimensional nuclear Overhauser effect (NOE) magnetic resonance spectrum (hereinafter 2D-NOESY spectrum) for compound 7-1R in table. 1.

In fig. Figure 6 shows the 2D-NOESY spectrum for compound 7-1S in Table. 1.

In fig. Figure 7 shows the 2D-NOESY spectrum for compound 11-5R in the table. 1.

In fig. Figure 8 shows the 2D-NOESY spectrum for compound 11-5S in the table. 1.

In fig. Figure 9 shows general approaches to synthesizing payload-containing linkers.

In fig. 10 shows the synthesis sequence of DIBAC-Suc-NHS (compound (V)).

In fig. 11 shows the synthesis sequence of DIBAC-Suc-PEG ₄ -acid/NHS (compound (VI)).

In fig. 12 shows the synthesis sequence of BCN-PEG4-Acid (compound (VII)).

In fig. 13 shows the synthesis sequence of DIBAC-Suc-PEG4-VC-pAB-PNP (compound (VIII)).

In fig. 14 shows the sequence for synthesizing a linker containing payload 1 (LP1).

In fig. 15 shows the synthesis sequence of linker containing payload 2 (LP2) and linker containing payload 3 (LP3).

In fig. 16 shows the sequence of synthesis of linkers containing payload 4-11 (LP4-LP11).

In fig. 17 shows the sequence for synthesizing a linker containing payload 12 (LP12).

In fig. 18 shows the sequence of synthesis of linker containing payload 12 (LP13) and linker containing payload 14 (LP14). In fig. Figure 19 shows the general process for the synthesis of an ALS conjugate via a [2+3] click reaction with the LP4 linker.

In fig. 20 shows a Coomassie-stained SDS-PAGE gel of an antiprolactin receptor antibody (anti-APR antibody), an azido-functionalized anti-APR antibody, and an anti-APR antibody-linker LP4 conjugate as described in Example 59.

In fig. 21 shows size exclusion chromatography (SEC) of an anti-APR antibody, an azido-functionalized antibody, and a 4DAR anti-APR antibody-LP4 linker conjugate as described in Example 59.

In fig. 22 shows electrospray ionization mass spectrometry (ESI/MS) of an anti-APR antibody, an azido-functionalized antibody, and an anti-APR antibody-linker LP4 conjugate as described in Example 59.

In fig. 23 shows selective activation of GCR in 293/PRLR/GRE-Luc cells (Fig. 23A) and 293/MMTV-Luc cells (Fig. 23B) by ALS steroid conjugates and budesonide control as described in Example 64.

In fig. 24 shows the involvement of the payload-containing linker in the activation of GCR by the steroid conjugate ALS and the control of budesonide tested in 293/PRLR/GRE-Luc cells as described in Example 65.

In fig. 25 shows the activation of the glucocorticoid receptor in the cell line HEK293/MMTVluc/IL-2Ry/IL7R by budesonide, 11-5 in table. 1, and ALS anti-IL2Ry ncADC conjugate for twenty-four (24), forty-eight (48), or seventy-two (72) hours as described in Example 66.

In fig. 26 shows the sequence for synthesizing the linker containing the payload (LP7).

In fig. 27 shows a synthetic process for the preparation of compound (27b).

In fig. 28 shows the sequence for synthesizing payload-containing linkers (LP15 and LP16).

In fig. Figure 29 shows the general process for the synthesis of an ALS conjugate via a [2+3] click reaction with a linker containing a cyclodextrin (CD) payload.

In fig. 30 shows ALS conjugates with and without cyclodextrin linkers as relative light units (RLU) versus Log ₁₀ [M].

In fig. 31 shows the sequence of synthesis of certain steroids (payloads 1-6) described herein.

In fig. 32 shows the sequence of synthesis of certain linker steroids (LP101LP116).

In fig. Figure 33 shows the general process for the synthesis of ALS conjugate via the [2+3] click reaction.

In fig. 34 shows ESI/MS of anti-APR antibodies, anti-PRLR Ab-N ₃ , and anti-PRLR-LPs.

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In fig. 35 shows ESI/MS of anti-Fel dl Ab, anti-Fel dl Ab-PEG ₃ -N ₃ , and anti-Fel dl Ab-LPs.

In fig. 36 shows the bioactivity of ALS steroid conjugates in antigen-positive cells (293_PRLR_PBind GR/UAS-Luc cells, Fig. 36A) compared to antigen-negative cells (293_PBind GR/UAS-Luc cells, Fig. 36B) as a function of relative light units (OSE) from Log10 [M].

In fig. 37A shows the average blood concentration time for compounds 4b and 6-I.

In fig. 37B shows the level of TNF-α in blood samples of payloads 4b and 6-I, as described in Examples 120-121.

Detailed description

A. Definitions.

As used herein, the term alkyl refers to a monovalent and saturated hydrocarbon radical moiety. Alkyl is optionally substituted and may be linear, branched or cyclic, i.e. cycloalkyl. Alkyl includes, but is not limited to, alkyls that have 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 _1-8 alkyl; 1-6 carbon atoms, i.e. C _1-6 alkyl; and 1-3 carbon atoms, i.e. C _1-3 alkyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 5-butyl, t-butyl, i-butyl, pentyl group, hexyl group, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Alkylene means divalent alkyl.

As used herein, the term haloalkyl refers to alkyl as defined above, where alkyl includes at least one substituent selected from halogen, for example, F, Cl, Br or I.

As used herein, the term alkenyl refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more non-aromatic carbon-carbon double bonds. Alkenyl is optionally substituted and may be linear, branched or cyclic. Alkenyl includes, but is not limited to, alkenyls that have 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. C _2-8 alkenyl; 2-6 carbon atoms, i.e. C _2-6 alkenyl; and 2-4 carbon atoms, i.e. C _2-4 alkenyl. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl and cyclohexenyl. Alkenylene is a divalent alkenyl.

As used herein, the term alkynyl refers to a monovalent hydrocarbon radical moiety containing at least two carbon atoms and one or more carbon-carbon triple bonds. The alkynyl is optionally substituted and may be linear, branched or cyclic. Alkynyl includes, but is not limited to, alkynyls that have 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. C _2-8 alkynyl; 2-6 carbon atoms, i.e. C _2-6 alkynyl; and 2-4 carbon atoms, i.e. C _2-4 alkynyl. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl and butynyl. Alkynylene is a divalent alkynyl.

As used herein, the term alkoxy refers to a monovalent and saturated hydrocarbon radical moiety in which the hydrocarbon includes a single bond to an oxygen atom and where the radical is located on the oxygen atom, for example CH3CH2-O for ethoxy. Alkoxy substituents bond to the compound they replace through this oxygen atom of the alkoxy substituent. Alkoxy is optionally substituted and may be linear, branched or cyclic, i.e. cycloalkoxy. Alkoxy includes, but is not limited to, alkoxy with 1-20 carbon atoms, i.e. C _1-20 alkoxy; 1-12 carbon atoms, i.e. C _1-12 alkoxy; 1-8 carbon atoms, i.e. C _1-8 alkoxy; 1-6 carbon atoms, i.e. C ₁₆ alkoxy; and 1-3 carbon atoms, i.e. C _1-3 alkoxy. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, i-butoxy, pentoxy, hexoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. .

As used herein, the term haloalkoxy refers to alkoxy as defined above, where the alkoxy includes at least one substituent selected from halogen, for example, F, Cl, Br or I.

As used herein, the term aryl refers to a monovalent moiety that is an aromatic radical in which the ring atoms are carbon atoms. Aryl is optionally substituted and may be monocyclic or polycyclic, for example bicyclic or tricyclic. Examples of aryl moieties include aryl moieties that contain from 6 to 20 ring carbon atoms, i.e. C _6-20 aryl; from 6 to 15 ring carbon atoms, i.e. With _6-15 aryl, and from 6 to 10 ring carbon atoms, i.e. From _6-10 aryl. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, and pyrenyl.

As used herein, the term arylalkyl refers to a monovalent moiety that is a radical of an alkyl-containing compound in which the alkyl-containing compound is replaced by an aromatic substituent, i.e. an aromatic compound includes a single bond with an alkyl group, and the radical is localized on the alkyl group. The arylalkyl group binds to the chemical structure shown through the alkyl group. Arilalkyl can be represented by the structure, for example, |sn ₂ . CH ₂ CH ₂

B ^x ' B^ ^CH ₂ B^ ' B^ A/ ' or ^{in CHa}

- 3 045579 where B is an aromatic moiety, for example phenyl. Arilalkyl is optionally substituted, i.e. an aryl group and/or an alkyl group may be substituted as described herein. Examples of arylalkyl include benzyl.

As used herein, the term aryloxy refers to a monovalent moiety that is a radical of an aromatic compound in which the ring atoms are carbon atoms, the ring is replaced by an oxygen radical, i.e. an aromatic compound involves a single bond to an oxygen atom, and the radical is localized on the oxygen atom, for example, in the case of phenoxy.

Aryloxy substituents bind to the compound they replace through this oxygen atom. Aryloxy is optionally substituted. Aryloxy includes, but is not limited to, aryloxy which have from 6 to 20 ring carbon atoms, i.e. C _6-20 aryloxy; from 6 to 15 ring carbon atoms, i.e. With _6-15 aryloxy, and from 6 to 10 ring carbon atoms, i.e. With _6-10 aryloxy. Examples of aryloxy groups include, but are not limited to, phenoxy, naphthoxy and anthroxy.

As used herein, the term R ^a R ^b N-aryloxy refers to a monovalent moiety that is an aromatic radical in which the ring atoms are carbon atoms, the ring is substituted with a R ^a R ^b N substituent and an oxygen radical, i.e. an aromatic compound includes a single bond with a R ^a RbN substituent and a single bond with an oxygen atom, and the radical R ^b R ^a N .

localized on the oxygen atom, for example,

The R ^a R ^b N-aryloxy substituents bond to the compound they replace through this oxygen atom. R ^a R ^b N-aryloxy is optionally substituted. R ^a R ^b N-aryloxy includes, including R ^a RbN-aryloxy, which have from 6 to 20 ring carbon atoms, from 6 to 15 ring carbon atoms; and 6-10 ring carbon atoms. Examples of R ^a R ^b N-aryloxy groups include 4-(dimethyl

amino)phenoxy,

As used herein, the term arylene refers to a divalent moiety of an aromatic compound in which the ring atoms are only carbon atoms. The arylene is optionally substituted and may be monocyclic or polycyclic, for example bicyclic or tricyclic. Examples of arylene moieties include, but are not limited to, arylene moieties that have from 6 to 20 ring carbon atoms, that is, C ₆ to ₂₀ arylene; from 6 to 15 ring carbon atoms, i.e. C ₆ - ₁₅ arylene, and from 6 to 10 ring carbon atoms, i.e. C ₆ - ₁₀ arylene.

As used herein, the term heteroalkyl refers to an alkyl in which one or more carbon atoms are replaced by heteroatoms. As used herein, the term heteroalkenyl refers to alkenyl in which one or more carbon atoms have been replaced by heteroatoms. As used herein, the term heteroalkynyl refers to an alkynyl in which one or more carbon atoms have been replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen and sulfur atoms. Heteroalkyl is optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.

As used herein, the term heteroaryl refers to 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. 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 from 5 to 10 ring atoms. Heteroaryl is optionally substituted.

As used herein, the term heteroarylene refers to an arylene in which one or more ring atoms of the aromatic ring are replaced by an oxygen, sulfur, nitrogen or phosphorus atom. Heteroarylene is optionally substituted.

As used herein, the term heterocycloalkyl refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen, oxygen and sulfur atoms. Heterocycloalkyl is optionally substituted. 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, the term N-containing heterocycloalkyl refers to a cycloalkyl in which one or more carbon atoms are replaced by heteroatoms and wherein at least one heteroatom is a nitrogen atom. Suitable heteroatoms, in addition to nitrogen, include, but are not limited to, oxygen and sulfur atoms. The N-containing heterocycloalkyl is optionally substituted. Examples N

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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, such as an optionally substituted alkyl, means that such moiety is optionally associated with one or more substituents. Examples of such substituents include, but are not limited to, halo, cyano, nitro, haloalkyl, azido, epoxide, optionally substituted heteroaryl, optionally substituted heterocycloalkyl,

LLC o N

I _orA -|-sr ^a -|-nr ^a r ^b R ^A -^OR ^a -NRArB -^. _nr c_U_ _r a _^_U_ _N rArB where R ^a , R ^b and R ^C in each case independently denote a hydrogen atom, alkyl, alkenyl, alkynyl, aryl, alkylaryl, arylalkyl, heteroalkyl, heteroaryl or heterocycloalkyl, or R ^a and R ^b , together with the atoms to which they are bonded, form a saturated or unsaturated carbocyclic ring, wherein the ring is optionally substituted and one or more ring atoms are optionally substituted with a heteroatom. In some embodiments, when the radical moiety is optionally substituted with an optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or an optionally substituted saturated or unsaturated carbocyclic ring, the substituents on the optionally substituted heteroaryl, optionally substituted heterocycloalkyl, or optionally saturated or unsaturated carbocyclic ring, if substituted, are not substituted with substituents which are further optionally substituted with additional substituents. In some embodiments, when a group described herein is optionally substituted, the substituent associated with the group is unsubstituted unless otherwise noted.

As used herein, the term binding agent refers to any bindable molecule having specificity for a given binding partner. In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof.

As used herein, the term linker refers to a divalent moiety that covalently binds a coupling agent to the steroid described herein.

As used herein, the term amide synthesis conditions refers to reaction conditions suitable for facilitating 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 examples, amide synthesis conditions refer to reaction conditions suitable to facilitate the formation of an amide bond between a carboxylic acid and an amine. In some of these examples, the carboxylic acid is first converted to an activated carboxylic acid before the activated carboxylic acid reacts with the amine to form the amide. Suitable conditions for effecting amide formation include, but are not limited to, conditions that employ reagents to react between a carboxylic acid and an amine, including but not limited to dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), (benzotriazol-1-yloxy) tris(dimethylamino)phosphonium hexafluorophosphate (BOP), (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP), (7-azabenzotriazol-1yloxy) 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[bis( dimethylαamino)methylene]1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide (HATU), 2-ethoxy-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 examples, the carboxylic acid is first converted to an activated carboxylic acid ester before reacting with an amine to form an amide bond. In certain embodiments, the carboxylic acid is reacted with a reagent. The reagent activates the carboxylic acid by deprotonating the carboxylic acid and then forming a product complex with the deprotonated carboxylic acid as a result of the nucleophilic attack of the deprotonated carboxylic acid on the protonated reagent. For some carboxylic acids, this activated ester is more sensitive to nucleophilic attack by the amine than the carboxylic acid before its conversion. This leads to the formation of an amide bond. As such, the carboxylic acid is described as activated. Typical reagents include DCC and DIC.

As used herein, the term therapeutically effective amount refers to 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 to delay the development or minimize one or more symptoms associated with a disease or disorder.

As used herein, the term pharmaceutically acceptable derivative refers to any form, such as an ester or prodrug, that provides the compound when administered.

- 5 045579 deniya to the patient.

As used herein, the term pharmaceutically acceptable salt refers to 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, incorporated herein by reference. Examples of salts include, but are not limited to, salts of acidic, basic, organic, inorganic origin

nia, salts of amines and alkali or alkaline earth metals, including, but not limited to, calcium, magnesium, potassium, sodium, 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, etc.

Some groups, moieties, substituents, and atoms are represented by a wavy line that crosses or covers the bond or bonds to indicate the atom through which the groups, moieties, substituents, and atoms are linked. For example, a phenyl group that is substituted with a propyl group, denoted _CH3CH ,

CH, CH ₃ as ³ or ' has the following structure:

Illustrations used herein showing substituents attached to a cyclic group (e.g., aromatic, heteroaromatic, fused ring, and saturated or unsaturated cycloalkyl or heterocycloalkyl) through a bond between ring atoms mean, unless otherwise indicated, that the cyclic group may be substituted by that substituent at any ring position in a cyclic group or in any ring in a fused ring group in accordance with the techniques set forth herein or known in the art to which ^the _present invention relates. For example, a group in which the subscript q means an integer from 0 to 4, the positions of the substituent R ¹ are described in general, i.e., they are not directly related to any vertex of the communication line structure, i.e. with a specific ring carbon atom, includes the following non-limiting examples from groups in which the R ¹ substituent is bonded to a specific ring carbon atom:

to 19, the positions of the substituent R ⁵ are described in general terms, i.e. are depicted as not directly associated with any vertex of the linkage structure, and include the following non-limiting examples of groups in which the R ⁵ substituent is bonded to a specific ring carbon atom:

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As used herein, the expression reactive linker or the abbreviation RL refers to a monovalent group that includes a reactive group and a linking group, illustrated as RG-L—where RL is a reactive group and L is a linking group. A linking group is any divalent moiety that connects a reactive group to a payload. Reactive linkers (RL), together with the payloads to which they are linked, contain intermediates (payload linkers) used as synthetic precursors to prepare the steroidal antibody conjugates described herein. The reactive linker contains a reactive group (RG), which is a functional group or fragment that reacts with a reactive portion of an antibody, modified antibody, or antigen-binding fragment thereof. The portion resulting from the reaction of a reactive group with an antibody, a modified antibody, or an antigen-binding fragment thereof, together with a linking group, comprises a linker binder (BL) conjugate portion described herein. 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 an antigen-binding moiety thereof. In certain embodiments, the reactive group is a functional group or group that is capable of undergoing a chemical click reaction. In some embodiments of the chemical click reaction, the reactive group is 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 alkynes that are suitable for strain-facilitated azidalkyne cycloadditions (SPAAC), cycloalkynes, such as cyclooctynes, benzanulated alkynes, and alkynes capable of undergoing 1,3 cycloaddition reactions with azides in the absence of copper catalysts . Suitable alkynes also include, but are not limited to, DIBAC, DIBO, BARAC, DIFO, substituted, for example, fluorinated alkynes, azacycloalkynes, BCN and derivatives thereof. Payload linkers 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 some embodiments, such a functionalized antibody is prepared by reacting an antibody containing at least one glutamine residue, such as a Q295 heavy chain (EC numbering), with a compound of the formula H ₂ N-LL-N ₃ , where LL represents a group of divalent polyethylene glycol, in the presence of the enzyme transglutaminase.

In some examples, the reactive group is an alkyne, e.g.

’ which can undergo click chemistry reactions, e.g.

_ _ _ _ _ N=N=N - _ ____ with an azide by click chemistry, for example, > forming a product ’ its regioisomer or a mixture of them. In some examples, the reactive group is an alkyne, e.g.

or ’ which can react with an azide by click chemistry, e.g. n-n-n to form a click chemistry product, e.g.

'gn

In some examples, the reactive group is an alkyne, for example, . which ____ ____ _ N=N=N _____ can react with an azide by click chemistry, for example, ’ forming a click chemistry product, for example,

• its regioisomer or mixture thereof. In some examples, reactive u

Υ ^ν 7 group means a functional group, for example, ⁰ ·, which reacts with the cisAL residue „

tein on its antibody or antigen-binding fragment, forming their bond, for example, ⁰ ' where Ab refers to the antibody or antigen-binding fragment, S refers to the S-atom on the cysteine residue through which the functional group is connected to the Ab. In some examples, the reactive group means a functional group, for example ^o

’ which reacts with os-

lysine bond on its antibody or antigen-binding fragment, forming a bond thereof, for example, where Ab refers to the antibody or antigen-binding fragment thereof, N refers to the N-atom on the lysine residue through which the functional group binds to the Ab.

As used herein, the term binding agent linker or BL refers to any divalent group or moiety that links a binding agent (eg, an antibody or an antigen-binding fragment thereof) to a payload compound described herein (eg, a steroid). In general, suitable coupling agent linkers for the antibody conjugates described herein are linkers that are stable enough to utilize the half-life of the antibody in the bloodstream, while still being capable of releasing its payload following 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, for example, cleavage by hydrolysis, reduction, or enzymatic reaction. Non-cleavable linkers are linkers that release the attached payload 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, reducing labile linkers, hyperflammable linkers, and non-cleavable linkers. Suitable linkers also include, but are not limited to, linkers that include or include glucuronides, succinimide thioesters, polyethylene glycol (PEG) units, hydrazones, mal-caproyl units, disulfide units (e.g., -SS-, -SC(R ¹ R ² )-, where R ¹ and R ² independently represent hydrogen or hydrocarbyl), carbamate units, para-amino-benzyl units (PAB), phosphate units, e.g. mono-, bis- or trisphosphate units, and peptide units, e.g. peptide units containing two, three, four, five, six, seven, eight or more amino acids, including, but not limited to, valine-citrulline units. In some embodiments, the binding agent linker (LA) includes a moiety that is formed by the reaction of a reactive group (RG) of a reactive linker (RL) and a reactive portion of a binding agent, such as an antibody, a modified antibody, or an antigen-binding fragment thereof.

In some examples, SL includes the following fragment: 1 of its regioisomers or a mixture thereof, where ⁵ means a connection with a coupling agent. In some examples, the SL includes the following fragment:

’ its regioisomer or mixture thereof, where r denotes a bond with a coupling agent. In some examples, the SL includes the following fragment:

' its regioisomer or mixture thereof, where means a connection with a coupling agent. In some examples, SL includes the following fragment: 1 > its regioisomer or mixture thereof, where ⁵ means a connection with a coupling agent. In some examples, the SL includes the following fragment: ^o > where " means the connection with the cysteine of the antibody or its antigen-binding fragment. In some examples, the SL includes the following fragment: ' where represents a bond to a lysine of an antibody or an antigen-binding fragment thereof. In these examples, communication with the coupling agent is realized directly or through a linker. In certain embodiments, the coupling agent is modified with an azide to facilitate binding to the SL. Examples are described below.

B. Steroids.

Here are compounds having a structure according to formula (A):

(A);

or a pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof, where R ¹ and R ²

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R ⁴

0^0 independently means -H, alkyl, alkylene-C(O)-O-, -OH or halo; or R ¹ and R ² together form > where

R ⁴ is alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl, wherein alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl are in each instance independently and optionally substituted by -NR ^a R ^b ;

R ³ is -OH, R ^Z -C(O)-X-, heteroalkyl, piperidinyl, -NR ^a R ^b , -hydroxyaryl-NR ^a R ^b , or -ZA(R ^p )t;

R ⁵ in each occurrence is independently -OH, halo, alkyl, or arylalkyl;

R ^z is alkyl;

X means O or NR ^a ;

Z means S, S(O), S(O) ₂ , SO2NR ^a , O, C(O)NR ^a , C(O), or NR ^a ;

A means aryl or heteroaryl;

R ^p in each instance is independently halo, optionally substituted alkyl, -OH, or -NR ^a R ^b ;

R ^a and R ^b are each independently -H or optionally substituted alkyl;

n means an integer from 0 to 19; and t means an integer from 1 to 3;

provided that (1) R ³ is not -OH (a) when R ¹ is -OH or (b) when R ¹ and R ² together are R ⁴

0^0 zuyut ' where R ⁴ means C _1-9 alkyl or

Fand (2) R ³ is not

In some embodiments, a compound of formula (A) has a structure of formula (A ¹ ):

r ^5A

(A ¹ ) where R1-R ³ are defined above and R ^5A and R ^5B in each case independently represent a halo or a hydrogen atom.

In some embodiments, compounds of formula (A ¹ ) R ^5A and R ^5B are hydrogen atoms. In some embodiments, compounds of formula (A ¹ ) R ^5A and R ^5B are fluorine. In some embodiments of a compound of formula (A ¹ ), R ^5A is a hydrogen atom and R ^5B is a fluorine atom.

In some embodiments of a compound of formula (A ¹ ), R ¹ is alkylene-C(O)-O or -OH and R ² is alkyl.

In some embodiments, compounds of formula (A ¹ ) R ¹ and R ² together form R ⁴ o^o, where R ⁴ is aryl, arylalkyl, or alkyl, wherein aryl, arylalkyl, and alkyl are optionally substituted with -NR ^a R ^b . In some embodiments, R ⁴ is -aryl-NR ^a R ^b . In some embodiments, R ⁴ is -phenyl-NR ^a R ^b .

In some embodiments, compounds of formula (A ¹ ) R ¹ and R ² together form R ⁴ o^o, where R ⁴ means

In some embodiments, a compound of formula (A ¹ ) R ³ is -OH, -NR ^a R ^b , R ^z °x 3 ^—NRaRb

C(O)-X- or >

where R ^p is halo, t is an integer from 0 to 2, R ^a is H, R ^b is H or alkyl, X is O or NH, R ^z is alkyl.

In some embodiments, compounds of formula (A ¹ ) R ³ are -OH, -NH ₂ , -NHCH3, -N(CH ₃ )2,

- 10 045579

F

In some embodiments, compounds of formula (A ¹ ) R ¹ and R ² together form

R ⁴ o^o where R ⁴ means aryl, arylalkyl, or alkyl, with aryl, arylalkyl, and alkyl optional

O-[|- —NR ^a R ^b are substituted by -NR ^a R ^b ; R ³ is -OH, -NR ^a R ^b , R ^Z -C(O)-X- or ' where Rp is halo, t is an integer from 0 to 2, R ^a is H, R ^b is H or alkyl, X is O or NH, R ^z is alkyl; and R ⁵ in each case independently represents fluorine or a hydrogen atom. Compounds with the formula (A ² ) are also given here:

(A ² ) _zz where n is an integer from 0 to 4, R ³ is -OH or R ^z -C(O)-O-; wherein R ^z means alkyl. In certain embodiments, n is 0 or 1. Also provided herein are compounds of formula ( ^A3 ):

(A ³ ) where n is an integer from 1-4, R ³ is -OH or R ^z -C(O)-O-; wherein R ^z means alkyl. In certain embodiments, n is 2. Also provided herein are compounds of formula ( ^A4 ):

(A ⁴ ) where R ³ means -NR ^a R ^b and R ⁴ means alkyl, and R ^a and R ^b in each case independently represent a hydrogen atom or alkyl, or R ^a and R ^b together form a 3-7 membered ring . In certain embodiments, R ⁴ is C1-4 alkyl. In some embodiments, R ⁴ is a cut. In certain embodiments, R ³ is -NH2, -NHCH ₃ , or -N(CH ₃ ) ₂ . Compounds with the formula (A ⁵ ) are also given here:

(A ⁵ ) wherein R ⁴ is alkyl, R ^P1 is halo or hydrogen, and R ^P2 is -NR ^a R ^b or -OH, with R ^a and R ^b in each case independently being hydrogen or alkyl. In some embodiments, R ⁴ is C1-4 alkyl, R ^P2 is -NH2.

Compounds with the formula (A ⁶ ) are also given here:

- 11 045579

where R ³ is either NR ^a , ® is aryl or heteroaryl, RP is halo, t is an integer from 0 to 2, R ^a and R ^b are each independently hydrogen or alkyl, R ^Z is alkyl, RQ is alkoxy ,

O-P- —NR ^a R ^b

R ⁴ is alkyl. In some embodiments, R ³ is Here also provided are compounds of the formula (A ⁷ ) where R ³ is

is aryl or heteroaryl, RP is halo, t is an integer from 0 to 2, R ^a and R ^b are each independently hydrogen or alkyl, R ^5A is hydrogen or fluorine, R ^5B is fluorine. In some variants_( ^RP )t

O-ij- 3—NR ^a R ^b max implementation R ³ means

In some examples, a compound having the structure of formula (I) is given here:

or a pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof, wherein R ¹ and R ²

R ⁴

0^0 independently means -H, alkyl, alkyl-C(O)-O-, -OH or halo; or R ¹ and R ² together form ' wherein R ⁴ is alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl, wherein alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl are in each instance independently and optionally substituted by -NR ^a R ^b ;

R ³ means -OH, alkyl-C(O)-O-, heteroalkyl, -NR ^a R ^b , -NR ^a R ^b -aryloxy, or R ^a R ^b N-aryloxy-, wherein alkyl-C(O) -O-, heteroalkyl, -NR ^a R ^b , and R ^a R ^b N-aryloxy- are optionally substituted with halo;

R ⁵ in each occurrence is independently -OH, halo, alkyl, or arylalkyl;

R ^a and R ^b are each independently H or alkyl;

n means an integer from 0 to 19;

provided that R ³ is not -OH, where either (a) or (b): (a) R ¹ is -OH or (b) R ¹ and R ² together

R ⁴ is formed and R ⁴ is C _1-9 alkyl or

In some of these examples, R ¹ and R ² are independently selected -H, alkyl, alkyl-C(O)-O-, -OH, and halo.

R ⁴ o^o

In other examples, R ¹ and R ² together form ·

In certain examples, R ¹ is -H. In other examples, R ¹ is alkyl. In some examples, R ¹ is alkyl-C(O)-O-. In other examples, R ¹ is -OH. In certain examples, R ¹ oz

- 12 045579 starts a halo. In other examples, R ¹ means -F. In some examples, R ¹ is -Cl. In other examples, R ¹ is -Br. In certain examples, R ¹ means -I. In other examples, R ² is -OH. In some examples, R ² means halo. In other examples, R ² means -F. In certain examples, R ² is -Cl. In other examples, R ² is -Br. In some examples, R ² means -I.

In some examples of formula (I), R ⁵ is -OH. In some examples, R ⁵ is a halo including -F, -Cl, -Br or -I. In some examples, R ⁵ means -F. In some examples, R ⁵ is Cl. In some examples, R ⁵ is -Br. In some examples, R ⁵ is -I. In some examples, R ⁵ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁵ is benzyl. In some examples of formula (I), R ³ is selected from -OH, alkylC(O)-O-, and R ^a R ^b N-aryloxy. In some of these examples, the alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some examples, R ³ is R ^a R ^b N-aryloxy. In some examples, R ³ means

In some examples, R ³ means

In some examples, R ³ means

In some examples, R ³ means

• In some

.sn

H ₃ (In examples R ³ means

In some examples of formula (I), R ³ is -OH, alkyl-C(O)-O-, heteroalkyl, -NR ^a R ^b , or R ^a R ^b N-aryloxy, wherein alkyl-C(O)- O-, heteroalkyl, -NR ^a R ^b , or R ^a R ^b N-aryloxy are optionally substituted with halo. R ^a and R ^b are each independently -H or alkyl. In some examples, R ³ is R ^a R ^b N-aryloxy, wherein R ^a and R ^b are each independently -H or alkyl.

In some examples, R ³ means

In some examples, R ³ means

- 13 045579 in some examples R ³ means

in some examples R ³ means h ₂ n

IN

• In some examples, R ³ means

In some examples, R ³ is R ^a R ^b N-aryloxy, where R ^a and

R ^{b is} in each case independently -H or alkyl.

In some examples of formula (I), R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b . In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is npropyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is sec-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl or nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some examples, R ⁴ is phenyl. In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo.

R ^b R ^a N,

In some examples, R ⁴ means ' where R ^a and R ^b are in each case independently o• In some examples, R ⁴ means • In some examples, R ⁴ means begin H or alkyl. In some examples, R ⁴ means R ^b R°N

In many examples, R means em

R ^b R ^a N

In some examples R ⁴

In some examples, R ⁴ means • In some examples, some

max R ⁴ means h ₂ n

In some examples, R ⁴ means • In some examples, R ⁴ means

- 14 045579

- 15 045579

HR ^a N

R ⁴ means

HR ^a N

In some examples, R ⁴ means • In some examples, R ⁴ means

In some examples, R ⁴ means

HR ^a N

• In some examples, R ⁴ means • In some examples

In some examples R

HR ^a N

means in some examples R ⁴ means

HR ^a N

• In some examples, R ⁴ means means

HR ^a N

IN

HR ^a N

0-4

In some examples R ⁴ • In some examples R 4 • In some examples R ⁴ means means

In some examples, R ⁴ means

HR ^a N

HR ^a N • In some examples, R ⁴ means

HR ^a N measures R ⁴ means

• In some examples, R ⁴ means

In some cases

In some examples

R ⁴ means

HR ^a N

F • In some examples, R ⁴ means

HR ^a N

F • In some examples, R ⁴ means

HR ^a N

F • In some cases - 16 045579

measures R ⁴ means

In some examples, R ⁴ is alkyl substituted with amino, including methyl amino, ethyl amino, propyl amino, butyl amino, pentyl amino, hexyl amino, heptyl amino, octyl amino, or nonyl amino. In some examples, R ⁴ is methyl amino. In some examples, R ⁴ is ethylamino. In some examples, R ⁴ is n-propyl-amino.

In some examples, R ⁴ is i-propyl-amino. In some examples, R ⁴ is n-butylamino. In some examples, R ⁴ is i-butyl-amino. In some examples, R ⁴ is t-butylamino. In some examples, R ⁴ is pentyl-amino. In some examples, R ⁴ is hexylamino. In some examples, R ⁴ is heptyl-amino.

In some examples, R ⁴ is octyl-amino. In some examples, R ⁴ is nonyl-amino.

In some examples, R ⁴ is ^H3S In some examples, R ⁴ is

In some examples, R ⁴ means

• In some examples, R ⁴ means

In some of the examples given herein, R ^a and R ^b are each independently selected from H and alkyl. In some examples, R ^a and R ^b are H. In some examples, R ^a and R ^b are methyl. In some examples, R ^a and R ^b are ethyl. In some examples, R ^a and R ^b represent a cut. In some examples, one of R ^a or R ^b is -H and the other is alkyl. In some examples, one of R ^a or R ^b is -H and the other is methyl.

In some examples, one of R ^a or R ^b is -H and the other is ethyl. In some examples, one of R ^a or R ^b is -H and the other is kerf.

In some examples, n means an integer from 0 to 19. In some examples, n means 0. In other examples, n means 1. In certain examples, n means 2. In other examples, n means 3. In certain examples, n means 4. In some examples n means 5.

In other examples, n is 6. In certain examples, n is 7. In other examples, n is 8. In certain examples, n is 9. In some examples, n is 10.

In other examples, n is 11. In certain examples, n is 12. In other examples, n is 13. In certain examples, n is 14. In some examples, n is 15.

In other examples, n is 16. In certain examples, n is 17. In other examples, n is 18. In certain examples, n is 19.

In some examples of Formula (I), R ³ is not -OH when R ¹ is -OH.

R ⁴ O^O

In some examples of formula (I), R ³ is not -OH when R ¹ and R ² together form > where R ⁴ is C _1-9 alkyl or 4-(dimethyl-amino)phenyl.

Some examples herein include a compound of formula (I) wherein R ¹ and R ² together form R ⁴

O^O ^L/

In some of these examples, R ⁴ is alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl. In certain examples, alkyl, aryl, heteroaryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b . In some of these examples, R ⁴ is alkyl. In some of these examples, R ⁴ is aryl. In some of these examples, R ⁴ is arylalkyl. In some of these examples, R ⁴ is N-containing heterocycloalkyl. In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is npropyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is sec-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl or nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some examples, R ⁴ is phenyl.

In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is heteroaryl-, including thiophene or phenol. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo. In some examples, a compound of formula (I) is given here, where R ¹ and R ² together

R ⁴ I* 9 ^9

Form ''' where R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl; and wherein alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b ; and where the stereochemistry of carbon, denoted by *, denotes the configuration R. B

R ⁴

I*

About jO some examples here is a compound of formula (I), where R ¹ and R ² together form where

R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl; and wherein alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b ; and where the carbon stereochemistry denoted by * denotes the S configuration.

In some examples, a compound of formula (I) is provided herein, wherein the compound has the structure of formula (PIa):

In some of these examples, R ¹ and R ² (Pla) are independently selected from -H, alkyl, alkyl-C(O)-O-, -OH and ha

lo. In other examples, R ¹ and R ² together form

In certain examples, R ¹ is -H. In other examples, R ¹ is alkyl. In some examples, R ¹ is alkyl-C(O)-O-. In other examples, R ¹ is -OH. In certain examples, R ¹ means halo. In other examples, R ¹ means -F. In some examples, R ¹ is -Cl. In other examples, R ¹ is -Br. In certain examples, R ¹ means -I. In other examples, R ² is -OH. In some examples, R ² means halo. In other examples, R ² means -F.

In certain examples, R ² is -Cl. In other examples, R ² is -Br. In some examples, R ² means -I.

In some examples of formula (PIa), R ⁵ is -OH. In some examples, R ⁵ is a halo, including -F, -Cl, -Br or -I. In some examples, R ⁵ means -F. In some examples, R ⁵ is Cl. In some examples, R ⁵ is -Br. In some examples, R ⁵ is -I. In some examples, R ⁵ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl.

In some examples of formula (PIa), R ³ is selected from -OH, alkyl-C(O)-O-, and R ^a RbN-aryloxy. In some of these examples, the alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some places

In some

Measures R ³ means R ^a R ^b N-aryloxy-. In some examples, R ³ means ^H3C O

n ₃ s.

examples R ³ means ²

In some examples, R ³ means

- 18 045579 o

In some examples, R ³ is ^CH3 In some examples, R ³ is

In some examples, R ³ is R ^a R ^b N-aryloxy-. In some examples, R ³ is -NR ^a R ^b aryloxy.

In some examples of formula (PIa), R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b . In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is npropyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl or nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some examples, R ⁴ is phenyl. In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo.

In some examples, R ⁴ means

In some examples R ⁴

- 19 045579 means h ₂ n

• In some examples • In some examples R ⁴ means

R ⁴ means

In some examples, R ⁴ means h ₂ n

• In some examples, R ⁴ means pax R ⁴ means h ₂ n

h ₂ n

In some examples

F is for

In some ₂ n examples of F

F

HR ^a N measures R ⁴ means in many examples • In some examples R ⁴

R ⁴ means

In the examples, R ⁴ means • In some examples, R ⁴ means

h ₂ n of some F

means

In some cases• In some means

HR'N

IN

R ⁴

measures R ⁴ means means

HR ^a N of some • In some examples R ⁴

In some examples R ⁴ examples R ⁴ means some examples R ⁴ means

In some examples, R ⁴ means

HR ^a N

• In some examples• In some examples R ⁴ means

HR°N

• In some examples, R ⁴ means

In some examples, R ⁴ means

HR'N

R ⁴ means

HR ^a N

In some examples, R ⁴ means

• In some examples

HR'N

In non-HR ^a N

• In some examples R ⁴ means which examples R ⁴ means

examples R ⁴ means • In some examples R ⁴ means

HR'N

In some

HR ^a N

In some cases

measures R ⁴ means • In some examples R ⁴ means

IN

In some examples, R ⁴ is alkyl substituted with amino, including methyl amino, ethyl amino, propyl amino, butyl amino, pentyl amino, hexyl amino, heptyl amino, octyl amino, or nonyl amino. In some examples, R ⁴ is methyl amino. In some examples, R ⁴ is ethyl amino. In some examples, R ⁴ is n-propyl-amino. In some examples, R ⁴ is i-propyl-amino. In some examples, R ⁴ is n-butyl-amino. In some examples, R ⁴ is i-butyl-amino. In some examples, R ⁴ is t-butyl-amino. In some examples, R ⁴ is pentyl-amino. In some examples, R ⁴ is hexyl-amino. In some examples, R ⁴ is heptyl-amino. In some examples, R ⁴ is octyl-amino. In some examples, R ⁴ is nonyl-amino.

In some examples, R ⁴ means ^H3C

• In some examples, R ⁴ means

- 21 045579

² In some examples R ⁴ means

In some of the examples given herein, R ^a and R ^b are each independently selected from -H or alkyl. In some examples, R ^a and R ^b are -H. In some examples, R ^a and R ^b are methyl. In some examples, R ^a and R ^b are ethyl. In some examples, R ^a and R ^b represent a cut. In some examples, one of R ^a or R ^b is -H and the other is alkyl. In some examples, one of R ^a or R ^b is -H and the other is methyl. In some examples, one of R ^a or R ^b is -H and the other is ethyl. In some examples, one of R ^a or R ^b is -H and the other is kerf.

In some examples of formula (PIa), n means an integer from 0 to 19. In some examples, n means 0. In other examples, n means 1. In certain examples, n means 2. In other examples, n means 3. In certain examples, n means 4. In some examples, n means 5. In other examples, n means 6. In certain examples, n means 7. In other examples, n means 8. In certain examples, n means 9.

In some examples, n means 10. In other examples, n means 11. In certain examples, n means 12. In other examples, n means 13. In certain examples, n means 14. In some examples, n means 15. In other examples, n means 16.

In certain examples, n means 17. In other examples, n means 18. In certain examples, n means 19.

In some examples of formula (PIa), R ³ is not -OH when R ¹ is -OH.

R ⁴

In some examples of formula (PIa), R ³ is not -OH when R ¹ and R ² together form > where R ⁴ is C _1-9 alkyl or 4-(dimethyl-amino)phenyl. In some examples, R ⁴ means

h ₂ n

In some examples, R ⁴ means

Some examples herein include a compound of formula (PIa), where the compound has a structure of formula (PIb-1) or (PIb-2):

(PIb-1) (Plb-2)

Some examples here include a compound of formula (PIa), where the compound has a structure of formula (PIc-1) or (PIc-2):

(PIc-1) (PIc-2)

Some examples here include a compound of formula (PIa), where the compound has a structure of formula (PId-1) or (PId-2):

(PId-1) (PId-2)

In some examples n means 0. In some examples n means 1. In some examples n means 2.

- 22 045579

Some examples herein include a compound of formula (I), where the compound has a structure of formula (PIe-1) or (PIe-2):

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc2), (PId-1), (PId-2), (PI(- 1) or (PIe-2), where R ³ is selected from alkyl-C(O)-O- or R ^a R ^b N-aryloxy-; where alkylC(O)-O-, or R ^a R ^b N-aryloxy - optionally substituted halo In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc-2), (PId-1), (PId- 2), (PIe-1) or (PI(-2), where R ³ is an alkyl-C(O)-O-optionally substituted halo. In some examples, a compound of formula (PIa), (PIb-1) is provided herein , (PIb-2), (PIc-1), (PIc-2), (PId-1), (PId-2), (PI(-1) or (PI(-2), where R ³ means

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc2), (PId-1), (PId-2), (PI(- 1) or (PI(-2), where R ³ means R ^a R ^b N-aryloxy-optionally substituted halo. In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc-2), (PId

1), (PId-2), (PI(-1) or (PIe-2), where R ³ means

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc

2), (PId-1), (PId-2), (PIe-1) or (PIe-2), where R ³ means

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc2), (PId-1), (PId-2), (PIt-1) is given here ) or (PIe-2), where R ³ is selected from -OH, alkyl-C(O)-O- and R ^a R ^b N-aryloxy-. In some of these examples, the alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some examples, R ³ is R ^a R ^b N-αryloxy-.

In some examples, R ³ means ^H3C o

n ₃ s. ό n ₂

• In some examples, R ³ means o pax R ³ means n ₃ s' • In some examples, R ³ means o ₃ s

CH ₃ n ₂ s.

n ₂

• In some examples, in some examples, R ³ means

In some examples, R ³ is R ^a R ^b N-aryloxy-. In some examples, R ³ is -NR ^a R ^b aryloxy.

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc2), (PId-1), (PId-2), (PI(- 1) or (PIe-2), where R ³ means R ^a R ^b N-aryloxy-, where R ^a and R ^b are in each case independently H or alkyl.

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc2), (PId-1), (PId-2), (PIe-1) is given here ) or (PIe-2), where R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl and N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b . In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is npropyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl or nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some examples, R ⁴ is phenyl. In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo.

- 24 045579 h ₂ n

R ⁴ other examples F

F stands for

In some examples, R ⁴ means • In some examples, R ⁴ means

HRN measures R ⁴ means

In some examples R ⁴

means h ₂ n

means

In some cases• In some R ⁴ means examples

HR ^a N

R ⁴

means measures R ⁴ means

In some examples, R ⁴ means

In some examples, R ⁴ means

In some examples

In some examples R ⁴ • In some cases HR ^a N

other examples R

HR ^a N . In some examples R ⁴

means • In some

r)4 examples R means means

HR ^a N

in some examples R ⁴ means

In some examples, R ⁴ means

HR ^a N • In some examples, R ⁴ means

• In some examples, R ⁴ means

HR ^a N

• In some examples, R ⁴ means

- 25 045579

In some examples, a compound of the formula (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc2), (PId-1), (PId-2), (PIe-1) is given here ) or (PIe-2), where R ⁴ is alkyl substituted with amino, including methyl amino, ethyl amino, propyl amino, butyl amino, pentyl amino, hexyl amino, heptyl amino, octyl amino or nonyl amino. In some examples, R ⁴ is methyl amino. In some examples, R ⁴ is ethylamino. In some examples, R ⁴ is n-propyl-amino. In some examples, R ⁴ is i-propylamino. In some examples, R ⁴ is n-butyl-amino. In some examples, R ⁴ is i-butylamino. In some examples, R ⁴ is t-butyl-amino. In some examples, R ⁴ is pentylamino. In some examples, R ⁴ is hexyl-amino. In some examples, R ⁴ is heptylamino. In some examples, R ⁴ is octyl-amino. In some examples, R ⁴ is nonylamino.

In some examples, R ⁴ is ^H3S In some examples, R ⁴ is

• In some examples, R ⁴ means

The compound of formula (I) is not one of the following:

'lk U=° \th * l x [ v ' \A

Ън f—/ Vo ⁰ he , or \=/ .

In some examples, a compound of formula (I) is provided herein, wherein the compound has the structure of formula (PII):

- 26 045579

(RP)

In formula (PII), R ³ is selected from -OH, alkyl-C(O)-O-, or R ^a R ^b N-aryloxy. In some of these examples, the alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some examples, R ³ means

ABOUT

R ^a R ^b N-aryloxy-. In some examples, R ³ means ^H3C o

n ₃ C.

Ό n ₂ tea ²

. In some examples, R ³ means in some examples, R ³ means n ₃ s• In some examples, R ³ means n ₃ s

dn ₃ 's n ₂ n ₂ .s _x

• In some examples, R ³ means • In some examples

In some examples, R ³ is R ^a R ^b N-aryloxy-.

In some examples of formula (PII), R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b .

In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is n-propyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is sec-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl or nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some examples, R ⁴ is phenyl. In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-aminophenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo.

In some examples, R ⁴ means h ₂ n

^(f) ^ ______ __ ____ „4______ • In some examples, R means h ₂ n means

• In some examples, R ⁴ means h ₂ n

In some examples, R ⁴ means

In some examples, R ⁴ • In some examples, R ⁴ means h ₂ n

• In some examples, R ⁴ means h ₂ n

• In some

- 27 045579

- 28 045579

HR ^a N examples R ⁴ means

In some examples F. F

R ⁴ means

In some examples, R ⁴ means

- 29 045579

In some examples, R ⁴ is alkyl substituted with amino, including methyl amino, ethyl amino, propyl amino, butyl amino, pentyl amino, hexyl amino, heptyl amino, octyl amino, or nonylamino. In some examples, R ⁴ is methyl amino. In some examples, R ⁴ is ethyl amino. In some examples, R ⁴ is p-propyl-amino. In some examples, R ⁴ is i-propyl-amino. In some examples, R ⁴ is n-butyl-amino. In some examples, R ⁴ is i-butyl-amino. In some examples, R ⁴ is t-butyl-amino. In some examples, R ⁴ is pentyl-amino. In some examples, R ⁴ is hexyl-amino. In some examples, R ⁴ is heptyl-amino. In some examples, R ⁴ is octyl-amino. In some examples, R ⁴ is nonyl-amino.

g ⁷ '

In some examples, R ⁴ means ^Hc In some examples, R ⁴ means ύ

In some examples, R ⁴ means ·

In some of the examples given herein, R ^a and R ^b are each independently selected from H or alkyl. In some examples, R ^a and R ^b are -H. In some examples, R ^a and R ^b are methyl. In some examples, R ^a and R ^b are ethyl. In some examples, R ^a and R ^b represent a cut. In some examples, one of R ^a or R ^b is -H and the other is alkyl. In some examples, one of R ^a or R ^b is -H and the other is methyl.

In some examples, one of R ^a or R ^b is -H and the other is ethyl. In some examples, one of R ^a or R ^b is -H and the other is kerf.

In some examples of formula (PII), n is an integer from 0 to 19. In some examples, n is 0. In other examples, n is 1. In certain examples, n is 2. In other examples, n is 3. In certain examples, n is 4. In some examples, n means 5. In other examples, n means 6. In certain examples, n means 7. In other examples, n means 8. In certain examples, n means 9. In some examples, n means 10. In other examples, n In certain examples, n means 12. In other examples, n means 13. In certain examples, n means 14. In some examples, n means 15. In other examples, n means 16. In certain examples, n means 17. In other examples, n means 18. In certain examples, n means 19.

Some examples herein include a compound of formula (I), wherein the compound has a structure of formula (PIIa) or (PIIb):

(RPa) _or (Piib)

In some examples, a compound of formula (PIIa) or (PIIb) is provided wherein R ⁴ is selected from 4-amino-phenyl, 4-amino-1-methyl-phenyl, 2-amino-ethyl, piperidinyl, or propyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-amino-1-methyl-phenyl. In some examples, R ⁴ is 2-amino-ethyl. In some examples, R ⁴ is piperidinyl. In some examples, R ⁴ means cut. In some examples, R ⁴ is n-propyl. In some examples, R ⁴ means i-cut.

In some examples, a compound of formula (PIIa) or (PIIb) is provided herein, wherein R ³ is selected from alkyl-C(O)-O- or R ^a R ^b N-aryloxy; wherein alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo.

In some examples, a compound of formula (PIIa) or (PIIb) is provided herein, wherein R ³ is

In some examples, a compound of formula (PIIa) or (PIIb) is given here, where R ³ is R ^b R ^a N—Oyet In some examples, a compound of formula (PIIa) or (PIIb) is given here, where h ₂ n

R ³ means

Some examples herein include a compound of formula (PIIa) or (PIIb), wherein the compound has the structure of formula (PIII):

- 30 045579

measures R ⁹ means -NR ^a R ^b , R ⁴ , R ⁴ and the subscript n are determined by formula I and noted above.

In formula (PIII), R ¹⁰ and R ¹¹ are in each case independently selected from H, F or -NR ^a R ^b .

Some examples herein include a compound of formula (III), wherein the compound has a structure of formula (PIIIa) or (PIIIb):

Some examples herein include a compound of formula (I), wherein the compound has the structure of formula (PIV):

In formula (PIV) -NR ^a R ^b , R ⁴ , R ⁵ and the subscript n are defined by formula I and noted above.

Some examples herein include a compound of formula (I), wherein the compound has a structure of formula (PV):

In formula (PV), R ⁴ , R ⁴ and the subscript n are defined by formula I and noted above.

Some examples here include a compound of formula (PV), where the compound has a structure of formula (PVa) or (PVb):

Some examples herein include a compound of formula (I), wherein the compound has a structure of formula (PVI):

- 31 045579

(PVI)

In formula (PVI), R ³ is selected from alkyl-C(O)-O- or R ^a R ^b N-aryloxy, wherein the alkyl-C(O)-O- or NR ^a R ^b -aryloxy is optionally substituted with halo.

In some examples, in formula (PVI), R ⁴ is selected from -H, -OH, halo or alkyl. In some examples, R ⁴ is a halo, including -F, -Cl, -Br or -I. In some examples, R ⁴ is -F. In some examples, R ⁴ is -Cl. In some examples, R ⁴ is -Br. In some examples, R ⁴ is -I. In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. The subscript n means an integer from 0 to 19. In some examples, n means 0. In other examples, n means 1. In certain examples, n means 2. In other examples, n means 3.

In certain examples, n is 4. In some examples, n is 5. In other examples, n is 6. In certain examples, n is 7. In other examples, n is 8. In certain examples, n is 9. In certain examples, n is 10. In in other examples, n is 11. In certain examples, n is 12. In other examples, n is 13. In certain examples, n is 14. In some examples, n is 15. In other examples, n is 16. In certain examples, n is 17. In others in certain examples, n is 18. In certain examples, n is 19. In certain examples, according to formula (PVI), R ³ is selected from -OH, alkyl-C(O)-O-, -NR ^a R ^b or NR ^a R ^b -aryloxy. In some of these examples, the alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some examples, R ³ is R ^a R ^b N-aryloxy.

In some examples, R ³ is -NR ^a R ^b . In some examples, R ³ is -NH2. In some examples, R ³ is -NH(CH ₃ ).

- 32 045579 in some examples R ³ means

Some examples herein include a compound of formula (I), wherein the compound has the structure of formula (PVII):

(PVII)

In formula (PVII), R ³ is selected from alkyl-C(O)-O- or R ^a R ^b N-aryloxy, wherein the alkyl-C(O)-O- or R ^a R ^b N-aryloxy is optionally substituted with halo.

In some examples of formula (PVII), R ⁴ is selected from -H, -OH, halo or alkyl. In some examples, R ⁴ is a halo, including -F, -Cl, -Br or -I. In some examples, R ⁴ is -F. In some examples, R ⁴ is -Cl. In some examples, R ⁴ is -Br. In some examples, R ⁴ is -I. In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. Subscript n means an integer from 0 to 19. In some examples, n means 0. In other examples, n means 1. In certain examples, n means 2. In other examples, n means 3. In certain examples, n means 4. In some examples, n In other examples, n means 6. In certain examples, n means 7. In other examples, n means 8. In certain examples, n means 9. In some examples, n means 10. In other examples, n means 11. In certain examples, n means 12. In other examples, n means 13. In certain examples, n means 14. In some examples, n means 15.

In other examples, n is 16. In certain examples, n is 17. In other examples, n is 18. In certain examples, n is 19. In some examples, in formula (PVII), R ³ is selected from -OH, alkyl-C(O)- O-, -NR ^a R ^b or R ^a R ^b N-aryloxy. In some of these examples, the alkyl-C(O)-Oyl or NR ^a R ^b -aryloxy is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some examples, R ³ is R ^a R ^b N-aryloxy.

In some examples, R ³ is -NR ^a R ^b . In some examples, R ³ is -NH ₂ .

In some examples, R ³ is -NH(CH ₃ ).

- 33 045579

a compound of formula (PVII), where the compound has a structure of formula (PVIIa):

R ³

oh he

In some examples, the structure according to formula (PVIIb) is given here:

(PVIIa) a compound of the formula (PVII), where the compound has

R ³

OH(PVIIb)

Some examples herein show a compound of formula (PVII), (PVIIa), or mean h ₃ c-nh

BH ₂ or R ^a R ^b N-aryloxy- is optionally substituted with halo.

(PVIIb), where

R ³

In some examples, a compound of formula (PVII), (PVIIa), or

R ^b R ^a N

ABOUT.

(PVIIb), where

R ³ means ·

In some examples, a compound of formula (PVII), (PVIIa), or (PVIIb) is provided herein, where

R ³ h ₂ n

ABOUT.

means ·

Some examples here show a compound structure with the formula (PVIIb-1) or (PVIIb-2):

according to formula (PVII), where the compound has

R ³

ABOUT

HE

OH (PVIIb-1) □ 3 or ^to (PVIIb-2) formula (PVII), (PVIIa), (PVIIb), (PVIIb-1)

In some examples, the compound at or (PVIIb-2) is given here, where R ³ is alkyl-C(O)-O- or R ^a R ^b N-aryloxy. In some examples, a compound of formula (I) is provided herein, wherein the compound has the structure of formula (PVIII):

- 34 045579

(PVIII)

In some examples, using any of the formulas (PI), (PIa), (PIb-1), (PIb-2), (PIc-1), (PIc-2), (PId-1), (PId2), ( PIe-1), (PIe-2), (PII), (PIIa), (PIIb), (PIIIa), (PIIIb), (PIV), (PV), (PVa), (PVb), (PVI) , (PVII), (PVIIa), (PVIIb), (PVIIb-1) or (PVIIb-2), where the halo, when present, means fluorine. In some examples, the compounds of formula (I) R ¹ and R ² are independently selected from -H, alkyl, alkyl-C(O)-O-, -OH, or halo. IN

R ⁴ about other examples R ¹ and R ² together form ·

In certain examples, R ¹ is -H. In other examples, R ¹ is alkyl. In some examples, R ¹ is alkyl-C(O)-O-. In other examples, R ¹ is -OH. In certain examples, R ¹ means halo. In other examples, R ¹ means -F. In some examples, R ¹ is -Cl. In other examples, R ¹ is -Br. In certain examples, R ¹ means -I. In other examples, R ² is -OH. In some examples, R ² means halo. In other examples, R ² means -F. In certain examples, R ² is -Cl. In other examples, R ² is -Br. In some examples, R ² means -I.

In certain examples of Formula (I), R ⁵ is in each instance independently selected from -OH, halo, alkyl, or arylalkyl. In some examples, R ⁵ is -OH. In some examples, R ⁵ is a halo, including -F, -Cl, -Br or -I. In some examples, R ⁵ means -F. In some examples, R ⁵ is -Cl. In some examples, R ⁵ is -Br. In some examples, R ⁵ is -I. In some examples, R ⁵ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁵ is benzyl.

In some examples of Formula (I), R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b . In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is npropyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is sec-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl or nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some examples, R ⁴ is phenyl. In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo.

In some examples, R ⁴ means

In some examples, R ⁴ means

In some examples, R ⁴ means

some examples - 35 045579 R ⁴ means h ₂ n

h ₂ n

• In some examples • In some examples R ⁴ means

R ⁴ means

In some examples, R ⁴ means h ₂ n

In some examples, R ⁴ means h ₂ n

R ⁴ means

• In some examples

in some examples R ⁴ means

In some examples, R ⁴ means

In some examples, R ⁴ means h ₂ n

In some examples, R ⁴ means

B • In some examples, R ⁴ means

some examples R ⁴

some

In the examples R ⁴

In some examples, R ⁴ means means

In some examples, R ⁴ means

- 36 045579 h ₂ n

In some examples, R ⁴ means

In some examples, R ⁴ is alkyl substituted with amino, including methyl amino, ethyl amino, propyl amino, butyl amino, pentyl amino, hexyl amino, heptyl amino, octyl amino, or nonylamino. In some examples, R ⁴ is methyl amino. In some examples, R ⁴ is ethyl amino. In some examples, R ⁴ is n-propyl-amino. In some examples, R ⁴ is i-propyl-amino. In some examples, R ⁴ is n-butyl-amino. In some examples, R ⁴ is i-butyl-amino. In some examples, R ⁴ is t-butyl-amino. In some examples, R ⁴ is sec-butyl. In some examples, R ⁴ is pentyl-amino. In some examples, R ⁴ is hexyl-amino.

In some examples, R ⁴ is heptyl-amino. In some examples, R ⁴ is octyl-amino. In some examples, R ⁴ is nonyl-amino.

In some examples, R ⁴ means ³ In some examples, R ⁴ means

In some examples, R ⁴ means

In some of the examples given herein, R ^a and R ^b are each independently selected from H or alkyl. In some examples, R ^a and R ^b are H. In some examples, R ^a and R ^b are methyl. In some examples, R ^a and R ^b are ethyl. In some examples, R ^a and R ^b represent a cut. In some examples, one of R ^a or R ^b is H and the other is alkyl. In some examples, one of R ^a or R ^b is H and the other is methyl. In some examples, one of R ^a or R ^b is H and the other is ethyl. In some examples, one of R ^a or R ^b is H and the other is kerf.

In some examples of formula (I), n is an integer from 0 to 19. In some examples, n is 0. In other examples, n is 1. In certain examples, n is 2. In other examples, n is 3. In certain examples, n is 4. In some examples n means 5. In other examples n means 6. In certain examples n means 7. In other examples n means 8. In certain examples n means 9. In some examples n means 10. In other examples n means 11 In certain examples, n means 12. In other examples, n means 13. In certain examples, n means 14. In some examples, n means 15. In other examples, n means 16. In certain examples, n means 17. In other examples, n means 18. In certain examples, n means 19.

In some examples of Formula (I), R ³ is not -OH when R ¹ is -OH. In some

R ⁴ 0^0 examples in formula (I) R ³ is not -OH when R ¹ and R ² together form , where R ⁴ is C1_ ₉ alkyl or 4-(dimethylamino)phenyl.

In some examples here is a compound of formula (I), where R ¹ and R ² together form R ⁴ o^o

In some of these examples, R ⁴ is alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl. In certain examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b . In some of these examples, R ⁴ is alkyl. In some of these examples, R ⁴ is aryl. In some of these examples, R ⁴ is arylalkyl. In some of these examples, R ⁴ is N-containing heterocycloalkyl. In some examples, R ⁴ is alkyl, including methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl or nonyl. In some examples, R ⁴ is methyl. In some examples, R ⁴ is ethyl. In some examples, R ⁴ is n-propyl. In some examples, R ⁴ means i-cut. In some examples, R ⁴ is n-butyl. In some examples, R ⁴ is i-butyl. In some examples, R ⁴ is t-butyl. In some examples, R ⁴ is sec-butyl. In some examples, R ⁴ is pentyl. In some examples, R ⁴ is hexyl. In some examples, R ⁴ is heptyl. In some examples, R ⁴ is octyl. In some examples, R ⁴ is nonyl. In some examples, R ⁴ is aryl, including phenyl or naphthyl. In some

- 37 045579 examples R ⁴ means phenyl. In some examples, R ⁴ is naphthyl. In some examples, R ⁴ is arylalkyl, including benzyl. In some examples, R ⁴ is N-containing heterocycloalkyl, including piperidinyl. In some examples, R ⁴ is 4-amino-phenyl. In some examples, R ⁴ is 4-aminophenyl, optionally substituted with halo. Some examples here are

R ⁴

O o a compound of formula (I), wherein R ¹ and R ² together form , where R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl and N-containing heterocycloalkyl; wherein alkyl, aryl, arylalkyl or N-containing heterocycloalkyl is optionally substituted -NR ^a R ^b ; and wherein the stereochemistry of carbon, denoted by *, is R. In some examples herein, a compound of formula (I) is provided, wherein

R ⁴

Oh oh

R ¹ and R ² together form ' where R ⁴ is selected from the group consisting of alkyl, aryl, arylalkyl and N-containing heterocycloalkyl; wherein alkyl, aryl, arylalkyl or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b , and wherein the carbon stereochemistry denoted by * is S. By

R ⁴

O In formula (I), R ³ is not -OH when R ¹ is -OH, or when R ¹ and R ² together form ' where R ⁴ is C1. ₉ alkyl or 4-(dimethyl-amino)phenyl.

In some examples, the payload provided herein is a derivative or analogue of budesonide or diflorazone. In certain examples, the derivative is an amine or aniline containing molecule that is structurally related to budesonide or diflorazone. As discussed herein, the payloads provided herein, as well as other steroids, can be conjugated to an antibody or antigen binding fragment thereof based on the methods set forth herein. As stated herein, the payloads provided herein, as well as other steroids, can be conjugated to an antibody or an antigen-binding fragment thereof, as well as a cyclodextrin moiety, based on the methods set forth herein. As discussed herein, stable payload linkers can be used with these conjugation methods to produce antibody-steroid conjugates. In some examples, the antibody-steroid conjugates also include a cyclodextrin moiety.

In some embodiments, compounds of formula (I ¹ ):

or a pharmaceutically acceptable salt, solvate, stereoisomer, or derivative thereof, wherein R ¹ and R ² are not

depending on -H, alkyl, alkyl-C(O)-O-, -OH or halo; or R ¹ and R ² together form wherein R ⁴ is alkyl, aryl, arylalkyl or N-containing heterocycloalkyl, wherein alkyl, aryl, arylalkyl and N-containing heterocycloalkyl are in each instance independently and optionally substituted by NR ^a R ^b ;

R ⁵ in each occurrence is independently -OH, halo, alkyl or arylalkyl;

R ³ means -OH, alkyl-C(O)-O- or -X-aryl-NR ^a R ^b , wherein X is selected from S, S(O), S(O) ₂ , SO ₂ NR ^a , CONR ^a , C(O) or NR ^a , wherein the alkyl-C(O)-O- and -X-αryl-NR ^a R ^b are optionally substituted with a halo or a prodrug.

R ^a and R ^b are in each case independently H or alkyl, aryl;

R ^c is -H or alkyl;

n means an integer from 0 to 19;

provided that R ³ is not -OH, where either (a) or (b): (a) R ¹ is -OH or (b) R ¹ and R ² together

R ⁴ 0^0 form , and r ⁴ means C ₁ . ₉ alkyl or

In some of these examples, alkyl-C(O)-O- or -X-aryl-NR ^a R ^b is optionally substituted with halo. In some examples, R ³ is -OH. In some examples, R ³ is alkyl-C(O)-O-. In some

- 38 045579 examples R ³ means R ^a R ^b N-aryloxy. In some examples, R ³ means

examples R ³ means

In some examples, R ³ means • In some

In some examples, R ³ means

means examples

In some examples, R ³ means

In some examples, R ³ is -X-aryl-NR ^a R ^b . In some examples R ³ h ₂ n

• In some examples, R ³ means h ₂ n

h ₂ n

• In some

R ³ means means h ₂ n

R ³

R ³ means • In some examples R ³ means in some examples h ₂ n

F

In some examples • In some • In some examples R ³ means

R ³ means

examples h ₂ n

' In some examples R ³ means measures R ³ means h ₂ n

• In some examples, R ³ means examples

In some cases h ₂ n

In some

R ³ means

IN

- 39 045579 in some examples R ³ means

In some examples, R ³ means h ₂ n

H ₂ N • In some examples, R ³ means

rah R ³ means h ₂ n

• In some examplesh ₂ n

F

In not• In some examples R ³ means which examples R ³ means

F • In some examples, R ³ means h ₂ n

In some examples, R ³ means F

0-4 h ₂ n

In some examples, R ³ means

In some examples

R ³ means

R ³ means h ₂ n

in some examples R ³ means • In some examples

in some examples R ³ means h ₂ n

F

R ³ means

In some examples, R ³ means

B h ₂ n

• In some examples • In some examples R ³ h ₂ n

F

F means • In some- 40 045579

- 41 045579

In some examples, a compound of formula (I) is provided herein, wherein R ³ has a structure selected from the following structures:

In some examples, R ³ means

In some examples R ³ means which examples R ³ means

In some examples, R ³ means In some examples, R ³ means

In these examples, q means an integer between 0 and 5.

In some examples, a compound of formula (I) is provided herein, wherein R ³ has a structure selected from the following structures:

In some examples, R ³ means

In some examples, R ³ means

S.

in which examples R ³ means

s

In some examples, R ³ means In some examples, R ³ means

In these examples, q means an integer between 0 and 5.

In some examples, a compound of formula (I) is provided herein, wherein R ³ has a structure selected from the following structures:

- 42 045579

in some examples R ³ means h ₂ nh ₂ n

In some examples, R ³ means

In some examples R ³ means /=\o /\_g

In some examples, R ³ means

IN

In some examples, R ³ means

Some examples herein include a compound of formula (I), where the compound has the structure of formula 1000:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof. According to formula 1000, R ¹ and R ² are independently selected from the group consisting of -H, -OH, alkyl, -O-C(O)-alkyl and halo; or R ¹ and R ² together R ⁴ o^o form ' where R ⁴ is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b , and N-containing heterocycloalkyl; wherein X is absent or means -N-, -CH2- or -O-; where Y is absent or means -CH2-. R ³ is selected from the group consisting of -OH, -O-C(O)-alkyl, -O-aryl, -NR ^a R ^b , -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -Xheteroarylene-Y-NR ^a R ^b , and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2 or -O-; where Y is absent or means -CH ₂ -. R ⁵ in each case is independently selected from a substituent consisting of -OH, halo and alkyl; n means an integer from 0 to 19; and each R ⁵ is located on any ring atom. R ^a and R ^b are in each case independently selected from the group consisting of -H and alkyl; or R ^a and R ^b cyclize to form a cycloheteroalkyl with 3-6 ring atoms, including one atom, which is N, to which they are attached. R ^a and R ^b are each independently optionally substituted with at least one substituent selected from the group consisting of -OH, -PO4H, NH2, -C(O)OH and -C(O)CH3. In certain embodiments, provided herein are compounds having formula 1000, wherein R ³ is selected from the group consisting of -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; wherein X is absent or means -N-, -CH2- or -O-; Y is missing or means -CH2-; R ⁴ is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is missing or means -CH ₂ -.

In certain embodiments, provided herein are compounds having formula 1000, wherein R ³ is selected from the group consisting of -OH, -O-C(O)-alkyl, -O-aryl, -NR ^a R ^b , -alkylene-NR ^a R ^b , -Charylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; wherein X is absent or means -N-, -CH2- or -O-; Y is missing or means -CH2-; R ⁴ is selected from the group consisting of -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is missing or means -CH2-.

In certain embodiments, provided herein are compounds having formula 1000, wherein R ³ is -NR ^a R ^b ; R ⁴ is alkyl. In certain embodiments, R ³ is -NH2. In certain embodiments, R ⁴ is n-propyl. In certain embodiments, R ³ is -NH ₂ and R ⁴ is n-propyl.

In certain embodiments, a compound of formula 1000 means a compound having formula 1010, 1020, 1030, or 1040:

- 43 045579

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

In certain embodiments, a compound of formula 1000 has formula 1110, 1120, 1130, or 1140:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

In certain embodiments of any of formulas 1000-1140, R ³ is -OH or -OC(O)-alkyl; R ⁴ means -alkylene-NR ^a R ^b , -X-arylene-NR ^a R ^b , -X-heteroarylene-NR ^a R ^b or N-containing heterocycloalkyl; in this case X is absent or means -CH2-.

In certain embodiments, R ⁴ is -alkylene-NH ₂ , -C6H5-NH2 or -CH ₂ -C6H5NH2.

In certain embodiments of any one of Formulas 1000-1140, R ³ is -O-aryl, -NR ^a R ^b , -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene- Y-NR ^a R ^b or N-containing heterocycloalkyl; where X is absent or means -N-, -CH ₂ - or -O-; Y is absent or means -CH ₂ -; R ⁴ is alkyl, aryl, alkylaryl, or arylalkyl. In certain embodiments, R ³ is -Oarylene-NR ^a R ^b , -O-heteroarylene-NR ^a R ^b ; wherein aryl or heteroaryl is optionally substituted with halogen, deuterium, hydroxyl, or methoxyl. In certain embodiments, R ³ is -O-phenyl-NR ^a R ^b , -O-heteroarylene-NR ^a R ^b ; wherein phenyl or heteroaryl is optionally substituted with halogen or deuterium. In certain embodiments of this paragraph, R ⁴ is npropyl.

In certain embodiments, compounds of any of formulas 1000-1140 are provided herein, wherein R ³ is -NR ^a R ^b ; R ⁴ is alkyl. In certain embodiments, R ³ is -NH2. In certain embodiments, R ⁴ is n-propyl. In certain embodiments, R ³ is -NH2 and R ⁴ is n-propyl.

According to any of the formulas 1000-1140, R ³ can be any of the specific R ³ presented above. In particular embodiments, R ³ means -NH2, -N(H)CH ₃ , -N(CH ₃ )2 or means

R ³

In private embodiments

In particular embodiments, R ³ means

- 44 045579

In particular embodiments, R ³ means

According to any of the formulas 1000-1140, R ⁴ can be any of the specific R ⁴ presented above. In particular embodiments, R ⁴ is selected from -CH2-CH2-NH2. In particular embodiments, R ⁴ is n-propyl. Compounds with the following structure are also presented here:

- 45 045579

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof.

Included within the scope of the present invention are pharmaceutically acceptable salts, solvates, crystalline forms, amorphous forms, polymorphic forms, regioisomers, stereoisomers, prodrugs, for example, phosphatase prodrugs, glucose prodrugs, ester prodrugs, etc., metabolites, and physiological adducts described herein steroid payloads, including formula (I), (I ¹ ), and (A1)-(A ⁷ ).

- 46 045579

C. Protein-steroid conjugates.

Provided herein are protein conjugates of the steroids described herein. Such conjugates include proteins, for example, antibodies or antigen-binding fragments thereof, which are covalently linked, for example, through the coupling agent linkers described herein, to the compounds described in section B above, for example, compounds of formula (A), (A ¹ ), ( A ² ), (A ³ ), (A ⁴ ), (A ⁵ ), (A ⁶ ), (A ⁷ ), (I), (I ¹ ), (PIa), (PIb-1), (PIb -2), (PIc-1), (PIc-2), (PId-1), (PId-2), (PIe-1), (PIe-2), (PII), (PIIa), (PIIb ), (PIII), (PIIIa), (PIIIb), (PIV), (PV), (PVa), (PVb), (PVI), (PVII), (PVIIa), (PVIIb), (PVIIb-1 ), (PVIIb-2), (PVIII) and (1000)-(1140).

The coupling agent linker may be linked to the steroid described herein, at any suitable moiety or position of the steroid, including, for example, through an amide, ether, ester, carbamate, or amine. For example, a linker coupling agent can be attached to compounds via R ¹ , R ³ or R ⁴ or a hydroxy group represented by formula (A ¹ ):

In certain embodiments, the steroids described herein are attached to a coupling agent linker by reacting the amino or hydroxyl group of the steroid with a suitable reactive group present on the linker. In some embodiments, the binding agent linker also includes a cyclodextrin moiety. For example, the cyclodextrin moiety may be linked to the backbone chemistry of the coupling agent linker.

In certain embodiments, compounds provided herein have the following structure:

BA-(L-PAY) _x where BA means the binding agent described herein; L is an optional linker described herein; PAY means the steroidal compound described herein; x is an integer from 1 to 30. In particular embodiments, each PAY is a radical obtained by removing an atom, such as a hydrogen atom, from a compound according to a Formula selected from the group consisting of formulas (A), (A ¹ ), ( A ² ), (A ³ ), (A ⁴ ), (A ⁵ ), (A ⁶ ), (A ⁷ ), (I), (I ¹ ), (PIa), (PIb-1), (PIb -2), (PIc-1), (PIc-2), (PId-1), (PId2), (PIe-1), (PIe-2), (PII), (PIIa), (PIIb), (PIII), (PIIIa), (PIIIb), (PIV), (PV), (PVa), (PVb), (PVI), (PVII), (PVIIa), (PVIIb), (PVIIb-1), (PVIIb-2), (PVIII) and (1000)-(1140). Examples of such compounds are described in detail below.

In certain embodiments, provided herein are compounds having the structure of formula (III):

where either (a) or (b):

(a) R ³ means -BL-, -BL-X- or

-BL-X-(CH ₂ )o. ₂

R ¹ and R ² are each independently -H, alkyl, alkyl-C(O)-O-, -OH or halo; or

R ¹ and R ² together form where R ⁴ is alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; wherein alkyl, aryl, arylalkyl and N-containing heterocycloalkyl are optionally substituted -NR ^a R ^b ; or (b) R ³ is -OH, alkyl-C(O)-O-, heteroalkyl, -NR ^a R ^b or aryloxy, wherein alkyl-C(O)-O-, heteroalkyl, or aryloxy is optionally substituted with -NR ^a R ^b , -NR ^a R ^b - aryloxy, or halo, and R ¹ and R ² together form

- 47 045579

R ⁴

0^0 is called ' where R ⁴ means -BL-, ^{BL X} ®~ ^(CH2)o · ² , -b-x-^nd^— or -BL-Y, where Y means N-containing divalent heterocyclic compound;

-BL- means divalent linker coupling agent;

R ⁵ in each occurrence is independently -OH, halo, alkyl, or arylalkyl;

R ^a and R ^b are each independently -H or alkyl;

RP in each case independently means halo;

BA means binding agent associated with -BL-;

X in each case independently means NR ^a or O;

Θ means aryl or heteroaryl;

t means an integer from 0 to 2;

x means an integer from 1 to 30;

n means an integer from 0 to 19.

In some examples, the subscript x is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some examples, the subscript x is 0. In some examples, the subscript x is 1. In some examples, the subscript x is 2. In some examples, the subscript the subscript x is 3. In some examples, the subscript x is 4. In some examples, the subscript x is 5. In some examples, the subscript x is 6. In some examples, the subscript x is 7. In some examples, the subscript x is 8. In some examples, the subscript x is 9. In some examples, the subscript x is 10. In some examples, the subscript x is 11. In some examples, the subscript x is 12. In some examples, the subscript x is 13. In some examples, the subscript x In some examples, the x index is 15. In some examples, the x index is 16. In some examples, the x index is 17. In some examples, the x index is 18. In some examples, the x index is 19. In some examples, the x index is 20 In some examples, the subscript of x is 21. In some examples, the subscript of x is 2 2. In some examples, the subscript of x is 23. In some examples, the subscript of x is 24. In some examples, the subscript of x is 25. In some examples, the subscript of x is 25. x is 26. In some examples, the subscript x is 27. In some examples, the subscript x is 28. In some examples, the subscript x is 29. In some examples, the subscript x is 30.

In some examples of formula (III), R ¹ and R ² are each independently -H, alkyl or -OH. In some examples of formula (III), one of R ¹ or R ² is -H, alkyl or -OH. In some examples of formula (III), R ¹ and R ² are either -H, alkyl, or -OH.

R ⁴

In some examples, according to formula (III), R ¹ and R ² together form

In some examples, R ⁴ is -RL. In some examples, R ⁴ is RL-NR ^a -aryl. In other examples, R ⁴ is alkyl. In certain examples, R ⁴ is arylalkyl. In some examples, R ⁴ is aryl. In other examples, R ⁴ is N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted.

In some examples of formula (III), R ⁵ is -H or halo. In some examples of formula (II), R ⁵ is -H or fluorine. In some examples of formula (III), one of R ⁵ is -H or halo. In some examples of formula (III), R ⁵ is -H or halo, n is 2. In some examples of formula (III), R ⁵ is -F, n is 1. In some examples of formula (II), R ⁵ is - F, n means 2.

In some examples of Formula (III), R ³ is BL. In some examples of formula (III), R ³ is RL-NR ^a -aryloxy-. In some other examples of formula (III), R ³ is -OH. In some other examples of formula (III), R ³ is alkyl-C(O)-O-. In some other examples of formula (III), R ³ is heteroalkyl. In some other examples of formula (III), R ³ is -NR ^a R ^b . In some other examples of formula (III), R ³ is aryl. In some other examples of formula (III), R ³ is aryloxy. In some other examples of formula (III) alkyl-C(O)-O-, heteroalkyl or aryloxy, optionally substituted -NR ^a R ^b or halo. In some examples of formula (II), R ³ is -OH. In some examples of formula (III), R ³ is alkyl-C(O)-O-. In some examples, R ³ means ^{H3 CH3}

In some examples of formula (III), R ³ is heteroalkyl. In some examples, R ³ means - 48 045579

? ^L

In some examples of formula (III), R ³ means ^H3< or

In some examples of Formula (III), R ³ is -NR ^a R ^b . In some examples, R ³ means ^V CH 3

-NR ^a R ^b -aryloxy. In some examples, R ³ means

R ^{a Ra} In some examples, R ³ means

Нз9 ^НзС In some examples R ³ means

R ^b • In some examples, R ³ is aryloxy. In some examples, R ³ means

IN

In some examples, R ³ means

According to formula (III), the subscript n means an integer from 0 to 19. In some examples, n means 0. In other examples, n means 1. In certain examples, n means 2. In other examples, n means 3. In certain examples, n means 4 In some examples, n means 5. In other examples, n means 6. In certain examples, n means 7. In other examples, n means 8. In certain examples, n means 9. In some examples, n means 10. In other examples, n means 11. In certain examples, n is 12. In other examples, n is 13. In certain examples, n is 14. In some examples, n is 15. In other examples, n is 16. In certain examples, n is 17. In other examples, n is 18. In In certain examples, n is 19. In certain examples, a compound having the structure of formula (IIIa) is given here:

- 49 045579

where BA means binding agent;

R ⁵ in each occurrence is independently -OH, halo, or alkyl;

R ³ is selected from -OH, alkyl-C(O)-O-, heteroalkyl, -NRaRb, -NR ^a R ^b -aryloxy or aryloxy, wherein alkyl-C(O)-O-, heteroalkyl or aryloxy is optionally substituted - NR ^a R ^b or halo; BL means binding agent linker;

Ra and R ^b are in each case independently selected from H, alkyl and alkyl-C(O); n means an integer from 0 to 19; x is an integer from 1 to 30. In some examples, a compound having the structure of formula (IIIa2) is given here:

where BA means binding agent;

R ⁵ in each occurrence is independently -OH, halo or alkyl;

R ³ means -OH, alkyl-C(O)-O-, heteroalkyl, -NR ^a R ^b , -NR ^a R ^b -aryloxy, or aryloxy, wherein alkyl-C(O)-O-, heteroalkyl, or aryloxy is optionally substituted with -NR ^a R ^b or halo;

BL means binding agent linker;

Ra and R ^{b are} in each case independently selected from H, alkyl, or alkyl-C(O);

n means an integer from 0 to 19; x is an integer from 0 to 30. In some examples of formula (IIIa2), R ³ is -OH.

In some examples of formula (IIIa2), R ³ is alkyl-C(O)-O-. In some examples R ³ oz

starts

In some examples of formula (IIIa2), R ³ is heteroalkyl. In some examples, R ³ means

teas or

In some examples of formula (IIIa2), R ³ is -NR ^a R ^b . In some examples, R ³ means

In some examples, R ³ is aryloxy. In some examples, R ³ means In some examples, R ³ means in some examples R ³ means

In some examples, R ³ means ^Hr1

• In nekoH ₃ C^

In some examples, R ³ means

In non- 50 045579

in which examples R ³ means (IIIa2) has the following structure:

in some examples, a compound of the formula wherein BA is a coupling agent;

R ³ means -OH or alkyl-C(O)-O-;

R ^5a and R ^5b are each independently -F or H;

BL means binding agent linker;

x means an integer from 1 to 30.

In some examples, a compound having the structure of formula (IIIb) is given here:

where BA means binding agent;

R ⁵ in each occurrence is independently -OH, halo, or alkyl;

R ⁴ is selected from alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl, wherein the alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted by -NR ^a R ^b ;

BL means binding agent linker;

R ^a and R ^{b are} in each case independently selected from H, alkyl and alkyl-C(O);

n means an integer from 0 to 19;

x means an integer from 0 to 30.

In some examples of formula (IIIb), R ⁵ is -H or halo. In some examples of formula (IIIb), R ⁵ is fluorine. In some examples of formula (IIIb), n is at least 2, and two R ⁵ is halo. In some examples of formula (IIIb), R ⁵ is -F, n is 1. In some examples of formula (IIIb), R ⁵ is -F.

In some examples of formula (IIIb), R ⁴ is alkyl. In some examples of formula (IIb), 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 examples of formula (IIIb), R ⁴ is n-propyl. In some examples, the compound of formula (IIIb) has the following structure:

where BA means binding agent;

R ⁴ is alkyl;

R ^5a and R ^5b are each independently -F or H;

BL means binding agent linker;

x means an integer from 1 to 30.

In some examples, a compound having the structure of formula (IIIc) is given here:

- 51 045579

(P1s);

where BA means binding agent;

R ¹ and R ² are independently -H, alkyl, alkyl-C(O)-O-, -OH or halo;

R ⁵ is in each case independently selected from -OH, halo or alkyl;

BL means binding agent linker;

n means an integer from 0 to 19;

x means an integer from 1 to 30.

In some examples of Formula (IIIc), R ⁵ is a halo. In some examples of formula (IIIc), R ⁵ is fluorine. In some examples of formula (IIIc), one of R ⁵ is a halo. In some examples of formula (IIIc), two R ⁵ means halo. In some examples of formula (IIIc), R ⁵ is -F, n is 2. In some examples of formula (IIIc), R ¹ is CH3. In other examples of formula (IIIc), R ¹ is OH. In some other examples of formula (IIIc), R ¹ is H. In some examples of formula (IIIc), R ² is CH3. In other examples of formula (IIIc), R ² is OH. In some other examples of formula (IIIc), R ² is H. In some examples of formula (IIIc), R ¹ is CH3, R ² is _CH3 .

In other examples of formula (IIIc), R ¹ is CH ₃ and R ² is OH.

In some examples of formula (IIIc), R ¹ is CH ₃ and R ² is H.

In some other examples of formula (IIIc), R ¹ is OH, R ² is CH ₃ .

In other examples of formula (IIIc), R ¹ is OH, R ² is OH.

In some examples of formula (IIIc), R ¹ is H, R ² is H.

In some other examples of formula (IIIc), R ¹ is H and R ² is OH.

In other examples of formula (IIIc), R ¹ is H, R ² is H.

In some embodiments, a compound of formula (IIIc) has the following structure:

(Шс) where BA means binding agent;

R ² means methyl;

R ^5a and R ^5b are each independently -F or H;

BL means binding agent linker;

x means an integer from 0 to 30.

In some embodiments, a compound of Formula (IIIc) has the following structure:

BA means binding agent;

RG means a reactive group residue;

CD means cyclodextrin;

SP ¹ means spacer group;

AA ⁴ means an amino acid residue;

AA ⁵ means a dipeptide residue;

- 52 045579

PEG means polyethylene glycol;

m means an integer from 0 to 4;

x means an integer from 0 to 30;

R ⁴ is alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl; wherein alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl are optionally substituted -NR ^a R ^b ;

R ^a and R ^b are each independently -H or alkyl;

BA means a coupling agent associated with -BL-;

SP ¹ and SP ² each independently mean the absence or remainder of a spacer group, where SP ¹ includes a trivalent linker; AA ⁴ means a trivalent linker comprising an amino acid residue; AA ⁵ means a dipeptide residue; PEG means polyethylene glycol residue; wherein indicates the atom through which the specified chemical group binds to the nearest group in the formula, CD in each case is independently absent or means a cyclodextrin residue, with at least CD present, the subscript m means an integer from 0 to 5; In these examples, the subscript m means 0, 1, 2, 3, 4, or 5. In some examples, the subscript m means 0. In some examples, the subscript m means 1. In some examples, the subscript m means 2. In some examples, the subscript m means 2. the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, any of AA ⁴ or AA ⁵ independently includes in each instance an amino acid selected from the group 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 selected from the group alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, an amino acid selected from the group 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 is lysine-valine-alanine. In some embodiments, AA ⁵ is lysine-valine-citrulline. In some embodiments, AA ⁵ is glutamate-valine-citrulline. In some examples, SP ¹ is in each instance independently selected from the group consisting of C1-6 alkylene, NH-, -C(O)-, (-CH2-CH2-O)e, -NH-CH2-CH2-(-O- CH2-CH2)e-C(O)-, -C(O)-(CH2)uC(O)-, -C(O)-NH-(CH2)v- and their combinations, where the subscript e means an integer a number from 0 to 4, the subscript u is an integer from 1 to 8, the subscript v is an integer from 1 to 8. In some examples, SP ² is in each instance independently selected from the group consisting of C1-6 alkylene, -NH- , -C(O)-, (-CH ₂ -CH ₂ -O) _e , -NH-CH ₂ CH2-(-O-CH2-CH2)e-C(O)-, -C(O)-( CH2)uC(O)-, -C(O)-NH-(CH2)v- and their combinations, where the subscript e means an integer from 0 to 4, the subscript and means an integer from 1 to 8, the subscript v means an integer from 1 to 8. Below are also compounds with the formula (B ² ):

(B ² ) _zz where n is an integer from 0 to 4, R ³ is -OH or R ^Z -C(O)-O-; wherein R ^Z is alkyl, BL is a coupling agent linker, BA is a coupling agent, x is an integer from 1 to 30. In certain embodiments, n is 0 or 1, x is an integer from 1 to 6. In certain embodiments x means 4. Below are also compounds with the formula (B ³ ):

(B ³ ) where n is an integer from 1 to 4, R ³ is -OH or R ^Z -C(O)-O-; where R ^Z is alkyl, BL is a linker coupling agent, BA is a coupling agent, x is an integer from 1 to 30. B

- 53 045579 in certain embodiments, n is 2, x is an integer from 1 to 6. In certain embodiments, x is 4.

Below are also compounds with the formula (B ⁴ ):

where R ⁴ is alkyl, R ^a is hydrogen or alkyl, BL is a coupling agent linker, and BA is a coupling agent. In certain embodiments, R ⁴ is C1-4 alkyl. In some embodiments, R ⁴ is a cut. In certain embodiments, R ³ is -NH2, -NHCH3, or -N(CH3) ₂ . In certain embodiments, x is an integer from 1 to 6. In certain embodiments, x is 4. Compounds of formula ( ^B5 ) are also provided below:

where R ⁴ means alkyl, RP ¹ means halo or

a hydrogen atom, X is NR ^a or O, wherein R ^a is a hydrogen atom or alkyl, BL is a coupling agent linker, BA is a coupling agent, x is an integer from 1 to 30. In some embodiments, R ⁴ is C _1-4 alkyl, X is NH, x is an integer from 1 to 6. In certain embodiments, x is 4. Compounds of formula ( ^B6A ) are also provided below:

where X is O or NRa, is aryl or heteroaryl, RP is halo, t is an integer from 0 to 2, R ^a is a hydrogen atom or alkyl, BL is a linker coupling agent, BA is a coupling agent, x is an integer from 1 up to 30, R ⁴ means alkyl. In some embodiments, X is O, R ⁴ is alkyl, x is an integer from 1 to 6. In certain embodiments, x is 4. Also listed below are compounds of the formula (B ^6B )

where Ra is a hydrogen atom or alkyl, BL is a coupling agent linker, BA is a coupling agent, x is an integer from 1 to 30. In some embodiments, x is an integer from 1 to 6. In some embodiments, x is 4.

As used herein, the term binding agent linker or BL refers to any divalent group or moiety that binds or connects a binding agent (eg, an antibody or an antigen-binding fragment thereof) to a payload-containing compound (eg, a steroid) as described herein. In general, suitable binding agent linkers for the antibody conjugates described herein are linkers that are stable enough to utilize the half-life of the antibody in the bloodstream while still being capable of releasing its beneficial

- 54 045579 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, for example, through hydrolysis, reduction, or an enzymatic reaction. Non-cleavable linkers are linkers that release the attached payload 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, reducing labile linkers, hyperflammable linkers and non-cleavable linkers. Suitable linkers also include, but are not limited to, linkers that include glucuronides, succinimide thioesters, polyethylene glycol (PEG) units, carbamates, hydrazones, mal-caproyl units, disulfide units (eg, -SS-, -SS-C(R1)() R ² )-, where R ¹ and R ² independently represent hydrogen or hydrocarbyl), para-amino-benzyl (PAB) units, phosphate units, for example mono-, bis- and trisphosphate units, peptides, for example 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-citrulline units, lysine-valinalanine units. In some embodiments, the linker moiety of the coupling agent of the conjugates described herein is obtained by reacting the reactive linker moiety of the payload-containing linker described herein with a reactive moiety of the antibody. Reactive linker group (RL) refers to a monovalent group, which includes a reactive group and a linking group, depicted as, where RG is a reactive RG-L— group, depicted as ' where RG is a reactive group, L is a linking group, wavy the line means communication with the payload. A linking group is any divalent moiety that connects a reactive group to a payload. The linking group can also be any trivalent moiety that links the reactive group, the payload, and the cyclodextrin moiety. In some examples, the linking group is trivalent and includes a cyclodextrin group linked to a trivalent group (eg, a lysine residue) in the linking group. Reactive linkers (RL), together with the payloads to which they are linked, contain intermediates (payload linkers) used as synthetic precursors to prepare the steroidal antibody conjugates described herein. The reactive linker contains a reactive group (RG), which is a functional group or fragment that reacts with a reactive portion of an antibody, modified antibody, or antigen-binding fragment thereof. The portion resulting from the reaction of a reactive group (RG) with an antibody, a modified antibody, or an antigen-binding fragment thereof, together with a linking group (L), contains the binder linker portion (BL) of the conjugate described herein.

IV n G ⁱⁿ ?

s RG ⁿ L s £ _x ⁵ * where “ means the linking agent, RGN means the fragment resulting from the reaction of the reactive moiety of the payload linker with the reactive part of the coupling agent, L means the linking group, ί means the payload linker.

In certain embodiments, the RGN fragment is derived from the reaction of RG with a cysteine or lysine residue of an antibody or an antigen binding fragment thereof. At certain resolutions, RGN is derived from a click chemical reaction. In some embodiments of said click chemistry, RGN is obtained from a 1,3-cycloaddition reaction between an alkyne and an azide. Non-limiting examples of such RGNs include those derived from strained alkynes, e.g., those suitable for strain-facilitated azide-alkyne cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzanulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with azides in the absence of copper catalysts. Suitable RGNs also include, but are not limited to, RGNs derived from DIBAC, DIBO, BARAC, substituted, for example, fluorinated alkynes, azacycloalkynes, BCN and derivatives thereof. Conjugates containing such RGN groups 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 H ₂ N-LL-N ₃ , where LL is a divalent polyethylene glycol group in the presence of a transglutaminase enzyme, such as microbial transglutaminase. Suitable antibody glutamine residues include Q295 or residues that are generated by insertion or mutation, such as the N297Q mutation. In some embodiments, the binding agent 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

- 55 045579 embodiments, the coupling agent of the conjugates described herein is

^w , where Ab is an antibody or an antigen-binding fragment thereof, n is an integer from 1 to 10, w is the number of linker payload fragments, is a linkage to one binder linker (BL), for example a linkage to a fragment resulting from reaction 1, 3cycloaddition between alkyne and azide. The conjugate contains 2 or 4 linker payload fragments.

In some embodiments, the coupling agent linker is a divalent moiety of the formula (BLA);

-RG ^N -(SP ¹ )q-(A)z-(NR ^a )s-(B)t-(CH ₂ )u-(O) _v -( SP ² ) _W (BL ^A );

where RG ⁿ is defined above;

A means amino acid or peptide;

R ^a is H or alkyl;

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

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

In some other embodiments, the binding agent linker is a trivalent moiety (BLB);

-RG ^N -(SP ¹ )q-(A)z-(NR ^a )s-(B) _t -(CH ₂ )u-(O)v-( SP ² ) _W (BL");

where RG ⁿ is defined above;

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

R ^a is H or alkyl;

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

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

In some examples, the cyclodextrin (CD) is directly linked to an amino acid residue, such as a lysine acid residue. This means that the CD is one bond position away from the covalent linker of the amino acid lysine. In some of these examples, the covalent linker is also linked directly to the payload moiety. This means that the covalent linker is one bond position away from the payload, such as, for example, the steroid payload described herein. In some of these examples, the covalent linker is also linked directly to the CD moiety. This means that the covalent linker is one bond position away from the CD, such as the CD described here. In some of these examples, the covalent linker is lysic acid or a derivative thereof.

In some examples, CD is linked indirectly to a covalent linker in the linking group (eg, BL). This means that the CD is more than one bond position away from the covalent linker. This also means that the CD is linked through another fragment to a covalent linker. For example, CD may be linked to a maleimide group, which is linked to a polyethylene glycol group, which is linked to a covalent linker. In some of these examples, the covalent linker is also indirectly associated with the payload moiety. This means that the covalent linker is at more than one bonding position from the payload, such as, for example, the steroid payload described herein. This also means that the covalent linker is linked through another group to the payload. For example, the covalent linker may be linked to a dipeptide, including Val-Ala or Val-Cit, which may be linked to a para-amino benzoyl, which may be linked to the payload. In some of these examples, the covalent linker is also indirectly linked to the cyclodextrin moiety. This means that the covalent linker is at more than one bond position from the cyclodextrin, such as the cyclodextrins described herein. This also means that the covalent linker is linked 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, which may be linked to a cyclodextrin. In some of these examples, the covalent linker is lysic acid or a derivative thereof.

In some embodiments, the coupling agent linker is -RG ^N -(SP1)q-(A)z-. In some embodiments, the coupling agent linker is -RG ^N -(SP ¹ )q-(A) ₂ -. In some

- 56 045579 embodiments, the coupling agent linker is a moiety of the formula (BL ^A1 ) —RG ⁿ -(SP ¹ )—

(BL ^A1 ) where R ^{AA 1} and R ^AA2 in each case independently represent amino acid side chains. In some examples of formula RL ^A1 SP ¹ is a divalent polyethylene glycol group, RG ^N is a 1,3 cycloadduct of the reaction between an alkyne and an azide.

In some embodiments, the coupling agent linker is -RG ^N -(SP ¹ )q-(A)z-. In some embodiments, the coupling agent linker is -RG ^N -(SP ¹ )q-(A) ₂ -. In some embodiments, the coupling agent linker is a moiety of the formula (BL)

O R^ ¹ _u o

RAA3 R ^AA2 (BL ^b1 ) where R ^aai and R ^aa2 in each case independently represent amino acid side chains. R ^AA3 denotes an amino acid side chain that is linked directly or indirectly to a cyclodextrin moiety. In some examples of formula RL ^B1 , SP ¹ is a divalent polyethylene glycol group, RG ⁿ is a 1,3-cycloadduct of the reaction between an alkyne and an azide.

In some embodiments, the coupling agent linker has the following structure:

-RG ^N -(SP ¹ )qZ ¹ -Z ² -Z ³ ₀ .i-, where RG ^N , SP ¹ are defined above; q means 0 or 1;

Z ¹ means a polyethylene glycol or caproyl group; Z ² means dipeptide or tripeptide; Z ³ means a para-aminobenzyl alcohol group.

In certain embodiments, RG ^N is a derivative of a click chemical reactive group, Z ¹ is a polyethylene glycol group. In certain embodiments, RGN-(SPl)q-Zl- means:

In some embodiments, the dipeptide is valine-citrulline or valine-alanine.

In some embodiments, the coupling agent linker is attached to the payload via a tertiary amine. For example, if the steroid is the following compound,

a reactive linker can bind to a tertiary amine as follows:

Some examples include the following connection: n

©R\©

BL-NR ^b where BL is a coupling agent linker as defined above; R ^a and R ^b are independent in each case

-57045579 simo means -H or alkyl.

In some examples herein, RGN means a derivative of a click chemical reactive group. In some examples, RGN means:

or a mixture thereof;

VA or a mixture thereof; where means the connection with the binding agent.

you m

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

RGN

means g

’ where * denotes a bond to the cysteine of a binding agent, such as an antibody. In some examples, RGN means

In some embodiments

SP ¹

In some examples SP ¹ means selected from:

In other examples SP ¹ means .o

In other examples SP ¹ means

ABOUT

In other examples SP ¹ means

• In other examples SP ¹ means

In any of the above examples, the subscripts a, b, and c each independently represent an integer from 1 to 20.

In some embodiments, R ^AA3 is selected from

CD ’ where CD means fragment

- 58 045579 cyclodextrin. In some embodiments, R ^AA3 is selected from

In any of the compounds of formula (II), (IIa), (IIIb) or (IIc), SP ¹ is selected from:

means

In some examples SP ¹ means

In some examples SP ¹

ABOUT

In some examples SP ¹ means

In some examples SP ¹ means measures SP ¹ means

In some examples SP ¹ means

In some examples SP ¹ means

O _{and CP} 1 begins ^g In some examples, SP means

In some cases

. In some examples SP ¹ means some examples SP ¹ means

In some variants

Explanation: In some examples, SP ¹ means implementation BL-SP ¹ means:

or or a mixture thereof;

or or a mixture thereof;

- 59 045579 or a mixture thereof;

or

In some of these examples, the subscripts b, c, and d each independently represent an integer from 1 to 20.

In any of the compounds of formula (II), (IIa), (IIb) or (IIc), BL-SP ¹ is selected from:

or mixtures thereof;

or mixtures thereof; or

- 60 045579

In some embodiments, A is a peptide selected from the group 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, glycinasparagine, asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid, citrulline-asparagine, asparagine-citrulline, alanine-asparagine, or asparagine-alanine.

IN

IN

IN

IN

IN

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

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

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

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

In some examples, A stands for valine.

In some examples, A stands for alanine.

In some examples, A is citrulline.

In some examples A means

In some of these examples, R ^AA1 is an amino acid side chain, RAA ² is an amino acid side chain.

In some examples A means

In some of these examples, R ^AA1 is an amino acid side chain, RAA ² is an amino acid side chain, and R ^AA3 is an amino acid side chain that is linked directly or indirectly to the cyclodextrin moiety.

In some examples A means

In some examples A means

In some examples A means

’ where represents a direct or indirect connection with the cyclodextrin moiety.

In some examples, including any of the following, the CD in each case is independently selected from

- 61 045579

In some examples, CD means

- 62 045579

In some examples, CD means 1 TO BUT-/ In some examples, CD means ι l ( l In some examples, CD means l· n In some examples, CD means BUT^l In some examples, CD means > with N- ^J but 0^ ^Vo Yn I BUT ⁷ . ⁿ % ^he ™ and but ⁷ θΎ NO ⁷ . > o N- ⁷ but <\^o bn ° ⁿ o ° HO ^Z NO _Dyu οΧ^°Η^ ξοΥ ⁰ no^on G o^° τ / G ON NO ΟΗ/χΟΗ O—ΓΤΌΗ but VV o^D^ o k/D-o / όΛ but / but

- 63 045579

In some examples A means

In some examples, R ^a is H.

In some examples, R ^a is alkyl.

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

In some embodiments, B is aryl.

In some examples, B is phenyl.

In some examples of compounds of formula (II), (IIa), (IIb) or (IIc), B is phenyl or pyridinyl.

In some of the examples given here, B means:

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

-|-or ^a -I-SO2R ⁷ ' NR ^a R ^b or azido. In these examples, the subscripts p and m are in each case independently selected from an integer from 0 to 4.

In some of the examples given here, B means:

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

In some embodiments, the formula (BL ^A ), -(NR ^a ) _s -(B) _t -(CH2)u-(O) _v -(SP ² )w means:

- 64 045579

The following are antibody-steroid conjugates having the following formulas:

- 65 045579

- 66 045579

- 67 045579

or a pharmaceutically acceptable salt or solvate thereof;

where BA means binder, x means an integer from 1 to 30.

In particular embodiments, BA means an antibody. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. The following are antibody-steroid conjugates having formula 1200:

or a pharmaceutically acceptable salt, solvate or stereoisomer thereof, where: BA is a binder; each L represents an optional linker; BA or L is covalently linked to R ³ or R ⁴ ; x means an integer from 1 to 30. The experienced reader will understand that if L is present, then L is related to R ³ or R ⁴ ; if L is absent, BA is bound to R ³ or R ⁴ . The R ³ or R ⁴ groups are described in detail below. In particular embodiments, BA means an antibody. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. In certain embodiments of Formula 1200, R ¹ and R ² are independently selected from

- 68 045579

R ⁴

0^0 group, including -H, -OH, alkyl, -O-C(O)-alkyl and halo; or R ¹ and R ² together form ·

In certain embodiments, R ³ is selected from the group consisting of -alkylene-NR ^a R ^b , -Charylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is absent or means absent or means -CH2-; R ⁴ is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, -alkylene-NR ^a R ^b , -X-arylene-YNR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is missing or means -CH ₂ -.

In certain embodiments of Formula 1200, R ³ is selected from the group consisting of OH, -O-C(O)-alkyl, -O-aryl, -NR ^a R ^b , -alkylene-NR ^a R ^b , -X-arylene- Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is absent or means absent or means -CH2-; R ⁴ is selected from the group consisting of -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means N-, -CH2- or -O-; where Y is absent or means absent or means -CH ₂ -.

In certain embodiments of Formula 1200, R ³ is -NR ^a R ^b ; and R ⁴ is alkyl.

In each embodiment of Formula 1200, BA or L is linked to a functional group on R ³ or R ⁴ . For example, if R ³ or R ⁴ includes an amine group, BA or L may be bonded to an amine group replacing a hydrogen atom. In each embodiment, R ⁵ is in each instance independently selected from a substituent on the group consisting of -OH, halo and alkyl; n means an integer from 0 to 19; each R ⁵ is located on any atom of the ring. In each embodiment, R ^a and R ^b are each independently selected from the group consisting of -H and alkyl; or R ^a and R ^b cyclize to form a cycloheteroalkyl with 3-6 ring atoms, including one atom that represents the N to which they are attached. In particular embodiments, BA means an antibody. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. The following are antibody-steroid conjugates having

or VA.

In certain embodiments of Formula 1210, 1220, 1230, or 1240, R ¹ and R ² are independently selected from the group consisting of -H, -OH, alkyl, -O-C(O)-alkyl, and halo; or R ¹ and R ² together form

In certain embodiments, R ³ is selected from the group consisting of -alkylene-NR ^a R ^b , -Charylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is missing or means -CH2-; R ⁴ is selected from the group consisting of alkyl, aryl, alkylaryl, arylalkyl, heteroaryl, -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N -containing heterocycloalkyl; where X is absent or means -N-, -CH2- or -O-; Y is absent or means absent or means -CH ₂ -. In certain embodiments, R ³ is NR ^a R ^b ; R ⁴ is alkyl. In each embodiment, BA or L is bonded to an amine group in R ³ , for example replacing a hydrogen atom. In each embodiment, R ^a and R ^b are each independently selected from the group consisting of -H and alkyl; or R ^a and R ^b cyclize to form a cycloheteroalkyl with 3-6 ring atoms, including one atom which is N, to which they attach

- 69 045579 secured. In particular embodiments, BA means an antibody. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. The following are antibody-steroid conjugates having the formula 1310, 1320, 1330, or 1340:

1310 1320

1330 1340 or a pharmaceutically acceptable salt, solvate or stereoisomer thereof; where R ⁴ is covalently linked to L or BA.

In certain embodiments of Formula 1310, 1320, 1330, or 1340, R ³ is selected from the group consisting of -OH, -O-C(O)-alkyl, -O-aryl, -NR ^a R ^b , -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH2 or -O-; where Y is absent or means absent or means -CH2-; R ⁴ is selected from the group consisting of -alkylene-NR ^a R ^b , -X-arylene-Y-NR ^a R ^b , -X-heteroarylene-Y-NR ^a R ^b and N-containing heterocycloalkyl; where X is absent or means -N-, -CH ₂ - or -O-; Y is missing or means or -CH ₂ -. In each embodiment, BA or L is bonded to an amine group in R ⁴ , for example replacing a hydrogen atom. In each embodiment, R ^a and R ^b are each independently selected from the group consisting of -H and alkyl; or R ^a and R ^b cyclize to form a cycloheteroalkyl with 3-6 ring atoms, including one atom that represents the N to which they are attached. In particular embodiments, BA means an antibody. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. The following are antibody-steroid conjugates having the following formulas:

or a mixture thereof;

- 70 045579

or a mixture thereof;

or a mixture thereof;

or a mixture thereof;

- 71 045579

or a mixture thereof;

or a mixture thereof;

or a mixture thereof;

- 72 045579

or a mixture thereof;

or a mixture thereof;

or a mixture thereof;

- 73 045579

wherein Ab is an antibody and x is an integer from 1 to 30. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. Also included below are antibody-steroid conjugates , having the following formulas:

or a mixture of them.

In particular embodiments, Ab is an antibody and x is an integer from 1 to 30.

In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2.

Below are also antibody-steroid conjugates having the following formula:

- 74 045579

Below are also antibody-steroid conjugates having the following formulas:

or a mixture thereof;

or a mixture thereof;

- 75 045579

or a mixture thereof;

- 76 045579

or a mixture thereof;

- 77 045579

or a mixture thereof;

or a mixture thereof;

- 78 045579

or a mixture thereof;

or a mixture thereof;

- 79 045579

or a mixture thereof;

or a mixture of them.

In particular embodiments, Ab is an antibody, x is an integer from 1 to 30. In some embodiments, x is an integer from 1 to 4. In some embodiments, x is 4. In some embodiments, x is 2. Conjugates are also provided herein budesonide or diflorazone binding agents. 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. As used herein, the term antibody means any antigen-binding molecule or molecular complex comprising 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 includes three domains, _CH1 , CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and _VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with regions that are

- 80 045579 are more conservative, called frame regions (FR). Each VH and V _L consists of three CDRs and four FRs, located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the framework regions of the antibody (or antigen-binding portion thereof) may be identical to the human germline sequence, or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more complementarity determining regions.

The term antibody as used herein also includes antigen-binding portions of the full antibody molecule. 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 an antigen to form a complex. Antigen-binding antibody fragments can be prepared, for example, from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibodies, variable and optionally constant domains. Such DNA is known and/or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to organize one or more variable and/or constant domains in a suitable configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc.

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

An antigen binding fragment of an antibody will typically contain at least one variable domain. The variable domain can be of any size or amino acid composition and will typically contain at least one complementarity determining region that is adjacent to or in a framework with one or more framework sequences. In antigen binding fragments having a _VH domain linked to a _VL domain, the VH and _VL domains may be located relative to each other in any suitable arrangement. For example, the variable region may be dimeric and contain dimers V _H -V _H , V _H -V _L or V _L -V _L . Alternatively, the antigen binding fragment of the antibody may comprise a monomeric V _H or V _L domain.

In certain embodiments, an antigen binding fragment of an antibody 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 found in an 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-CH ₂ -Ch3; (vi) Vh-CH ₂ -Ch3; (vii) VhCl; (viii) Vl-Ch1; (ix) Vl-Ch2; (x) Vl-Ch3; (xi) Vl-Ch1-Ch2; (xii) Vl-Ch1-CH2-Ch3; (xiii) Vl-SSH-SnZ; and (xiv) V _l -C _l . In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may either be directly linked to each other or may be linked by a full or partial hinge or linker region. The hinge region may consist of at least 2 (eg, 5, 10, 15, 20, 40, 60 or more) amino acids that result in flexible or semi-flexible linkage between adjacent variable and/or constant domains in one polypeptide of the molecule. In addition, the antigen binding fragment of an antibody of the present invention may contain a homodimer or heterodimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with each other and/or with one or more monomeric V _H or V _L domains (eg, disulfide bond(s)).

As with full antibody molecules, antigen-binding fragments can be monospecific or multispecific (eg, bispecific). A polyspecific antigen-binding antibody fragment will typically contain 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 exemplary bispecific antibody formats disclosed herein, can be adapted for use in the context of an antigen binding antibody fragment of the present invention using routine techniques available in the art.

The antibodies of the present invention may function through complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). The term complement-dependent cytotoxicity (CDC) refers to 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 (FcRs) (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and thereby lead to lysis of the target cells CDC and ADCC can be measured using 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 antibody's ability to fix complement and mediate cell-dependent cytotoxicity. Thus, the antibody isotype can be selected based on whether the antibody is desired to mediate cytotoxicity.

Antibodies useful for the compounds of the present invention 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 human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or somatic mutation in vivo), for example, in the complementarity determining region and, in particular, in CDR3. However, as used herein, the term human antibody is not intended to include antibodies in which complementarity determining region sequences derived from germline sequences 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 a natural, unmodified living organism.

In some embodiments, the antibodies may be recombinant human antibodies. As used herein, the term recombinant human antibody is intended to include all human antibodies that are produced, expressed, created or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a human recombinant combinatorial antibody library (described below), antibodies isolated from an animal (eg, mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res. 20: 6287- 6295), or antibodies produced, expressed, created or isolated by any other method that involves splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from germline human immunoglobulin sequences. However, under certain circumstances, such recombinant human antibodies are subject to mutagenesis in vitro (or, when an animal transgenic for human Ig sequences is used, somatic mutagenesis in vivo) and thus the amino acid sequences of the VH and _VL regions of the recombinant antibody are sequences that, although derived from and associated with germline VH and VL sequences, cannot naturally exist in the human germline antibody spectrum 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 heavy chain disulfide bond. In the second form, the dimers are not linked by interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, consisting of a covalently linked light and heavy chain (half-antibody). These forms are extremely difficult to separate, even after affinity purification.

The frequency of occurrence of the second form in different intact IgG isotypes is due to, but not limited to, structural differences associated with the isotype of the antibody hinge region. A single amino acid substitution in the hinge region of the human IgG4 loop can significantly reduce the occurrence of the second form (Angal et al. (1993) Molecular Immunology 30: 105) to levels typically observed using the human IgG1 hinge. The present disclosure covers antibodies having one or more mutations in the hinge, CH2 or CH3 region, which may be desirable, for example, in manufacturing, to improve the yield of the desired form of the antibody.

Antibodies useful for the compounds of the present invention may be isolated antibodies. The term isolated antibody, as used herein, means an antibody that has been identified and separated and/or recovered from at least one component thereof.

- 82 045579 natural environment. For example, an antibody that has been isolated or separated from at least one component of the body or from a tissue or cell in which the antibody naturally exists or is produced is an isolated antibody for purposes of the present disclosure. The isolated antibody also includes the antibody in situ in the recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. In certain embodiments, the isolated antibody may be substantially free of other cellular material and/or chemicals.

Antibodies useful for the compounds disclosed herein may include 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 antibodies were obtained. Such mutations can be easily identified by comparing the amino acid sequences described here with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof that are derived from any amino acid sequence described herein, wherein one or more amino acids in one or more framework and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was produced, either to 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 referred to herein collectively as germline mutations). One of ordinary skill in the art, starting with the heavy and light chain variable region sequences described herein, can readily produce numerous antibodies and antigen binding fragments that contain one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and/or complementarity determining region residues in the VH and/or _VL domains are mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, for example, only the mutated residues found in 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 residues and/or CDRs are mutated to the corresponding residue(s) of a different germline sequence (ie, a germline sequence that is different from the germline sequence from which the antibody was originally derived). In addition, antibodies of the present invention may contain any combination of two or more germline mutations within framework regions and/or complementarity determining regions, for example, where certain individual residues are mutated to the corresponding residue of a particular germline sequence, while certain other residues that differ from the original germline sequence are retained or mutate into the corresponding residue of another germline sequence. Once produced, antibodies and antigen binding fragments that contain one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as appropriate) , reduced immunogenicity, etc.

In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, an antibody fragment (Fab, Fab' and F(ab) ₂ , mini-antibody, diabody, tribody, etc.), or a bispecific antibody. The antibodies herein may be humanized using the methods described in US Patent No. 6,596,541 and US Publication No. 2012/0096572, each of which is incorporated by reference in its 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 that may be glycosylated or phosphorylated.

Suitable targets to which the coupling agent binds include any target to which delivery of steroids is desired. In some cases, the binding agent is an antibody, modified antibody, or antigen-binding fragment that binds a target selected from the following: AXL, BAFFR, BCMA, BCR-listed components, BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3 , CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD168, CD177, CD19, 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, 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,

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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, Siglec10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, TCR List Components/Helpers, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4, and VTCN1 .

Binding agent linkers can be linked to a binding agent, such as an antibody or antigen binding molecule, by attaching to a specific amino acid in the antibody or antigen binding molecule. Exemplary amino acid linkages that may be used in the context of this aspect of the 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 eg WO 2012/05982). Linkers can be conjugated via glutamine via transglutaminase-based chemotherapeutic conjugation (see, for example, Dennler et al., Bioconjugate Chem. 2014, 25, 569-578). Linkers can also conjugate to antigen binding protein by attaching 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 examples, the linker is an antibody, and the antibody is linked to the linker via a lysine residue. In some embodiments, the antibody is linked to the linker via a cysteine residue.

D. Methods for preparing compounds.

The conjugates described herein can be synthesized by coupling the payload linkers 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; Int. J. Mol. Sci. 2016, 17, 561, incorporated herein by reference in its entirety). Payload linkers are synthetic intermediates comprising the payload of interest and a coupling moiety that ultimately serves as the moiety (or part thereof) that links the coupling agent to the payload. Payload linkers contain a reactive group that reacts with a coupling agent to form the conjugates described herein. When the linking agent is an antibody, the antibody may be linked to a linker payload through one or more cysteine, lysine, or other residues of the antibody. Linker payloads can be linked to cysteine residues, for example, by exposing the antibody to a reducing agent, such as dithiotherite, to cleave the antibody's disulfide bonds, purifying the reduced antibody, such as by gel filtration, and then reacting the antibody with a linker payload containing the reactive fragment, for example, the maleimido group. Suitable solvents include, but are not limited to, water, DMA, DMF and DMSO. Linker payloads 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 technologies, including, for example, size exclusion chromatography, dialysis and ultrafiltration/diafiltration.

Binding agents, such as antibodies, can also be conjugated via a chemical click reaction. In some embodiments of the chemical click reaction, the payload linker 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, e.g., alkynes that are suitable for strain-facilitated azide-alkyne cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzanulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with azides in the absence of copper catalysts. Suitable alkynes also include, but are not limited to, DIBAC, DIBO, BARAC, DIFO, substituted, for example, fluorinated alkynes, azacycloalkynes, BCN and derivatives thereof. Payload linkers 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, with a compound of the formula H ₂ N-LL-N ₃ , where LL is a divalent group

- 84 045579 polyethylene glycol in the presence of the enzyme transglutaminase. For convenience, in some of the formulas herein, the Ab antibody is a modified antibody with one or more covalently linked -LL-N3 groups or residues thereof. Preferably, each -LL-N3 is covalently linked to an amino acid side chain of a glutamine residue of the antibody. Also preferably, -LL-N3 is or can react with a reactive RG group to form a covalent bond with the payload linker. Again, for convenience, the -LL-N3 groups are clearly labeled in some of the formulas here.

Set forth below are methods for synthesizing the conjugates described herein, which involve contacting a coupling agent, such as an antibody, with a payload linker described herein. In certain embodiments, the payload linker includes a cyclodextrin moiety.

In some embodiments, the payload linker is a compound of formula (II):

R ³ (P) (R ^p )t

RL—X—(CH ₂ ) ₀ . ₂ —^A^-X— (a) R ³ means RL-, RL-X-, or '

R ¹ and R ² are each independently -H, alkyl, alkyl-C(O)-O-, -OH or halo; or

R ⁴ O^O

R ¹ and R ² together form ' where R ⁴ is alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; wherein alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl are optionally substituted with -NRaRb; or (b) R ³ is -OH, alkyl-C(O)-O-, heteroalkyl, -NRaRb or aryloxy, wherein alkyl-C(O)-O-, heteroalkyl or aryloxy is optionally substituted with -NRaRb or halo, and R ¹ and

R ⁴ oAe rl-x-^a^-(CH ₂ )o. ₂ —

R ² together form > where R ⁴ means -RL-, '

BL-X-(CH ₂ ) _1-4 - or RL-Y, Y is an N-containing divalent heterocycle;

RL means reactive linker;

R ⁵ in each occurrence is independently -OH, halo, alkyl or arylalkyl;

Ra and R ^b are each independently -H or alkyl;

R ^P in each case independently means halo;

X in each case independently means NRa or O;

O is aryl or heteroaryl;

n means an integer from 0 to 19.

The compounds of formula (II) are linker payloads that are useful as synthetic intermediates in the synthesis of the conjugates described herein. These payload linkers include a reactive group that can react with the antibody to form the conjugates described herein.

In some examples of formula (II), R ¹ and R ² are each independently -H, alkyl or -OH. In some examples of formula (II), one of R ¹ or R ² is -H, alkyl or -OH. In some examples of formula (II), R ¹ and R ² are either -H, alkyl or -OH.

R ⁴

0^0

In some examples, according to formula (II), R ¹ and R ² together form

In some examples, R ⁴ is -RL. In some examples, R ⁴ is RL-NRa-aryl. In other examples, R ⁴ is alkyl. In certain examples, R ⁴ is arylalkyl. In some examples, R ⁴ is aryl. In other examples, R ⁴ is N-containing heterocycloalkyl. In some of these examples, alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl is optionally substituted.

In some examples of Formula (II), R ⁵ is halo. In some examples of formula (II), R ⁵ is fluorine. In some examples of Formula (II), one of R ⁵ is halo. In some examples of formula (II), R ⁵ is halo, n is 2. In some examples of formula (II), R ⁵ is -F, n is 1. In some examples of formula (II), R ⁵ is -F, n means 2.

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In some examples of Formula (II), R ³ is RL. In some examples of formula (II), R ³ is RL-NR ^a -aryloxy-. In some other examples of formula (II), R ³ is -OH. In some other examples of formula (II), R ³ is alkyl-C(O)-O-. In some other examples of formula (II), R ³ is heteroalkyl. In some other examples of formula (II), R ³ is -NR ^a R ^b . In some other examples of formula (II), R ³ is aryl. In some other examples of formula (II), R ³ is aryloxy. In some other examples of formula (II), alkyl-C(O)-O-, heteroalkyl or aryloxy is optionally substituted with -NR ^a R ^b or halo.

In some examples of formula (II), R ³ is -OH. In some examples of formula (II), R ³ y is alkyl-C(O)-O-. In some examples, according to formula (II) R ³ means ^Нз ^ ^Нз ·

In some examples of formula (II), R ³ is heteroalkyl. In some examples according to formula (II) R ³ means ^H3C ^ or

? ^L /K/

N (5

In some examples of formula (II) R ³ means ^H3C or ³ ·

In some examples of formula (II), R ³ is -NR ^a R ^b . In some examples, according to formula (II) ^R 4 J

R ³ means ^R

H39

In some examples of formula (II), R ³ is

R ^a • In some examples of formula (II), R ³ is ^Rb . In some examples of formula (II), R ³ is aryloxy. In some examples of formula (II) R ³ means

In some examples, according to formula (II) R ³ η ₂ ν' means ⁴

R.L.

In some examples of formula (II) R ³ means

In some examples, according to formula (II) R ³ means ^ΗζΝ

In some examples of formula (II) R ³ means

In some examples of formula (II) R ³ is ^H2N

examples according to formula (II) R ³ means

In some

In some examples of formula (II) R ³ means

In some examples of formula (II), R ³ is

. At some point

In many examples according to formula (II) R ³ means

According to formula (II), the subscript n means an integer from 0 to 19. In some examples, n means 0. In other examples, n means 1. In certain examples, n means 2. In other examples, n means 3. In certain examples, n means 4 In some examples, n means 5. In other examples, n means 6. In certain examples, n means 7. In other examples, n means 8. In certain examples, n means 9. In some examples, n means 10. In other examples, n means 11. In certain examples, n is 12. In other examples, n is 13. In certain examples, n is 14. In some examples, n is 15. In other examples, n is 16. In certain examples, n is 17. In other examples, n is 18. In In certain examples, n is 19. In certain examples, a compound having the structure of formula (IIa) is given here:

- 86 045579

where R ⁵ in each case is independently -OH, halo or alkyl;

R ³ is selected from -OH, alkyl-C(O)-O-, heteroalkyl, -NR ^a R ^b or aryloxy, wherein alkyl-C(O)-O-, heteroalkyl or aryloxy is optionally substituted with -NR ^a R ^b or halo ;

RL means reactive linker;

R ^a and R ^{b are} in each case independently selected from H, alkyl and alkyl-C(O);

n means an integer from 0 to 19.

In some examples, a compound having the structure of formula (IIa2) is given here:

RL—I

ABOUT

R ³ (Pa2);

where R ⁵ in each case is independently -OH, halo or alkyl;

R ³ is -OH, alkyl-C(O)-O-, heteroalkyl, -NR ^a R ^b or aryloxy, where alkyl-C(O)-O-, heteroalkyl or aryloxy is optionally substituted with -NR ^a R ^b or halo;

RL means reactive linker;

R ^a and R ^b are in each case independently selected from H, alkyl, or alkyl-C(O);

n means an integer from 0 to 19.

In some examples of formula (IIa2), R ³ is -OH. In some examples, according to formula (IIa2) 0^0^

R ³ means alkyl-C(O)-O-. In some examples, R ³ is ^{H3 CH3.} In some examples, in n H ₃ SG formula (IIa2), R ³ is heteroalkyl. In some examples, R ³ means • In some sn ₃ .N n ₃ C/ examples according to formula (IIa2) R ³ means -NR ^a R ^b . In some examples, R ³ is • In some examples of formula (IIa2), R ³ is aryloxy. In some examples, according to formula (IIa2) R ³ means

In some examples, according to formula (IIa2) R ³ means

In some examples, according to formula (IIa2) R ³ means ^H2 '

F

ABOUT.

In some examples, according to formula (IIa2) R ³ h ₂ n means

• In some examples, using formula (IIa2), R ³ means

In some examples, the compound of formula (IIa2) has the following structure:

where R ³ means -OH or alkyl-C(O)-O-;

R ^5a and R ^5b are each independently -F or H;

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RL means reactive linker.

In some examples, a compound having the structure of formula (IIb) is given here:

(p');

where R ⁵ in each case is independently -OH, halo or alkyl;

R ⁴ is selected from alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl, wherein the alkyl, aryl, arylalkyl or N-containing heterocycloalkyl is optionally substituted with -NR ^a R ^b ;

RL means reactive linker;

R ^a and R ^{b are} in each case independently selected from H, alkyl and alkyl-C(O);

n means an integer from 0 to 19.

In some examples of formula (IIb), R ⁵ is a halo. In some examples of formula (IIb), R ⁵ is fluorine. In some examples of formula (IIb), n is at least 2 and two R ⁵ is halo. In some examples of formula (IIb), R ⁵ is F, n is 1. In some examples of formula (IIb), R ⁵ is -F.

In some examples of formula (IIb), R ⁴ is alkyl. In some examples of formula (IIb), 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 examples of formula (IIb), R ⁴ is n-propyl.

In some examples, the compound of formula (IIb) has the following structure:

where R ⁴ means alkyl;

R ^5a and R ^5b are each independently -F or H;

RL means reactive linker.

In some examples, a compound having the structure of formula (IIc) is given here:

(Ps);

where R ¹ and R ² independently mean -H, alkyl, alkyl-C(O)-O-, -OH or halo;

R ⁵ is in each case independently selected from -OH, halo or alkyl;

RL means reactive linker;

n means an integer from 0 to 19.

In some examples of formula (IIc), R ⁵ is halo. In some examples of formula (IIc), R ⁵ is fluorine. In some examples of formula (IIc), one of R ⁵ is a halo. In some examples of formula (IIe), two R ⁵ means halo. In some examples of formula (IIc), R ⁵ is -F, n is 2.

In some examples of formula (IIc), R ¹ is CH3.

In other examples of formula (IIc), R ¹ is OH.

In some other examples of formula (IIc), R ¹ is H.

In some examples of formula (IIc), R ² is CH3.

In other examples of formula (IIc), R ² is OH.

In some other examples of formula (IIc), R ² is H.

In some examples of formula (IIc), R ¹ is CH3, R ² is CH3.

In other examples of formula (IIc), R ¹ is CH3, R ² is OH.

In some examples of formula (IIc), R ¹ is CH3, R ² is H.

In some other examples of formula (IIc), R ¹ is OH, R ² is CH3.

In other examples of formula (IIc), R ¹ is OH, R ² is OH.

In some examples of formula (IIc), R ¹ is H, R ² is H.

In some other examples of formula (IIc), R ¹ is H, R ² is OH.

In other examples of formula (IIc), R ¹ is H, R ² is H.

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In some embodiments, a compound of formula (IIc) has the following structure:

(Ps)

R ² means methyl;

R ^5a and R ^5b are each independently -F or H; And

RL means reactive linker.

In certain embodiments, provided herein is a compound having the structure of

R ¹ and R ² are each independently -H, alkyl, alkyl-C(O)-O-, -OH or halo; or R ⁴

O^O

R ¹ and R ² together form ' where R ⁴ is alkyl, aryl, arylalkyl, or N-containing heterocycloalkyl; wherein alkyl, aryl, arylalkyl and N-containing heterocycloalkyl are optionally substituted -NR ^a R ^b ;

R ⁵ in each occurrence is independently -OH, halo, alkyl or arylalkyl;

R ^a and R ^b are each independently -H or alkyl;

RP in each case independently means halo;

(e) o means aryl or heteroaryl;

t means an integer from 0 to 2;

x means an integer from 1 to 30;

wherein RL means a reactive linker as defined below; SP ¹ and SP ² each independently mean the absence or remainder of a spacer group, where SP ¹ includes a trivalent linker; AA ¹ means a trivalent linker comprising an amino acid residue; AA ² means a dipeptide residue; PEG means polyethylene glycol residue; indicates the atom through which the specified chemical group is linked to the nearest group in the formula, CD in each case is independently absent or means a cyclodextrin residue in which at least one CD is present, the subscript m means an integer from 0 to 5; In these examples, the subscript m means 0, 1, 2, 3, 4, or 5. In some examples, the subscript m means 0. In some examples, the subscript m means 1. In some examples, the subscript m means 2. In some examples, the subscript m means 2. the subscript m is 3. In some examples, the subscript m is 4. In some examples, the subscript m is 5. In some examples, any of AA ¹ or AA ² independently includes in each case an amino acid selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or citrullinaline, valine, leucine 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 selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, glycine, 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. In certain embodiments, AA ¹ is lysine or a lysine derivative. 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

- 89 045579 glycine. In some embodiments, AA ² is glycine-valine. In some embodiments, AA ² is glutamate-valine-citrulline. In some embodiments, AA ² is glutamine-valine-citrulline. In some embodiments, AA ² is lysine-valine-alanine. In some embodiments, AA ² is lysine-valine-citrulline. In some embodiments, AA ² is glutamate-valine-citrulline. In some examples, SP ¹ is in each instance independently selected from the group consisting of C1-6 alkylene, -NH-, -C(O)-, (-CH2-CH2-O)e, -NH-CH2-CH2-(-OCH2 -CH2)eC(O)-, -C(O)-(CH2)uC(O)-, -C(O)-NH-(CH2)v- and their combinations, where the subscript e means an integer from 0 to 4, the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. In some examples, SP ² is in each instance independently selected from the group consisting of C1-6 alkylene, -NH-, - C(O)-, (-CH3-CH3-O^, -NH-CH2-CH2-(-O-CH2-CH2)eC(O)-, -C(O)-(CH2)UC(O)- , -C(O)-NH-(CH ₂ ) _v - and combinations thereof, where the subscript e means an integer from 0 to 4, the subscript u means an integer from 1 to 8, and the subscript v means an integer from 1 to 8. In certain embodiments, provided herein is a compound having the structure of formula (IIIc-R):

RL means reactive linker;

CD means cyclodextrin;

SP ¹ means spacer group;

AA ⁴ means an amino acid residue;

AA ⁵ means a dipeptide residue;

PEG means polyethylene glycol;

m means an integer from 0 to 4;

x means an integer from 0 to 30;

R ⁴ is alkyl, aryl, arylalkyl or N-containing heterocycloalkyl; wherein the alkyl, aryl, arylalkyl, and N-containing heterocycloalkyl are optionally substituted -NR ^a R ^b ; R ^a and R ^b are each independently -H or alkyl;

SP ¹ and SP ² each independently mean the absence or remainder of a spacer group, where SP ¹ includes a trivalent linker; AA ⁴ means a trivalent linker comprising an amino acid residue; AA ⁵ means a dipeptide residue; PEG means polyethylene glycol residue;

indicates the atom through which the specified chemical group is bonded to the nearest group in the formula, CD in each case is independently absent or means a cyclodextrin residue in which at least one CD is present, the subscript m denotes an integer from 0 to 5. In these examples, the subscript m subscript m means 0, 1, 2, 3, 4., or 5. In some examples, subscript m means 0. In some examples, subscript m means 1. In some examples, subscript m means 2. In some examples, subscript m means 3. In some examples, the subscript m means 4. In some examples, the subscript m means 5. In some examples, any one of AA ⁴ or AA ⁵ in each case independently includes an amino acid 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 selected from alanine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine, proline, glycine, 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 is lysine-valine-alanine. In some embodiments, AA ⁵ is lysine-valine-citrulline. In some embodiments, AA ⁵ is glutamate-valinecitrulline. In some examples, SP ¹ is in each case independently selected from the group consisting of C _1-6

- 90 045579 alkylene, -NH-, -C(O)-, (-CH2-CH2-O)e, -NH-CH2-CH2-(-O-CH2-CH2)eC(O)-, -C( O)-(CH2)uC(O)-, -C(O)-NH(CH ₂ ) _v - and their combinations, where the subscript e means an integer from 0 to 4, the subscript and means an integer from 1 to 8, the subscript v denotes an integer from 1 to 8. In some examples, SP ² is in each instance independently selected from the group consisting of C _1-6 alkylene, -NH-, -C(O)-, (-CH ₂ -CH ₂ -О) _е , -NHCH2-CH2-(-O-CH2-CH2)eC(O)-, -C(O)-(CH2)uC(O)-, -C(O)-NH-(CH2 )v- and combinations thereof, where the subscript e is an integer from 0 to 4, the subscript u is an integer from 1 to 8, and the subscript v is an integer from 1 to 8. As used herein, the phrase reactive linker or the abbreviation RL refers to a monovalent group which includes a reactive group and an RG-L—linking group, depicted as ' where RG is a reactive group and L is a linking group. A linking group is any divalent moiety that connects a reactive group to a payload. A linking group also includes any trivalent group that links the reactive group, the cyclodextrin group and the payload. Reactive linkers (RL), together with the payloads to which they are linked, contain intermediates (payload linkers) used as synthetic precursors to prepare the steroidal antibody conjugates described herein. The reactive linker contains a reactive group (RG), which is a functional group or fragment that reacts with a reactive portion of an antibody, modified antibody, or antigen-binding fragment thereof. The fragment obtained by reacting a reactive group with an antibody, a modified antibody, or an antigen-binding fragment thereof, together with a linking group, contains a fragment of a linker binder (BL) conjugate described herein. 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 moiety thereof. In certain embodiments, a reactive group means a functional group or group that is capable of undergoing a chemical click reaction. In some embodiments of the chemical click reaction, the reactive group is 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, e.g., alkynes that are suitable for strain-facilitated azide-alkyne cycloadditions (SPAAC), cycloalkynes, e.g., cyclooctynes, benzanulated alkynes, and alkynes capable of undergoing 1,3-cycloaddition reactions with alkynes in absence of copper catalysts. Suitable alkynes also include, but are not limited to, DIBAC, DIBO, BARAC, substituted, for example, fluorinated alkynes, azacycloalkynes, BCN and derivatives thereof. Payload linkers 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, with a compound of the formula H2N-LL-N ₃ , where LL is, for example, a divalent polyethylene glycol group, or where LL is a trivalent group, which includes polyethylene glycol and a cyclodextrin group, in the presence of the enzyme transglutaminase. In some studies, antibody means a functionalized antibody having the following structure:

where Ab is an antibody, R is a hydrocarbon radical, n is an integer from 1 to 10, w is an integer from 1 to 10. In certain embodiments, R is ethylene. In certain embodiments, n is 3. In certain embodiments, w is 2 or 4. In certain examples, the reactive group is an alkyne, for example, 'co-_ __ _____ _ ______ ____ ... Ν=Ν=ΐΛ _____ _ _ _____ __ which can enter into a click reaction with an azide, for example, forming a click reaction product, naCHa

Arb _{in b is} an example, 'its regioisomer or a mixture of them. In some examples, the reactive group is an alkyne, for example,

’ which can enter into a click reaction with an azide, for example N=N=N _ mer, ’ forming a click reaction product, for example,

sn

In some examples, the reactive group is an alkyne, for example, > which „ ________ _ _ „„ ______ _ _ N=N=N ________ _ _ _ _ _ _ can undergo a click reaction with an azide, for example, ' forming a click reaction product, For example,

' its regioisomer or mixture thereof. In some examples, the reactive group means a functional group, for example, ^O

’ which reacts with the cysteine residue on anAb„

antibody or its antigen-binding fragment, forming a bond between them, for example, ' where Ab refers to the antibody or its antigen-binding fragment, and S refers to the S atom in the cysteine residue through which the functional group binds to the Ab. In some examples, reactionary

o o y group means a functional group, e.g. ^O ', which reacts with a cysteine residue on an antibody or antigen-binding fragment thereof, forming a bond between them, o e.g.

AB

’ where Ab refers to an antibody or an antigen-binding fragment thereof, and NH- refers to the end of the lysine residue through which the functional group binds to the Ab. In some examples, this N atom is located in a lysine residue through which the functional group bonds

indicated here by the letter N above the connection, for example,

In some embodiments, RL is a monovalent moiety of the formula (RLA);

RG-(SP ¹ )q-(A)z-(NR ^a )s-(B)t-(CH ₂ ) _u -(O) _v -(SP ² ) _w -(RL ^A ); where RG denotes a reactive group; A means amino acid or peptide; R ^a is H or alkyl;

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

In some embodiments, RL is RG-(SP ¹ )q-(A)z-. In some embodiments, RL is RG-(SP ¹ )q-(A)2-. In some embodiments, RL is a fragment of the formula (RLA ¹ )

RG—(SP ¹ )—R'

O (RL ^A1 ) where R ^aa1 and R ^AA2 in each case independently represent amino acid side chains. In some examples of formula RL ^A1 SP ¹ is a divalent polyethylene glycol group, RG is a group comprising an alkyne capable of 1,3-cycloaddition with an azide.

In some embodiments, the RL has the following structure:

RG-(SP ¹ )qZ ¹ -Z2-Z ³ _0-1 -, where RG, SP ¹ and q correspond to the definition given here;

- 92 045579

Z ¹ means a polyethylene glycol or caproyl group;

Z ² means dipeptide; And

Z ³ means a para-amino-benzyl alcohol group.

In some other embodiments, BL is a trivalent moiety of the formula (BLB);

-RG ^N -(SP ¹ )q-(A)z-(NR ^a )s-(B) _t -(CH ₂ ) _u -(O) _v -(SP ² ) _w -(BL ^B ); where RG ^N corresponds to the definition given here;

A means a tripeptide in which at least one of the amino acids in the tripeptide is linked directly or indirectly to a cyclodextrin moiety; R ^a is H or alkyl;

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

In some examples, the cyclodextrin (CD) is linked directly to an amino acid residue, such as a lysine acid residue. This means that the CD is located one bond position away from the covalent linker of the amino acid lysine. In some of these examples, the covalent linker is also linked directly to the payload moiety. This means that the covalent linker is one bond position away from the payload, including such as the steroid payload described herein. In some of these examples, the covalent linker is also linked directly to the CD moiety. This means that the covalent linker is one bond position away from the CD, such as the CD described here. In some of these examples, the covalent linker is lysic acid or a derivative thereof.

In some examples, the CD is linked indirectly to a covalent linker in the binding group (eg, BL). This means that the CD is more than one bond position away from the covalent linker. This also means that the CD is linked through another fragment to a covalent linker. For example, CD may be linked to a maleimide group, which is linked to a polyethylene glycol group, which is linked to a covalent linker. In some of these examples, the covalent linker is also indirectly associated with the payload moiety. This means that the covalent linker is more than one bond position away from the payload, including the steroid payload described herein. This also means that the covalent linker is linked through another group to the payload. For example, the covalent linker may be linked to a dipeptide, including Val-Ala or Val-Cit, which may be linked to a para-amino benzoyl, which may be linked to the payload. In some of these examples, the covalent linker is also indirectly linked to the cyclodextrin moiety. This means that the covalent linker is more than one bond position away from the cyclodextrin, such as the cyclodextrins described herein. This also means that the covalent linker is linked 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, which may be linked to a cyclodextrin. In some of these examples, the covalent linker is lysic acid or a derivative thereof. In some embodiments, BL is -RG ^N -(SP ¹ )q-(A)z-. In some embodiments, BL is -RG ^N -(SP ¹ )q-(A) ₂ -. In some embodiments, BL is a group of the formula (BL ^B1 ) o r ^aa1 _u o

II | p || where RAA ¹ and r ^AA2 in each case independently represent amino acid side chains. R ^AA3 means an amino acid side chain that is linked directly or indirectly to the cyclodextrin moiety. In some examples of formula RL ^B1 SP ¹ is a divalent polyethylene glycol group, RG ⁿ is a 1,3-1,3-cycloadduct of the reaction between an alkyne and an azide.

In some examples A means

About RAA ¹ _s About

II I and II

In some of these examples, R ^AA1 is an amino acid side chain, RAA ² is an amino acid side chain, and RAA ³ is an amino acid side chain that is linked directly or indirectly to the cyclodextrin moiety.

In some examples A means

- 93 045579

where -CD represents a direct or indirect connection with the cyclodextrin moiety. In some examples, including any of the above, the CD in each case is independently selected from

In some examples, CD means

- 94 045579

In some examples, CD means

- 95 045579

In some embodiments, RL is coupled to a tertiary amine. For example, if steroid means the following compound,

RL can bind to a tertiary amine as follows:

Some examples here include the following connection:

- 96 045579

where RL is a reactive linker as defined herein;

R ^a and R ^b are each independently -H or alkyl.

In some of the examples given here, RG is selected from the chemical click reaction group.

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

In some embodiments, RG means

In some examples, RG means • In other examples, RG means /O • In other examples, RG means

Γ ^ν 7 • In some examples RG means ^o In other examples RG means

In other examples, RG means

In some embodiments, SP ¹ may be selected from:

O means

In some examples SP ¹

In other examples SP ¹ means o

means

In other examples SP ¹ means 'o'

. In other examples SP ¹ • In other examples SP ¹ means

In any of the above examples, the subscripts a, b, and c each independently represent an integer between 1 and 20.

In any of the compounds of formula (II), (IIa), (IIb), or (IIc), SP ¹ may be selected from:

- 97 045579

In some examples SP ¹ means o

In some

In some examples SP ¹

ABOUT

In some examples SP ¹ means in examples SP ¹ means o means

In some examples SP ¹ means

In some examples SP ¹ means tea:

. In some examples SP ¹ means O

In some examples SP ¹ means O

IN

In some examples SP ¹ in some examples SP ¹ means means

In some embodiments, RL-SP ¹ may be selected from the group including

In some of these examples, the subscripts b, c, and d each independently represent an integer from 1 to 20.

- 98 045579 o

• In some examples, RL-SP ¹ means

In some examples, RL-SP ¹ means but

In some examples RL-SP ¹

In any of the compounds of formula (II), (IIa), (IIb) or (IIc), RL-SP ¹ is selected from:

means

In some embodiments, A is a peptide 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, glycinasparagine, asparagine-glycine, glutamic acid-asparagine, asparagine-glutamic acid, cyt

- 99 045579 rollin-asparagine, asparagine-citrulline, alanine-asparagine or asparagine-alanine.

In some examples, A is valine-citrulline or citrulline-valine. In some examples, A is valine-alanine or alanine-valine. In some examples, A is valine. In some examples, A is alanine. In some examples, A is citrulline.

In some examples A means

In some of these examples, R ^AA1 is the amino acid chain.

In some examples A means

stands for amino acid side chain, RAA ² stands for side chain. In some examples, A stands for

In some examples, Ra is H

In some examples, Ra is alkyl

In some examples, Ra is methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, or pentyl.

In some embodiments, B is aryl.

In some examples, B is phenyl.

In some examples of compounds of formula (II), (IIa), (IIb) or (IIc), B is phenyl or pyridinyl.

In some of the examples given here, B means:

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

-hO ₂ RA _ . _ ..... . _____ _____ ''' NR R ^b or azido. In these examples, the subscripts p and m are in each case independently selected from an integer from 0 to 4. In some of the examples given here, B means:

(R ¹ ) _P

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

In some embodiments, the formula (RL ^A ), -(NR ^a )s-(B) _t -(CH ₂ ) _u -(O) _v -(SP ² )w means: o

Linkers containing budesonide and diflorazone payloads are also provided here. In some embodiments, a linker with a payload having the following structure is provided:

where RL denotes a reactive linker.

- 100 045579

Examples of payload linkers include:

- 101 045579

- 102 045579

- 103 045579

- 104 045579

- 105 045579

- 106 045579

and their salts.

E. Pharmaceutical compositions and methods of treatment.

The present disclosure includes methods of treating diseases, conditions or disorders, such as inflammatory diseases and autoimmune disorders, or treating symptoms thereof, comprising administering a therapeutically effective amount of one or more compounds disclosed herein

- 107 045579 neniya. Included are any diseases, disorders or conditions associated with the glucocorticoid receptor, glucocorticoid binding and/or glucocorticoid receptor signaling. Such methods include administering to a patient a steroid payload or a protein conjugate thereof described herein. Thus, included in this disclosure are methods of treating a disease, disorder, or condition associated with a glucocorticoid receptor, comprising administering a compound of formula (I), (I) ¹ or a protein conjugate thereof, e.g., a compound of formula (III), to a patient having disease, disorder or condition. Disclosed herein are methods of treating a disease, disorder or condition associated with a glucocorticoid receptor, comprising administering a protein conjugate of a compound of formula selected from the group consisting of (A), (A ¹ ), (A ² ), (A ³ ), (A ⁴ ), (A ⁵ ), (A ⁶ ), (A ⁷ ), (I), (I ¹ ), (PIa), (PIb-1), (PIb-2), (PIc-1) , (PIc-2), (PId-1), (PId-2), (PIt-1), (PIe-2), (PII), (PIIa), (PIIb), (PIII), (PIIIa) , (PIIIb), (PIV), (PV), (PVa), (PVb), (PVI), (PVII), (PVIIa), (PVIIb), (PVIIb-1), (PVIIb-2), ( PVIII) and their combinations.

In some embodiments, the disorder or condition means an allergic condition, including, but not limited to, asthma, atopic dermatitis, contact dermatitis, drug hypersensitivity reactions, perennial or seasonal allergic rhinitis, and serum sickness; dermatological diseases, including bullous dermatitis herpetiformis, exfoliative erythroderma, mycosis fungoides, pemphigus and severe erythema multiforme (Stevens-Johnson syndrome); endocrine disorders including, but not limited to, primary or secondary adrenal insufficiency, congenital adrenal hyperplasia, cancer-associated hypercalcemia, and non-autoimmune thyroiditis; 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; nervous system disorders, including, but not limited to, acute attacks of multiple sclerosis, cerebral edema associated with a primary or metastatic brain tumor, craniotomy or head trauma; ophthalmological diseases, including, but not limited to, sympathetic ophthalmia, temporal arteritis, uveitis and inflammatory diseases of the eyes that are not treatable with topical corticosteroids; kidney disease, including, but not limited to, to induce diuresis or remission of proteinuria in idiopathic nephrotic syndrome or due to lupus erythematosus; respiratory diseases, including, but not limited to, berylliosis, fulminant or disseminated pulmonary tuberculosis when used simultaneously 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 help the patient during acute episodes or exacerbations) for acute gouty arthritis, acute rheumatic carditis, ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, including juvenile rheumatoid arthritis and for use in dermatomyositis, polymyositis and systemic lupus erythematosus.

Some examples set forth herein provide a method of treating a disease, disorder, or condition selected from the following: autoimmune disease, allergies, arthritis, asthma, respiratory disorders, blood disorders, cancer, collagen disease, connective tissue diseases, dermatological diseases, diseases eye, endocrine problems, immunological diseases, inflammatory diseases, intestinal disorders, gastrointestinal diseases, neurological disorders, organ transplant conditions, rheumatoid disorders, skin disorders, edema conditions, wound healing conditions, and a combination thereof involving administration of a payload of a steroid or its conjugate described here.

In some cases, the autoimmune disease is selected from the following: multiple sclerosis, autoimmune hepatitis, herpes zoster, systemic lupus erythematosus (i.e., lupus), myasthenia gravis, Duchenne muscular dystrophy, and sarcoidosis. In some examples, the breathing disorder is selected from the following: asthma, chronic obstructive pulmonary disease, bronchitis, and acute bronchitis. In some examples, the cancer is selected from the following: leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), and multiple myeloma. In some examples, collagen disease is systemic lupus erythematosus. In some examples, the eye disease is keratitis. In some examples, the endocrine problem is selected from Addison's disease, adrenal insufficiency, adrenocortical and congenital adrenal hyperplasia. In some examples, the inflammatory disease is selected from joint inflammation, tendon inflammation, bursitis, epicondylitis, Crohn's disease, inflammatory bowel disease, lipid pneumonitis, thyroiditis, urticaria (urticaria), pericarditis, nephrotic syndrome, and uveitis. In some examples, intestinal disorders are selected from ulcerative colitis, Crohn's disease, and inflammatory bowel disease. In some cases, rheumatoid disease selected from rheumatoid arthritis, polymyalgia rheumatica, psoriatic

- 108 045579 arthritis, ankylosing spondylitis and systemic lupus erythematosus. In some examples, the skin disease is selected from psoriasis, eczema, and dermatitis. In some examples, the neurological disorder is chronic inflammatory demyelinating polyradiculoneuropathy.

In some cases, the provided 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 methods of treatment, 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.

Some examples set forth herein provide 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. Certain examples set forth herein provide 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. Certain examples set forth herein provide 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. Certain examples set forth herein provide 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. Some examples set forth herein describe a method of treating hypercalcemia caused by cancer, Meniere's disease, migraine, cluster headache, 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 payload of a steroid or a conjugate thereof described herein. In some examples, the compounds disclosed herein may be used to treat inflammatory bowel disease, Crohn's disease, or ulcerative colitis. In some examples, the disease, disorder, or condition is a chronic inflammatory condition, including, but not limited to, asthma, skin infections, and eye infections. In some examples, the compounds described herein are used for immunosuppression in patients who have undergone organ transplantation.

In some embodiments, the steroid payload and conjugates thereof described herein are administered to a patient for the treatment of a disorder associated with GR signaling, including, but not limited to, psychiatric disorders such as schizophrenia, drug addiction, post-traumatic stress disorder (PTSD), and mood disorders , substance abuse, stress and anxiety. In some embodiments, the steroid payloads and conjugates thereof 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. Some studies describe the steroid payloads and steroid conjugates described herein for the treatment of cardiovascular disease. In some embodiments, the steroid payloads and conjugates thereof described herein are administered to a patient to treat a disorder of glucose and/or liver metabolism. In some embodiments, the steroid payloads and conjugates thereof described herein are administered to a patient to treat a musculoskeletal disorder. In some cases, the steroid payloads and their conjugates described herein are administered to a patient to treat skin inflammatory conditions such as eczema and psoriasis.

The protein conjugates described herein provide a means for targeting its steroid payload to specific cells or organ systems, thereby reducing or preventing the side effects that result from administration of the free, unconjugated steroid payload. Thus, provided herein are methods of treating a disease, disorder or condition associated with a glucocorticoid receptor, comprising administering a conjugate of formula (I) or (I) ¹ to a patient having said disease, disorder or condition, wherein the side effects associated with by introducing the payload of free steroids of the specified conjugate are reduced. Further provided herein are methods of delivering a compound of Formula (I) or (I) ¹ to a cell, comprising contacting said cell with a protein conjugate compound of Formula (I) or (I) ¹ , wherein the protein conjugate comprises an antibody or an antigen-binding fragment that binds a surface antigen of a specified cell.

The compounds described herein may be administered alone or together with one or more additional therapeutic agents. One or more additional therapeutic agents may be administered immediately before, simultaneously with, or shortly after administration of the compounds described herein. The present disclosure also includes pharmaceutical compositions containing any of the compounds described herein in combination with one or more additional therapeutic agents, and methods of treatment including administering such

- 109 045579 combinations for subjects in need.

Suitable additional therapeutic agents include, but are not limited to, a second glucocorticoid, an autoimmune therapeutic agent, a hormone, a biological or a monoclonal antibody. Suitable therapeutic agents also include, but are not limited to, any pharmaceutically acceptable salts, acids or derivatives of a compound set forth herein.

The compounds described herein may also be administered and/or combined in combinations with antivirals, antibiotics, analgesics, corticosteroids, steroids, oxygen, antioxidants, COX inhibitors, cardioprotectors, metal chelators, IFN-gamma and/or NSAIDs.

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 therapeutic agents mentioned herein) can be administered to a subject via a certain period of time. Methods in accordance with this aspect of the disclosure 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 disclosure 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 optionally followed by 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 starting dose is the dose that is administered at the beginning of the treatment regimen (also called the base dose); secondary doses are 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 contain the same amount of the compound described herein, but, as a rule, may differ from each other in terms of frequency of administration. In certain cases, the amount of compound contained in the initial, secondary and/or tertiary doses varies depending on each other (eg, adjusted depending on the situation) during the course of treatment. In certain conditions, two or more (eg, 2, 3, 4, or 5) doses are given at the beginning of the treatment regimen as loading doses, followed by subsequent doses that are administered less frequently (eg, maintenance doses).

In some exemplary embodiments of the present disclosure, each secondary and/or tertiary dose is administered from 1 to 26 (for example, 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 a sequence of multiple doses of a compound that is administered to a patient before the very next dose in the sequence, with no intervening doses.

Methods according to this aspect of the disclosure 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, the patient is administered two or more (eg, 2, 3, 4, 5, 6, 7, 8 or more) secondary doses. Likewise, the patient is given only one tertiary dose. 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 schedule may be continued indefinitely during the life of a particular subject or until such treatment is no longer therapeutically necessary 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 multiple tertiary doses, each tertiary dose can be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain aspects of the disclosure, the frequency with which secondary and/or tertiary doses are administered to a patient may vary over the course of treatment. The frequency of administration may also be adjusted by the physician during the course of treatment depending on the needs of the individual patient after clinical examination.

The present disclosure includes dosing regimens in which 2 to 6 loading doses are administered to a patient at a first frequency (e.g., once a week, once every two weeks, once every three weeks, once a month, once every two months, and etc.) followed by administration of two or more maintenance doses to the patient less frequently. For example, according to this aspect of the disclosure, if loading doses are administered at a frequency of once per month, then maintenance doses may be administered to the patient once

- 110 045579 every six weeks, once every two months, once every three months, etc.

The present disclosure includes pharmaceutical compositions of the compounds and/or conjugates described herein, for example, compounds of formula (I) and (II), for example, compositions containing the compound described herein, a salt, stereoisomer, polymorph and a pharmaceutically acceptable carrier, diluent and/or excipient. Examples of suitable carriers, diluents and excipients include, but are not limited to, buffers for maintaining the pH of the appropriate composition (e.g., citrate buffers, succinate buffers, acetate buffers, phosphate buffers, lactate buffers, oxalate buffers, etc.), carrier proteins (e.g. human serum albumin), nanoparticles, saline, polyols (e.g. trehalose, sucrose, xylitol, sorbitol, etc.), surfactants (e.g. polysorbate 20, polysorbate 80, polyoxolate, etc. ), antimicrobials and antioxidants.

In certain examples set forth herein, a method of treating a disease, disorder, or condition is provided, comprising administering to a patient with said disorder a therapeutically effective amount of a compound of Formula I, III, or a pharmaceutical composition thereof.

In certain examples set forth herein, a method of treating a disease, disorder, or condition is provided, comprising administering to a patient having said disorder a therapeutically effective amount of a compound provided herein or a pharmaceutical composition thereof.

Some examples set forth herein provide a method of treating a disease, disorder, or condition selected from the group consisting of an immunological disease, an autoimmune disease, inflammation, asthma or inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

Certain examples set forth herein provide a method of treating a disease, disorder, or condition by targeting an antigen, such as a cell surface expressing antigen, for which delivery of steroids can achieve a therapeutic effect, including administration of the conjugates described herein. In some embodiments, the antigen means AXL, BAFFR, BCMA, BCR list components, BDCA2, BDCA4, BTLA, BTNL2 BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137 , CD138, CD14, CD168, CD177, CD19, 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, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2 , TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4 or VTCN1. In some embodiments, the antigen is IL2R-y.

Some examples set forth herein provide a method of treating a disease, disorder, or condition selected from an immunological disease, an autoimmune disease, an inflammatory disease, a dermatological disease, or a gastrointestinal disease.

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

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

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

Some examples set forth herein provide a method of reducing or ameliorating the side effects of chemotherapy, which comprises administering to a patient having the disorder a therapeutically effective amount of a compound or composition described herein.

Certain examples set forth herein provide a method of reducing or ameliorating the side effects of immunosuppressive therapy, which comprises administering to a patient having the disorder a therapeutically effective amount of a compound or composition described herein.

Certain examples set forth herein provide a method of treating cancer where

- 111 045579 This method includes administering to a patient having the specified disorder a therapeutically effective amount of a compound or composition described herein. In some examples, the cancer is selected from acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), or multiple myeloma, as well as others.

F. Examples.

Some 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 from other suppliers unless expressly stated otherwise.

The 1H NMR spectrum 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 parts per million (ppm) proton shifts from the internal tetramethylsilane standard.

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

Method A for HPLC-MS measurements included as mobile phase: A: water (0.01% trifluoroacetic acid, trifluoroacetic acid) and B: acetonitrile (0.01% trifluoroacetic acid). The gradient phase was 5% of B and increased to 95% of B over a period of 15 minutes (min) at a flow rate of 1.0 ml/min. The column used was SunFire C18, 4.6 x 50 mm, 3.5 µm. The column temperature was 50°C. Detectors included analog-to-digital converter ELSD (evaporative light scattering detector, hereafter 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% of B and increased to 95% of B over a period of 15 minutes at a flow rate of 1.0 ml/min. The column used was XBridge C18, 4.6 x 50 mm, 3.5 µm. The column temperature was 50°C. Detectors included FVS ELSD, DAD (214 nm and 254 nm) and a mass sensitive detector (MSD ES-API).

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

Method A for LC-MS measurements was performed on a WATERS 2767 instrument. The column was a Shimadzu Shim-Pack, PRC-ODS, 20x250 mm, 15 µm, two connected in series. The mobile phase was A: water (0.01% TFA) and B: acetonitrile. The gradient phase was 5% of B and increased to 95% of B over a period of 3 minutes 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 ELSD (Evaporative Light Scattering Detector), DAD (Diode Array Detector) (214 nm and 254 nm) and ES-API.

Method B for LC-MS measurements was performed on a Gilson GX-281 instrument. The column was an Xbridge Prep C18 10 µm OBD, 19x250mm. The mobile phase was A: Water (10 mmol NH ₄ HCO ₃ ) and B: Acetonitrile. The gradient phase was 5% of B and increased to 95% of B over a period of 3 minutes at a flow rate of 1.8-2.3 ml/min. The column used was XBridge C18, 4.6 x 50 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 on a Gilson GX-281 instrument. Two solvent systems were used: one acidic and one basic. The acidic solvent system included a Waters SunFire 10 µm C18 column (100 A, 250x19 mm). Preparative HPLC solvent A contained 0.05% TFA in water, and solvent B contained acetonitrile. The elution conditions were a linear gradient that increased the solvent B content from 5% to 100% over a period of 20 minutes at a flow rate of 30 ml/min. The main solvent system included a Waters Xbridge 10 µm C18 column (100 A, 250x19 mm). Preparative HPLC solvent A was 10 mmol ammonium bicarbonate (NH4HCO3) in water and solvent B was acetonitrile. The elution condition was a linear gradient that increased the solvent B content from 5% to 100% over a period of 20 minutes at a flow rate of 30 ml/min.

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

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

Abbreviation Meaning ADC (ALS conjugate) Antibody-drug conjugate AglycosHnated antibody Antibody lacking glycan residues API Ionization at atmospheric pressure aq Water Vos N-tert-butoxycarbonyl BupH™ Thermo Scientific Prod #28372 containing 100 mM sodium phosphate and 150 mM sodium chloride, no potassium, pH adjusted to 7.2-7.6-7.8 MQ unless otherwise noted. CD Cyclodextrin COT Cyclooctynol Da (Yes) Dalton DAD Diode array detector DAR Drug to Antibody Ratio DCM Dichloromethane DIBAC Dibenzocyclooctine or 11,12-didehydro-5,6-dihydrodibenz[b,1]azocine; or Dibenz[b,1]azocine-5(6H)-butanoic acid, 11,12-didehydro DIBAC-Suc 11,12-didehydro-5,6-dihydro-Dibenz[Z>/|azocine succinic acid DIBAC-Suc-PEG ₄ -VCpAB-PNP {4-[(2S)-2-[(2S)-2-[l-(4-{2- azatricyclo[10.4.0.0 ⁴ ' ⁹ ]hexadeca-1(12),4(9),5,7 ,13,15hexaen-10-in-2-yl }-4-oxbutanamido) -3,6,9,12tetraoxapentadecane-15-amido]-3-methylbutane amido]-5(carbamoylamino)pentanamido]phenyl} methyl 4-nitrophenyl carbonate DIBACT 3/7-Benzo[s]-1,2,3-triazolo[4,5-e][1]benzazocine, 8,9dihydro-; or Dibenzocyclooctine triazole DIPEA Diisopropylethylamine DMF N,N-dimethylformamide DMSO Dimethyl sulphate oxide E.C. Enzyme Commission ELSD Evaporative light scattering detector ESI Electrospray ionization Fmoc Fluorenylmethyloxycarbonyl chloride Fmoc-vcPAB-PNP N-Fmoc-L-valine-E-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate G(r) Gram HATU 2-(7-Aza-1 N-benztriazol-1 -yl)-1,1,3,3tetramethyluronium hexafluorophosphate H.C. Immunoglobulin heavy chain HEK Human embryonic kidney cells HPLC (HPLC) High performance liquid chromatography hr or hrs (h) watch

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L.C. Liquid chromatography HPLC High Pressure Liquid Chromatography MALDI Matrix-assisted laser desorption/ionization M.C. Maleimidocaproyl mg (mg) milligrams min (min.) minutes mL (ml) milliliters mmh tus-tus-hexahistidine (marker) pL (µl) microliters mM (mmol) millimolar μΜ (μmol) micromolar MMAE Monomethyl auristatin E MS (MS) Mass spectrometry MsCl Methanesulfonyl chloride MSD Mass selective detector MTG Microbial transglutaminase (MTG EU 2.3.2.13, Zedira, Darmstadt, Germany) MW Molecular mass ncADC Noncytotoxic antibody-drug conjugate NHS N-hydroxy succinimide nM (nmol) nanomolar ΝΜΚ(ΉΜΡ) Nuclear magnetic resonance NOESY Spectroscopy of the nuclear Overhauser effect

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RAV Para-amino-benzyl alcohol / U ON RAVS Para-aminobenzyloxy(carbonyl) O ^/ PBS 10 mmol sodium phosphate buffer and 150 mmol sodium chloride PBSg 10 mmol phosphate, 150 mmol sodium chloride, 5% glycerol P.E.G. Polyethylene glycol PNP p-nitrophenyl MC-VC-PAB-PNP z-A / O :. ΐ about z z o Yu PPm Parts per million (chemical shift) R.P. Reversed phase RT Room temperature SDS-PAGE (DNS-PAGE) Polyacrylamide gel electrophoresis SEC Size exclusion chromatography Sue succinic acid TCEP Tris(2-carboethyl)phosphine hydrochloride TEA Triethylamine

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T.F.A. Trifluoroacetic acid TG Transglutaminase THF Tetrahydrofuran TOF Time of flight UPLC Ultra-high performance liquid chromatography UV Ultraviolet V.A. Valine-alanine V.C. B aline-citrulline VC-PABC Valine-citrulline-p-aminobenzyloxy(carbonyl) CD Cyclodextrin HATU 2-(7-Aza-1 N-benzotriazol-1 -yl)-1,1,3,3tetramethyluronium hexafluorophosphate M.C. Maleimidocaproyl COT SKZHH Cyclooctynol Supercritical liquid chromatography Abbreviation Name of classification of the International Union of Fundamental and Applied Chemistry Structure Boc-vcPAB-PNP (L2a) mpew-butyl (5)-3-methyl-1-((5)1 -(4-(((4-nitrophenoxy)carbonyloxy)methyl)phenylamino)-1-oxo-5ureidopentan-2-ylamino)-1 oxobutan- 2-ylcarbamate ΟγΝΗ ₂ ΝΗ 9 Г Н bochn^A^A^n^^ Σ th ПЦ |1 АЧ ⁰ Υ 11

- 116045579

Fmoc-vcPAB- PNP (L2b) (9R-fluoren-9-yl)methyl (5)-3methyl-1 -((5)-1 -(4-(((4nitrophenoxy)carbonyloxy)methyl)phenylamino)-1-oxo-5ureidopentane-2-ylamino) -1oxobutan -2-ylcarbamate o^nh ₂ NH U\n FmocHN. A : th P G || >4 O ^A/°xx ⁰ ^^ T Ϊ 1 O H Ax Boc-Val-Ala-OH (L3a) (5)-2-((5)-2-(from/?eot-butoxy carbonylamino)-3-methylbutane amido)propionic acid FROM Oh oh.....Ah oh I Fmoc-Val-Ala- OH (L3b) (5)-2-((5)-2-(((9R-fluoren-9yl)methoxy)carbonylamino)-3 methylbutanamido)propionic acid Η P = Boc-Val-Cit-OH (L3c) (65',95)-1-amino-9-isopropyl13,13-dimethyl-1,8,11-trioxo12-oxa-2,7,10- triazatetradecane-6-carboxylic acid from o o / / ^O= \ ' ZI ° /—/ $=o § t yu Fmoc-D-Lys- COT (L5) (27?)-2-(((9R-fluoren-9yl)methoxy)carbonylamino)-6(2-(cyclooct-2-ynyloxy)acetamido)hexanoic acid O FmocHN^A _OH

- 117 045579

CD-N ₃ (L7a) 5-(azidomethyl)-10,15,20,25,30pentakis(hydroxymethyl)2,4,7,9,12, 14,17,19,22,24,27,29- dodecaoxyhepta cyclorb.g.g.g ³ ' ⁶ ^ ⁸ ' ¹¹ ^ ¹³ ' ¹⁶ ^ ¹⁸ ' ²¹ . 2 ²³ ' ²⁶ ] dotetracontan31,32,33,34,35,36,37, 3 8,39,40,41,42-dodecol but ⁷ N3-PEG4- CONHCH2CH2S EP (L7b) 1-azido-15-oxo-3,6,9,12tetraoxa-16-azaoctadecane-18sulfonic acid O O II II ³ n O BCN-PEG ₄ -acid (L9a) (Endo)-1 -(bicyclo[6.1.0]non-4in-9-yl)-3-oxo-2,7,10,13,16pentaoxa-4-azanonedecane-19- oic acid ,, O O g ^A^o^^o^^A DIBAC-PEG4acid (L9b) 1-(4-{2-azatricyclo[10.4.0.0 ⁴ ' ⁹ ] hecad eca-1 (12),4(9), 5,7,13,15 hexaen-10-in-2-yl }-4oxobutanamido) -3,6,9,12tetraoxapetradesan-15-oic acid O sch ) ^{0 0} BCN-PEG4-NHS (LlOa) (Endo)-2,5-d ioxopyrrolidin1-yl1 1-(bicyclo[6.1.0]non-4in-9-yl)-3-oxo-2,7,10,13,16pentaoxa-4-azanonedecane-19- oat n ^{0 0} °Ul\\A ^{N n 0}

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DIBAC-PEG4- NHS (LIOB) 2,5-dioxopyrrolidin-1-yl 1-(4-{2-azatricyclo[10.4.0.0 ⁴ , ⁹ ]hexade ka-1 (12),4(9), 5,7,13,15-hexaene10-yne -2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-oate 0 U ^{0 n} ° MAL-PEG ₄ -NHS (LlOc) 2,5-dioxopyrrolidin-1-yl 1(2,5-dioxo-2,5-dihydro-1Hpyrrol-1-yl)-3,6,9,12tetraoxapentadecane-15-oate DIBAC-PEG4vcPAB-PNP (LH) {4-[(20>2-[(2A)-2-[1-(4-{2azatricyclo[10.4.0.0 ⁴ ' ⁹ ]hexade ka-1 (12),4(9), 5,7,13 ,15 -hexaen10-in-2-yl}-4oxobutanamido)-3,6,9,12tetraoxapentadecane -15tetraoxapentadecane]-3methylbutanamido] -5 (carbamoyl amino)amido] phenyl} methyl 4-nitrophenyl carbonate ⁰ GT Ο ^Λ ΝΗ, Lk-DIBAC O 2 n, . J

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Lk-BCN - A ΐ IZ O \ Lk-MAL - O ⁴ Lk-CCK - O A 0 nh ₂ aCDCCK ° ⁿ %k HO ^Z 0 ' _0H SulCCK ) O O dualSulCCK ZI oA A J ^ZI ° ZI 1 _O S o ¹

Methods of obtaining

Example 1.

This example demonstrates one method for preparing desonide chemical derivatives with stereochemical control at the ^C22 position. In fig. 1 and 2, position C ²² is indicated for connections 7, 8 and 11 with an asterisk, i.e. *. The synthesis of steroids with stereochemical control at the ^C22 position was carried out according to the synthesis route shown in Fig. 1 and 2.

Desonide (1), which is the common name for (1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-8(2-hydroxyacetyl)-6,6,9,13-tetramethyl-5 ,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16one reacted with isobutyric anhydride (compound 2) to form intermediate 3 by esterification at the primary alcohol position compound 1. Compound 3 was reacted with a series of aldehydes (4-1; 4-2; 4-3; and 4-4, each of which differs with respect to the R-CHO group shown to the right of these numerical values) by transacetalation at conditions of strong acid HClO ₄ to obtain alcohols 5 and esters 6. As shown in FIG. 1, these aldehydes differed from each other with respect to the R group indicated in FIG. 1.

Alcohols 5 and ester 6 were separated by column chromatography.

Each alcohol 5 or ester 6 was individually reacted with diethylamine to remove the Fmoc group or with Fe/NH ₄ Cl to reduce the nitro to give epimeric compounds 7 and 8 having R/S stereochemistry at C ²² , respectively.

As described in detail below, the R and S epimers were separated and their R and S configurations were identified. For example, the R epimers of compounds 7 and 8 in FIG. 1 were isolated and confirmed as the predominant stereoisomer by more than 90% by 1H NMR. The C ²² configuration of each epimer was determined using 2D-NOESY spectroscopic studies.

In table 1 below presents steroids obtained using the methods described herein.

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Table 1

Structure and chemical and physical properties of compounds

Connection no. From ²² Structure Molecules, formula Mol. mass (calc.) ms (M+N) Pure value by HPLC (%) cLogP 7-1 5 1 / l /=\ W >0 but^H* _n C28H33NO6 479.6 480.2 96 2.53 7-1 R ^H ^ ^M —χΑ У ⁰ no-Arkh - C28H33NO6 479.6 480.3 100 2.53 8-1 R Well Η ₂ Ν—JlT/ \ ⁴⁼⁷ ° 1Α λ~Ά /=Ο ° he C32H39NO7 549.7 550.3 96 4.22 7-2 R/S ζ to (°/ 0* o C29H35NO6 493.6 494.3 98 2.59 8-2 R/S ο ΙΜ ^Ι <7>/° ^Χ Ο/) / ^5th ζ I C33H41NO7 563.7 564.3 98 4.28 8-3 R/S n ₂ y-^ody-^ts/ , ι °ΊΛλ/Ι^° V ^{1 1} /γ O he 0 C27H37NO6 471.6 472.2 96 1.63 7-4 R U__Η Η ,—. ( Τ ί/ ^V / ' 0 ⁰ he C27H37NO6 471.6 472.2 96 1.63

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11-1 R/S o\fx/° A C25H ₃₅ NO ₅ 429.6 429.9 100 2.63 11-2 R/S \_/ [ U-U h >= Tv/7\ / ⁰ ΜβΗΝ^ΧθΑ,Η C2 ₆ H ₃₇ NO ₅ 443.6 444.2 96 3.06 11-3 R/S OH C2?H ₃ 9NO5 457.6 458.2 100 3.44 11-4 R/S -lo4-<dLL /—\JU '—'— (__OH from ₃₀ h ₄₃ no ₅ 497.3 498.2 94 4.29 11-5 R/S ,— _h O l7\ Z~A ^^0-°^° Vh C ₃ iH ₃₉ NO6 521.6 522.3 100 4.24 11-5 S /=\si/ )-/ )=o ^Η2Ν 0 ^Ο Λ4; C ₃ 1H ₃₉ NO ₆ 521.6 522.2 99.8 4.24 11-5 R ^••< l/vK. /=\ oDH U-K >=o h ₂ nC - ^υ OH C ₃ 1H ₃₉ NO ₆ 521.6 522.2 99.1 4.24 11-6 S -lrХ-хД^Л ^F \ / А AN / ° /А /Ά ^Z '—VH *'— h ₂ n—c V- 0 θ OH C ₃ 1H ₃₈ FNO ₆ 539.6 540.3 98 4.38

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11-6 R Η H Hy—i F. WT a ^- / /° ΛΑ*'—СН*'—' h ₂ n—e Fo about it C3iH ₃₈ FNO6 539.6 540.3 100 4.38 11-7 R _Λ ...Μ^Χ f MA λ-K l° LA* h ₂ n—e /—o ⁰ OH C ₃ iH ₃₈ FNO6 539.6 540.2 100 4.38 11-8 R HH Hy—. зтТОпСл ⁰ \ /Ά —<н ¹ — HN—/ О О ОН C ₃₂ H ₄ iNO6 535.7 518.2 (M+H -H ₂ O) 100 4.54 11-10 R/S ODA LK /=° ζτ^. ΛΑ*'—(η * '— ^/ F—Υ V- 0 ⁰ OH C ₃ iH ₃₇ FO ₆ 524.3 525.3 100 5.21 11-11 R/S U NN ΛΑ ° HN—C /~O ° OH C ₃₃ H ₄ iNO ₇ 563.7 564.4 100 4.31 11-12 R/S ll ⁰ ovI/KZ/ ⁰ C ₃ iH ₃₇ F ₂ NO ₆ 557.6 558.3 97 3.94 11-12 R C ₃ iH ₃₇ F ₂ NO ₆ 557.6 558.2 100 11-13 R H ₂ N.^O^k _o ^A _OH C ₃ iH ₃₇ F ₂ NO ₆ 557.6 558.2 100 3.94 11-14 R/S ^/TSAUrS. ^F yA^ ⁰ ΗνΙ/τΟ^ ⁰ Η ₂ Ν—/ y—O^Ur? X ² \—/ O he C ₃ iH ₃₆ F ₃ NO ₆ 575.6 576.2 100 4.09

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11-15 R/S t u Z bd 1 Lx o*d<D1 ¹ V ' o C32H39F2NO6 554.2 555.2 100 3.90 11-16 R/S Η N N,— _h / ^O D^-DnD= _x ⁰ 'on nsg ⁴ ^ С31Н38О7 522.3 523.5 100 4.76 11-17 R/S /nuD Υο-ρμμ ^ο C30H36F2N2O6 558.6 559.2 100 3.91 11-19 R/S n ^F Ън C25H33F2NO5 465.2 466.2 98.5 2.33 11-19 R 100 11-20 R/S N _n /—. g / \ \__/ 1 / f\ G /=\ ι ^o D/\ ⁿ /X / ⁰ CD^oDn C34H43F2NO6 599.2 600.3 100 4.71 11-21 R/S l / O O \ \—UI θ' O*D<D t VJ ή MJ o C26H3 ₅ F ₂ NO ₅ 479.3 480.2 100 2.76 14-2 F σ /-D/x n md / ⁰ MeIgO ^- Un C ₂ 6H3 ₅ F ₂ NO ₅ 479.6 480.2 98 2.81 15-5 ι ^Η ν№ Ιλλ \=/ (/but'v_/ /\=/ OH C26H3 ₅ F ₂ NO ₅ 479.6 480.2 98 2.81 16-5 ⁿ . .m Well—/—. /к ^F /=4 h ₂ n-/ У-оУТХ m/ >=о \=/ tfH0/V_/ i\=/ he C28H33F2NO5 483.6 484 98 2.85

In table 2 below presents steroids obtained using the methods described herein.

- 124 045579

table 2

Structure and chemical and physical properties of compounds

Connection no. Structure Molecules, formula ms (mass/charge) 100% Naive. peak mass/charge Cleanliness by HPLC (%) 4b /—nh ₂ MV < Η 1 I ^{F n} F C25H33F2NO5. C2HF3O2 466.2 (M+H) 466.2 (M+H) 98.5 4s νη ₂ ho ί H ....... 1 Tu “ C24H31F4NO6 392.2 (M+H) 392.2 (M+H) >99 4d H ₂ N 0 \ OH HO^lV . ] h | h ί TFJH F C24H30F5NO6 410.2 (M+H) 410.2 (M+H) 98 4e h ₂ n^ ho V HO^jjZ' ⁰ i 1 ^H £ / ^0H Г Tf JH C ₂ 1H ₂₈ FNO ₅ .C2 HF3O2 394.2 (M+H) 394.2 (M+H) >99 4f 0 HO\\il ^H L/ ^NH2 C22H31NO4 374.3 (M+H) 374.3 (M+H) >99 4h H ₂ N o ^λ -\ o^^/ BUT. /\1У I i 1 ^H Ia° h 1 I f 1 ^H о^У>+ C25H34FNO5.C2 HF3O2 448.2 (M+H) 448.2 (M+H) >99 5-1 N- ^NH 2 HO. /\1V T ί Ρ4λ° h Г T f J and F C31H38F2N2O5.C 2HF3O2 557.1 (M+H) 557.1 (M+H) >99 6-1 -Ο-ΛΛ-ΝΗ2 °4 oJ^ 1 1 RHF° n 1 I F I and F C31H37F2NO6 522.3 (M+H) 522.3 (M+H) 97

- 125 045579

/g-6-ι ο^^νη ₂ ⁰ \ ОХХ” no. 1 1 1 ⁿ 1l° n I 1 ^F 1 n F C31H37F2NO6 522.2 (M+H) 522.2 (M+H) >99 5-6-1 °=\ οχ HO^ U I TTk° 1 1 ^F 1 ⁿ F C31H37F2NO6 522.2 (M+H) 522.2 (M+H) 97 6-1 D :¼ / O o \ \ LT /lt/\ O r'Y Ag "o" C31H37F2NO6 297.6 (M/2+H) 558.2 (M+H) (10%) 98.4 6-11 0 /= n ο^ι Ya/ ³ ^, #~ ^{n Hz} ιΥ ΤΑ-οΎ^ ° ^_ F C32H39F2NO7.C2 HF3O2 558.3 (M+H) 558.3 (M+H) >99 6-SH _ ⁿ °x°^o^-mn ₂ ^o= C3aZXi% ? f y+H ⁰ C31H36F3NO6 558.3 (M+H) 558.3 (M+H) >99 6-VI Ο^θ_ΝΗ ₂ ^o= \ o^yX but. ^iv t .YhTV j I ^H J ^H C31H39NO6 576.2 (M+H) 576.2 (M+H) >99 R-6- VI _r o^y _N H ₂ ⁰⁼ \ oYY no./\iy i iY^Ia ⁰ h JTFJ AND F C31H39NO6 588.3 (M+H) 588.3 (M+H) >99 S-6- VI Y. °°v> O C31H39NO6 587.2 (M-55) 665.2 (M+Na) (25%) >99 6-VII ^HO II C28H35NO5 587.3 (M+H) 587.3 (M+H) >99

In table 3 below shows linker payloads made using the methods described herein.

- 126 045579

Table 3

Examples of linkers with payloads

- 127 045579

- 128 045579

In table 4 below shows payload linkers obtained using the methods described herein.

- 129 045579

Table 4

Examples of linkers with payloads

- 130 045579

- 131 045579

Example 2.

This example demonstrates methods for preparing the chemical derivatives of budesonide, dexamethasone and flumethasone. These methods are illustrated generally as shown in FIG. 2, 3 and 4.

As shown in FIG. 2, mesylate analogues of budesonide (9) or its difluoro analogue (9B) were reacted with alkylamines or substituted phenols (10) to form aniline or amine containing compounds (11), such as compounds 11-1 to 11-23 in FIG. . 2.

As shown in FIG. 3, mesylate analogues of dexamethasone (12) reacted with alkylamines or substituted phenols (10) to form aniline- or amine-containing compounds (14) or (15) in FIG. 3.

As shown in FIG. 4, mesylate analogues of flumethasone (13) reacted with alkylamines or substituted phenols (10) to form aniline- or amine-containing compounds (16) in FIG. 4.

As described in detail below, the stereochemically pure epimers 11-5S and 11-5R in Table. 1 were obtained by chiral separation from a mixture of their corresponding R/S isomers. The absolute stereochemistry for each compound was determined by 2D-NOESY. The 2D-NOESY spectra showed that H ²² and H ¹⁸ were correlated at 11-5R, and that there was no correlation between H ²² and H ¹⁸ at 11-5S. Similarly, chiral centers at position ^C22 were identified for compounds 7-1S, 7-1R, 7-4R, 8-1R, 11-6S, 11-6R, 11-7R, 11-8R, 11-12R, 11- 13R and 11-19R in table. 1 by 2D-NOESY method.

Example 3.

This example demonstrates the method for preparing compounds 7-1S and 7-1R in Table. 1. This example refers to the connections numbered in FIG. 1.

2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-6-(4-nitrophenyl)-16-oxo-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ⁴ ' ⁸ .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-methylpropanoate (5-1) and (1S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-(4-nitrophenyl)5,7-dioxapentacyclo[10.8.0.0 ² ' ⁹ .0 ⁴ ' ⁸ . 0 ¹³ ' ¹⁸ ]icosa-14,17-dien-16-one (6-1).

Step 1: Compound 3 was synthesized according to the procedures described in US 2007/135398, the entire contents of which are incorporated herein by reference, by reacting desonide (1) with isobutyric acid in acetone.

Step 2: To a solution of compound 3 (320 mg, 0.657 mmol) in nitropropane (20 ml) was added dropwise

- 132 045579 aqueous perchloric acid (70%, 1.90 g, 1.33 mmol) at 0°C, followed by the addition of 4-nitrobenzaldehyde (4-1, 151 mg, 1.00 mmol). The resulting mixture was stirred at room temperature overnight and then diluted with ethyl acetate (80 ml). The resulting mixture was washed with saturated aqueous sodium bicarbonate (30 mlx3), then with brine (30 mlx2). The resulting solution was then dried over sodium sulfate and concentrated in vacuo. The residue was then purified by flash chromatography eluting with 0-35% ethyl acetate/petroleum ether to give compound (51) as a yellow solid (120 mg, 32% yield), which was a 3/1 mixture of 5R/5S epimers based on 1H NMR, and further eluting with 60-70% ethyl acetate in petroleum ether to obtain compound (6-1) as a yellow solid (150 mg, 36% yield), which was a mixture of 6R/6S epimers in a ratio of 5/1 per based on 1H NMR (R/S not determined).

Compound (5-1): NMR mass/charge: 580 (M+H)+.

1H NMR (CDCl ₃ , 400 MHz, epimers A and B with ratio = 3) δ 8.27 and 8.25 (d, J=8.8 Hz, 2H), 7.62 and 7.55 (d, J =8.8 Hz, 2H), 7.28-7.21 (m, 1H), 6.33-6.23 (m, 1H), 6.03 and 6.05 (s, 1H), 5, 62 and 6.16 (s, 1H), 5.12 and 5.43 (d, J=5.4 Hz, 1H), 4.97 and 4.77 (d, J=17.6 Hz, 1H) , 4.88 and 4.33 (d, J=17.6 Hz, 1H), 4.52 (br s, 1H), 2.80-2.50 (m, 2H), 2.44-2, 29 (m, 1H), 2.29-2.05 (m, 3H), 2.01-1.84 (m, 2H), 1.80-1.67 (m, 2H), 1.51 and 1.59 (br s, 1H), 1.46 and 1.48 (s, 3H), 1.29-1.07 (m, 7H), 1.03 and 1.05 (s, 3H) ppm. Compound 6-1: NMR mass/charge: 510 (M+H) ⁺ .

1H NMR (DMSO _d6 , 400 MHz, epimers A and B with ratio = 5) δ 8.26 and 8.24 (d, J=8.8 Hz, 2H), 7.77 and 7.57 (d, J =8.8 Hz, 2H), 7.32 (d, J=10.0 Hz, 1H), 6.17 and 6.18 (dd, J=10.0 Hz, 1.8 Hz, 1H), 5.93 and 5.95 (s, 1H), 5.63 and 6.28 (s, 1H), 5.14 and 5.03 (t, J=6.0 Hz, 1H), 4.99 and 5.35 (d, J=6.3 Hz, 1H), 4.82 (d, J=3.2 Hz, 1H), 4.644.13 (m, 3H), 2.64-2.51 (m , 1H), 2.37-2.24 (m, 1H), 2.20-1.99 (m, 2H), 1.94-1.57 (m, 5H), 1.40 (s, 3H ), 1.140.98 (m, 2H), 0.88 (s, 3H) ppm.

Step 3: Preparation of (1S,2S,4R,6R,8S,9S,11S,12S,13R)-6-(4-aminophenyl)-11-hydroxy-8-(2hydroxyacetyl)-9,13-dimethyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (7-1R) in table. 1) and (1S,2S,4R,6S,8S,9S,11S,12S,13R)-6-(4-aminophenyl)-11-hydroxy-8-(2-hydroxyacetyl)-9,13dimethyl-5,7 -dioxapentacyclo[10.8.0. 0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (7-1S) in table. 1).

Iron powder (56.0 mg, 1.00 mmol) and ammonium chloride (53.5 mg, 1.00 mmol) were simultaneously added to a solution of compound 5-1 (51.0 mg, 0.100 mmol) in a combined ethanol solution (3 ml) and water (0.5 ml). The suspension was stirred at 80°C for one hour and filtered through celite to remove solids. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC (Method B) to provide 7-1R (30 mg, 63% yield) as a white solid and 7-1S (8 mg, 17% yield) as a white solid. .

2D-NOESY spectroscopy was used to determine the stereochemical configurations of the chiral centers of compound 7-1R and compound 7-1S. 2D - NOESY spectra confirmed the presence of a correlation between H ²² and H ²¹ in compound 7-1R, indicating that it has an R configuration of the chiral center. No correlation was observed between ^H22 and ^H21 in compound 7-1S, indicating that it has an S configuration of the chiral center. The NMR study also showed that the ^H22 shift of compound 7-1R (5.33 ppm) was much higher than that of compound 7-1S (6.01 ppm), indicating that the ^H22 of compound 7-1R was more difficult. The 2D-NOESY spectra of compound 7-1-22R and compound 7-122S are shown in FIG. 5 and 6.

Compound 7-1R in table. 1: Mass/charge at NMR: 480 (M+H) ⁺ .

^1H NMR (MeODd4, 400 MHz) δ 7.46 (d, J=10.1 Hz, 1H), 7.17 (d, J=8.4 Hz, 2H), 6.67 (d, J= 8.4 Hz, 2H), 6.27 (dd, J=10.1, 1.8 Hz, 1H), 6.04 (s, 1H), 5.33 (s, 1H), 5.00 ( d, J=5.4 Hz, 1H), 4.61 (d, J=19.4 Hz, 1H), 4.504.39 (m, 1H), 4.31 (d, J=19.4 Hz, 1H), 2.78-2.61 (m, 1H), 2.47-2.35 (m, 1H), 2.35-2.22 (m, 1H), 2.22-2.10 ( m, 1H), 2.04-1.94 (m, 1H), 1.91-1.66 (m, 4H), 1.51 (s, 3H), 1.25-1.11 (m, 1H), 1.07 (dd, J=11.2 Hz, 3.5 Hz, 1H), 0.99 (s, 3H) ppm.

Compound 7-1S in table. 1: Mass/charge at NMR: 480 (M+H) ⁺ .

1H NMR (MeODd4, 400 MHz) δ 7.47 (d, J=10.1 Hz, 1H), 7.02 (d, J=8.4 Hz, 2H), 6.65 (d, J=8 .5 Hz, 2H), 6.27 (dd, J=10.1, 1.8 Hz, 1H), 6.03 (s, 1H), 6.01 (s, 1H), 5.36 (d , J=6.2 Hz, 1H), 4.46-4.31 (m, 2H), 4.12 (d, J=19.2 Hz, 1H), 2.75-2.61 (m, 1H), 2.47-2.35 (m, 1H), 2.27-2.11 (m, 2H), 2.08-1.97 (m, 1H), 1.96-1.73 ( m, 4H), 1.51 (s, 3H), 1.33-1.17 (m, 2H), 1.17-1.09 (m, 1H), 1.01 (s, 3H) ppm.

Example 4.

This example demonstrates the method for preparing compound (8-1R/S) and compound (8-1R) in table. 1. This example refers to the connection numbered in FIG. 1. 2-[(1S,2S,4R,8S,9S,11S,12S,13R)6-(4-aminophenyl)-11-hydroxy-9,13-dimethyl-16-oxo-5,7-dioxapentacyclo[ 10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa 14,17-dien-8-yl]-2-oxoethyl 2-methylpropanoate (8-1R).

Iron powder (56.0 mg, 1.00 mmol) and ammonium chloride (53.5 mg, 1.00 mmol) were simultaneously added to a solution of compound (6-1) (58.0 mg, 0.100 mmol) in a combined ethanol solution (3 ml) and water (1 ml). The resulting suspension was stirred at 80°C for one hour and filtered through celite to remove solids. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC (Method B) to give compound (8-1R) and its enantiomer (i.e. S-stereochemistry at ^C22 ) (26

- 133 045579 mg, yield 45%) in the form of a white solid. The ratio of R-epimer to S-epimer is 4:1 according to HPLC and 1H NMR. Mass/charge at NMR: 550 (M+H)+.

Next, the R-epimer was isolated and the configuration was determined by 2D NMR.

Compound (8-1R): NMR mass/charge: 550 (M+H) ⁺ .

^1H NMR (MeODd4, 500 MHz) δ 7.46 (d, J=10.0 Hz, 1H), 7.19 (d, J=8.5 Hz, 2H), 6.69 (d, J= 8.4 Hz, 2H), 6.27 (dd, J=10.0 Hz, 2.0 Hz, 1H), 6.05 (s, 1H), 5.44 (s, 1H), 5.07 (d, J=17.5 Hz, 1H), 4.96 (d, J=5.5 Hz, 1H), 4.88 (d, J=17.5 Hz, 1H), 4.48-4 .44 (m, 1H), 2.73-2.64 (m, 2H), 2.42-2.39 (m, 1H), 2.32-2.24 (m, 1H), 2.19 -2.15 (m, 1H), 2.03-1.99 (m, 1H), 1.95-1.92 (m, 1H), 1.90-1.83 (m, 2H), 1 .76-1.69 (m, 1H), 1.52 (s, 3H), 1.27-1.12 (m, 7H), 1.09-1.05 (m, 1H), 1.02 (s, 3H) ppm.

Example 5.

This example demonstrates the method for preparing the compound (7-2R/S) in table. 1. This example refers to the connection numbered in FIG. 1.

Step 1: 1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-[(4nitrophenyl)methyl]-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (5-2).

To a solution of compound (3) (226 mg, 0.464 mmol) in nitropropane (10 ml), aqueous perchloric acid (70%, 985 mg, 6.90 mmol) was added dropwise at 0°C, followed by the addition of 2-(4nitrophenyl)acetaldehyde (4-2, 115 mg, 0.696 mmol) according to the synthesis in Synthesis, 2011, 18, 2935-2940, the entire contents of which are incorporated herein by reference for all purposes. The resulting mixture was stirred at room temperature overnight and then diluted with ethyl acetate (60 ml). The mixture was washed with saturated aqueous sodium bicarbonate (50 mlx3), then brine (50 mlx3), and then dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography eluting with 0-35% ethyl acetate/petroleum ether to give compound (6-2) as a brown solid (95 mg, 34% yield, including >10/1 22R/S epimers) according to ^1H NMR) and further eluting with 60-70% ethyl acetate in petroleum ether to give compound (5-2) (145 mg, 60% yield) as a brown solid.

Compound (5-2): NMR mass/charge: 524 (M+H) ⁺ .

^1H NMR ( _CDCl3 , 400 MHz) δ 8.09 (d, J=8.7 Hz, 2H), 7.39 (d, J=8.7 Hz, 2H), 7.17 (d, J =10.1 Hz, 1H), 6.31 (dd, J=10.1 Hz, 1.8 Hz, 1H), 6.02 (s, 1H), 4.92 (d, J=5.3 Hz, 1H), 4.86 (t, J=3.6 Hz, 1H), 4.52-4.39 (m, 2H), 4.28-4.17 (m, 1H), 3.08 (d, J=3.5 Hz, 2H), 2.96 (t, J=4.9 Hz, 1H), 2.53-2.40 (m, 1H), 2.32-2.19 ( m, 1H), 2.041.95 (m, 1H), 1.95-1.82 (m, 2H), 1.60-1.46 (m, 3H), 1.38 (s, 3H), 1 .34 (br s, 1H), 0.91-0.77 (m, 4H), 0.76-0.62 (m, 2H) ppm.

Step 2: (1S,2S,4R,8S,9S,11S,12S,13R)-6-[(4-aminophenyl)methyl]-11-hydroxy-8-(2-hydroxyacetyl)9,13-dimethyl-5 ,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (7-2R/S).

Iron powder (78.0 mg, 1.40 mmol) and ammonium chloride (75.0 mg, 1.40 mmol) were simultaneously added to a solution of compound (5-2) (75.0 mg, 0.143 mmol) in a combined ethanol solution (4 ml) and water (0.5 ml). The suspension was stirred at 80°C for 1.5 hours and filtered through celite to remove solids. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC (Method B) to give compound (7-2R/S) (26 mg, 37% yield) as a white solid. Mass/charge at NMR: 494 (M+H) ⁺ .

^1H NMR (MeOD _d4 , 400 MHz) δ 7.44 (d, J=10.1 Hz, 1H), 6.93 (d, J=8.3 Hz, 2H), 6.48 (d, J =8.3 Hz, 2H), 6.30 (dd, J=10.1 Hz, 1.9 Hz, 1H), 6.07 (s, 1H), 4.85-4.77 (m, 2H ), 4.51 (d, J=19.4 Hz, 1H), 4.35-4.29 (m, 1H), 4.24 (d, J=19.4 Hz, 1H), 2.87 -2.72 (m, 2H), 2.62-2.47 (m, 1H), 2.38-2.28 (m, 1H), 2.08-1.93 (m, 1H), 1 .90-1.78 (m, 2H), 1.67-1.58 (m, 1H), 1.53-1.37 (m, 5H), 0.91-0.77 (m, 5H) , 0.74 (dd, J=11.2 Hz, 3.4 Hz, 1H) ppm.

Example 6.

This example demonstrates the method for preparing the compound (8-2R/S) in table. 1. This example refers to the connection numbered in FIG. 1.

Step 1: 2-[(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-6-[(4-nitrophenyl)methyl]-16oxo-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-methylpropanoate (62).

The synthesis of compound 6-2 was described in example 5 above. Compounds 6-2: NMR mass/charge: 594 (M+H) ⁺ .

¹ H NMR (CDCl ₃ , 400 MHz) δ 8.15 (d, J=8.7 Hz, 0.1H) and 8.09 (d, J=8.7 Hz, 1.9H), 7.40 (d, J=8.6 Hz, 2H), 7.20 (d, J=10.1 Hz, 1H), 6.31 (dd, J=10.1 Hz, 1.8 Hz, 1H), 6.02 (s, 1H), 4.94 (t, J=3.6 Hz, 1H), 4.87 (d, J=5.1 Hz, 1H), 4.81 (d, J=17 ,6 Hz, 1H), 4.71 (d, J=17.6 Hz, 1H), 4.46 (s, 1H), 3.09 (d, J=3.5 Hz, 2H), 2, 73-2.61 (m, 1H), 2.53-2.41 (m, 1H), 2.31-2.21 (m, 1H), 2.07-1.96 (m, 1H), 1.94-1.84 (m, 2H), 1.84-1.76 (m, 1H), 1.63-1.43 (m, 3H), 1.39 (s, 3H), 1. 22 (t, J=7.0 Hz, 6H), 0.92-0.82 (m, 4H), 0.76-0.61 (m, 2H) ppm.

Step 2: 2-[(1S,2S,4R,8S,9S,11S,12S,13R)-6-[(4-aminophenyl)methyl]-11-hydroxy-9,13-dimethyl-16oxo-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-methylpropanoate (82R/S).

To a solution of compound 6-2 (65.0 mg, 0.109 mmol) in a combined solution of ethanol (5 ml) and water (1 ml), iron powder (61.0 mg, 1.09 mmol) and ammonium chloride (58, 4 mg, 1.09

- 134 045579 mmol). The suspension was stirred at 80°C for one hour and filtered through celite to remove solids. The filtrate was concentrated in vacuo and the residue was purified by preparative HPLC (Method B) to give compound (S-2R/S) (30 mg, 49% yield) as a white solid. Mass/charge at NMR: 564 (M+H)+.

^1H NMR (CDCl3, 400 MHz) δ 7.25 (d, J=10.2 Hz, 1H), 6.95 (d, J=8.3 Hz, 2H), 6.44 (d, J= 8.3 Hz, 2H), 6.31 (dd, J=10.1, 1.8 Hz, 1H), 6.05 (s, 1H), 4.92-4.84 (m, 2H), 4.80 (d, J=5.2 Hz, 1H), 4.73 (d, J=17.7 Hz, 1H), 4.41 (s, 1H), 3.48 (br s, 1H) , 2.85 (d, J=2.7 Hz, 2H), 2.75-2.62 (m, 1H), 2.56-2.41 (m, 1H), 2.31-2.19 (m, 1H), 2.051.91 (m, 2H), 1.88-1.80 (m, 1H), 1.77-1.70 (m, 1H), 1.55-1.41 (m , 3H), 1.39 (s, 3H), 1.29-1.18 (m, 8H), 0.910.74 (m, 5H) ppm.

Example 7.

This example demonstrates the method for preparing the compound (8-3R/S) in table. 1. This example refers to the connection numbered in FIG. 1.

Step 1: 2-[(1S,2S,4R,8S,9S,11S,12S,13R)-6-(2-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}ethyl)-11hydroxy-9 ^{( _ _} 6-3).

To a solution of compound 3 (240 mg, 0.493 mmol) in nitropropane (5 ml), aqueous perchloric acid (70%, 214 mg, 1.49 mmol) was added dropwise at 0°C, followed by the addition of Fmoc-3-amino-1propanal ( 4-3, 236 mg, 0.799 mmol) according to synthesis in J. Am. Chem. Soc., 2006, 128(12), 4023-4034, the contents of which are incorporated herein by reference in their entirety for all purposes. The resulting mixture was stirred at room temperature overnight and then diluted with ethyl acetate (80 ml). The mixture was washed with saturated aqueous sodium bicarbonate (50 mlx3), then water (50 mlx2), then brine (50 ml) and then dried over sodium sulfate and concentrated in vacuo. The residue was purified by preparative TLC (silica gel, methanol/methylene chloride, v/v = 1/25) to give compound (6-3) (200 mg, 56% yield, 6R/6S epimers) as a yellowish solid. Mass/charge at NMR: 724 (M+H) ⁺ .

1H NMR (CDCl ₃ , 400 MHz) δ 7.76 (d, J=7.6 Hz, 2H), 7.56 (d, J=7.2 Hz, 2H), 7.40 (d, J= 7.2 Hz, 1H), 7.327.20 (m, 3H), 6.28-6.25 (m, 2H), 6.00 (s, 1H), 5.28-5.04 (m, 2H ), 4.87-4.76 (m, 1H), 4.46-4.35 (m, 3H), 4.18 (t, J=6.8 Hz, 1H), 3.49 (s, 1H), 3.39-3.24 (m, 2H), 2.77-2.49 (m, 2H), 2.37-2.26 (m, 1H), 2.23-1.96 ( m, 3H), 1.961.47 (m, 6H), 1.45-1.41 (m, 3H), 1.28-1.06 (m, 10H), 1.02-0.94 (m, 3H) ppm.

Step 2: 2-[(1S,2S,4R,8S,9S,11S,12S,13R)-6-(2-aminoethyl)-11-hydroxy-9,13-dimethyl-16-oxo-5,7dioxapentacyclo[ 10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethyl 2-methylpropanoate (8-3R/S).

A solution of compounds (6-3) (40.0 mg, 55.3 μmol) in diethylamine (1 ml) and methylene chloride (1 ml) was stirred at room temperature overnight. Volatiles were removed in vacuo and the residue was purified by preparative HPLC (Method B) followed by preparative TLC (silica gel, methylene chloride/methanol, v/v = 75/10) to give compound (8-3R/S) (3 mg, 11% yield) as a yellowish solid. Mass/charge at NMR: 502 (M+H) ⁺ .

^1H NMR (MeODd4, 400 MHz) δ 7.36 (d, J=10.1 Hz, 1H), 6.16 (dd, J=10.1 Hz, 1.8 Hz, 1H), 5.91 (s, 1H), 5.23 (t, J=4.4 Hz, 1H), 5.08-4.90 (m, 1H), 4.75-4.65 (m, 1H), 4, 38-4.28 (m, 1H), 2.83-2.50 (m, 2H), 2.33-2.23 (m, 1H), 2.13-2.00 (m, 2H), 1.90-1.46 (m, 6H), 1.39 (s, 3H), 1.24-1.12 (m, 2H), 1.23-0.78 (m, 11H) ppm.

Example 8.

This example demonstrates the method for preparing compound 7-4R in table. 1. This example refers to the connection numbered in FIG. 1.

(1S,2S,4R,6R,8S,9s,11S,12S,13R)-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-(piperidin-4yl)-5,7- dioxapentacyclo[10.8.0.00 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (7-4R).

To a solution of desonide (1, 0.10 g, 0.25 mmol) in nitropropane (5 ml), aqueous perchloric acid (70%, 0.11 g, 0.75 mmol) was added dropwise at 0°C, followed by the addition of 1 -Boc-4piperidinecarbaldehyde (4-4, 64 mg, 0.30 mmol). After stirring at room temperature overnight, the suspension was concentrated in vacuo. The residue was made alkaline by adding a solution of ammonia in methanol (7 M, 10 ml). The resulting mixture was concentrated in vacuo and the crude product was purified twice by preparative HPLC (Method B) to give compound 7-4R (15 mg, 13% yield) as a white solid. Mass/charge at NMR: 472 (M+H) ⁺ .

^1H NMR (MeOD _d4 , 500 MHz) δ 7.47 (d, J=10.0 Hz, 1H), 6.27 (dd, J=10.0 Hz, 2.0 Hz, 1H), 6, 03 (s, 1H), 4.90 (d, J=4.0 Hz, 1H), 4.50 (d, J=19.0 Hz, 1H), 4.46-4.43 (m, 1H ), 4.41 (d, J=4.0 Hz, 1H), 4.29 (d, J=19.0 Hz, 1H), 3.13-3.09 (m, 2H), 2.71 -2.60 (m, 3H), 2.42-2.38 (m, 1H), 2.27-2.13 (m, 2H), 1.99-1.96 (m, 1H), 1 .85-1.64 (m, 7H), 1.52 (s, 3H), 1.51-1.38 (m, 2H), 1.14-0.99 (m, 2H), 0.96 (s, 3H) ppm. The stereometric R configuration for compound 7-4R was determined by 2D NMR.

Example 9.

This example demonstrates the method for preparing the compound (11-1R/S) in table. 1. This method is generally illustrated as shown in FIG. 2.

Step 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-oxoethyl methanesulfonate (9).

- 135 045579

General Procedure A for the synthesis of mesylates from its alcohol. To a solution of alcohol (1.0 equiv.) in DCM (10 ml per gram of starting material) was added triethylamine or 4-dimethylaminopyridine (2 equiv.) and methanesulfonyl chloride (1.2 equiv.). After stirring at 0°C for half an hour or until the starting material was consumed according to TLC, silica gel (100-200 mesh) was added to the reaction mixture and concentrated in vacuo. The silica gel residue was purified by silica gel column chromatography (0-50% ethyl acetate in petroleum ether) to give the mesylate product. Alternatively, the mixture was washed with dilute water hydrochloride (1 N) and brine, dried over sodium sulfate and concentrated. The crude product was purified by flash chromatography (0-2% MeOH in DCM) to give the corresponding mesylate product.

An alternative method for preparing compound 9: to a solution of budesonide (0.28 mg, 0.65 mmol) in pyridine (5 ml) was added 4-dimethylaminopyridine (0.16 g, 1.3 mmol), then methanesulfonyl chloride (0. 11 g, 0.97 mmol) at 0°C. After stirring at room temperature for 2 hours, the resulting mixture was poured into ethyl acetate (100 ml). The mixture was washed with dilute aqueous hydrochloride (1 N), then brine, dried over sodium sulfate and concentrated. The crude product was purified by flash chromatography (0-1% methanol in methylene chloride) to give compound (9) (0.26 g, 85% yield) as a white solid. Mass/charge at NMR: 509 (M+H)+.

^1H NMR (CDCl3, 400 MHz) (epimers) δ 7.25 and 7.22 (d, J=2.0 Hz, 1H), 6.30-6.27 (m, 1H), 6.03- 6.02 (m, 1H), 5.17-5.11 (m, 1.5H), 5.06-4.96 (m, 1.5 H), 4.87-4.86 (m, 0.5 N), 4.59 (d, J=4.5 Hz, 0.5 N), 4.52-4.50 (m, 1H), 3.24 (s, 3H), 2.60 -2.53 (m, 1H), 2.36-2.33 (m, 1H), 2.24-2.00 (m, 3H), 1.86-1.62 (m, 4H), 1 .53-1.33 (m, 8H), 1.21-1.09 (m, 2H), 1.02-0.96 (m, 3H), 0.94-0.91 (m, 3H) chnm.

Step 2: (1S,2S,4R,8S,9S,11S,12S,13R)-8-(2-aminoacetyl)-11-hydroxy-9,13-dimethyl-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-1R/S) Compound 9 was added to a solution of ammonia in MeOH (7 M, 15 ml) at room temperature (0.10 g, 0.20 mmol). The solution was sealed and stirred at 40°C overnight. Volatiles were removed in vacuo and the crude product was purified by preparative HPLC (Method B) to give compound (11-1R/S) (8.0 mg, 9% yield) as a yellowish solid. Mass/charge at NMR: 429.9 (M+H) ⁺ .

1H NMR (MeOD _d4 , 400 MHz) δ 7.46 (d, J=10.0 Hz, 1H), 6.26 (d,J=10.0 Hz, 1H), 6.02 (s, 1H) , 5.22-5.15 (m, 1.5 N), 4.88 (m, 0.6 N), 4.58 (m, 0.5 N), 4.42 (m, 1 H), 3, 96-3.81 (m, 0.7H), 3.50-3.41 (m, 0.7H), 2.70-2.63 (m, 1H), 2.40-2.37 (m , 1H), 2.22-1.94 (m, 3H), 1.87-1.25 (m, 11H), 1.17-0.80 (8H) ppm. Anal. HPLC: > 95%, Holding time: 7.63 minutes (method B).

Example 10.

This example demonstrates the method for preparing compound 11-2R/S in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12S,13R)-11-hydroxy-9,13-dimethyl-8-[2-(methylamino)acetyl]-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-2R/S) Solution of compound 9 (51 mg, 0.10 mmol) in methylamine (2 M solution in THF, 0.5 ml) in a sealed tube was stirred at 20-25°C for 4 hours, then stirred at 40°C overnight. Volatiles were removed in vacuo and the residue was purified by preparative HPLC (Method A) and then preparative HPLC (Method B) to give compound (11-2R/S) (15 mg, 33% yield) as a white solid. Mass/charge by NMR : 444.3 (M+H) ⁺ .

1H NMR (CDCl·, 400 MHz) δ 7.26-7.23 (d, J=10.8 Hz, 1H), 6.30-6.26 (m, 1H), 6.03-6, 02 (m, 1H), 5.205.16 (m, 1H), 4.90-4.89 (d, J=4.8 Hz, 0.5 N), 4.69-4.66 (t, J =4.8 Hz, 0.5H), 4.49-4.51 (m, 1H), 3.50-3.29 (m, 2H), 2.61-2.52 (m, 1H), 2.37-2.32 (m, 1H), 2.17-2.16 (d, J=3.6 Hz, 3H), 2.14-2.08 (m, 3H), 1.86- 1.74 (m, 3H), 1.59-1.48 (m, 2H), 1.45 (s, 3H), 1.42-0.89 (m, 12H) ppm.

Example 11.

This example demonstrates the method for preparing compound 11-3R/S in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12S,13R)-8-[2-(dimethylamino)acetyl]-11-hydroxy-9,13-dimethyl-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-3R/S) To a solution of compound 9 (51 mg, 0.10 mmol) in THF (3 ml ) a solution of dimethylamine in THF (2 M, 0.75 ml, 1.5 mmol) was added dropwise at room temperature. The reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated and the crude product was purified by preparative HPLC (Method B) to give 11-3R/S (15 mg, 33% yield) as a white solid. Mass/charge at NMR: 458.2 (M+H) ⁺ .

1H NMR (MeODd4, 400 MHz) δ 7.46 (d, J=10.4 Hz, 1H), 6.26 (d, J=10.0 Hz, 1H), 6.02 (s, 1H), 5.21 (t, J=4.8 Hz, 0.6H), 5.17 (d, J=7.2 Hz, 0.6H), 4.58 (d, J=4.4 Hz, 0 ,4H), 4.44-4.41 (m, 1H), 3.80-3.57 (m, 1H), 3.26 (d, J=18.8 Hz, 0.7H), 3, 08-2.91 (m, 0.7H), 2.70-2.61 (m, 1H), 2.49-2.33 (m, 7H), 2.26-2.11 (m, 2H ), 2.02-1.95 (m, 1H), 1.85-1.55 (m, 5H), 1.49 (s, 3H), 1.49-1.30 (m, 3H), 1.09-1.00 (m, 2H), 0.98-0.90 (m, 6H) ppm. Anal. HPLC: >95%, Hold time: 8.34 minutes (method B).

Example 12.

This example demonstrates the method for preparing compound 11-5R/S in table. 1. This example is from

- 136 045579 is attached to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12S,13R)-8-[2-(4-aminophenoxy)acetyl]-11-hydroxy-9,13-dimethyl-6-propyl5,7-dioxapentacyclo[10.8 .0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-5R/S) General Procedure B for the preparation of a substituted phenolic ester from its mesylate precursor. To hot acetonitrile or acetone (60-65°C) the mesylate precursor (1 eq.), substituted phenol (2.0-2.5 eq.) and potassium carbonate or cesium carbonate (2.0-3.0 eq.) were added. ). The resulting suspension was refluxed for 2-3 hours, the reaction was monitored using LCMS and/or TLC. After cooling the reaction mixture to room temperature, the volatiles were removed in vacuo and water was added to the residue. The aqueous mixture was extracted with ethyl acetate. The combined organic solution was washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The crude product was used directly for the next step or purified by flash chromatography or prep-HPLC.

Step 1: mixture of compound 9 (0.13 g, 0.26 mmol), 4-nitrophenol (10-5, 72 mg, 0.52 mmol) and potassium carbonate (72 mg, 0.52 mmol) in acetone (10 ml) was refluxed (60°C) overnight. After filtration to remove solids, the filtrate was concentrated in vacuo. The crude product was purified by flash chromatography (0-1% methanol in methylene chloride) to give the nitro intermediate (0.11 g, 77% yield) as a brown oil. Mass/charge at PER: 552 (M+H)+.

^1H NMR ( _CDC13 , 500 MHz) (with epimers) δ 8.23-8.15 (m, 2.4H), 7.26-7.23 (m, 1H), 6.97-6.91 (m, 2.4H), 6.31-6.28 (m, 1H), 6.05-6.04 (m, 1H), 5.22-5.18 (m, 1.4H), 5 .10-5.07 (m, 0.6H), 4.93 (d, J=5.0 Hz, 0.6H), 4.834.77 (m, 1H), 4.67 (d, J=5 .0 Hz, 0.6H), 4.56-4.53 (m, 1H), 2.62-2.55 (m, 1H), 2.38-2.5 (m, 1H), 2. 24-2.07 (m, 3H), 1.88-1.56 (m, 5H), 1.46-1.40 (m, 6H), 1.20-1.13 (m, 2H), 1.05-0.99 (m, 3H), 0.97-0.94 (m, 3H) ppm

Step 2: Iron powder (0.10 g, 1.9 mmol) and ammonium chloride (0.10 g, 1.9 mmol) were simultaneously added to a solution of the nitro intermediate (0.10 g, 0.19 mmol) in combined solution of ethanol (20 ml) and water (2 ml). The suspension was stirred at 80°C for 2 hours and filtered through celite to remove inorganic salts. The filtrate was concentrated in vacuo and the residue was purified by prep-HPLC (Method B) to give compound (11-5R/S) (50 mg, 50% yield) as a white solid. Mass/charge at NMR: 522 (M+H) ⁺ .

1H NMR (MeOD _d4 , 500 MHz) (with epimers) δ 7.47 (d, J=10.0 Hz, 1H), 6.78-6.70 (m, 4H), 6.29-6.26 (m, 1H), 6.04 (br s, 1H), 5.25 (t, J=5.0 Hz, 0.4H), 5.20 (d, J=7.0 Hz, 0.4H ), 5.06 (d, J=18.0 Hz, 0.4H), 4.98 (d, J=18.0 Hz, 0.6H), 4.90-4.87 (m, 0. 6 H), 4.75-4.66 (m, 1.6H), 4.46-4.44 (m, 1H), 2.71-2.64 (m, 1H), 2.42-2 .38 (m, 1H), 2.28-2.18 (m, 2H), 2.06-2.00 (m, 1H), 1.87-1.83 (m, 1H), 1.76 -1.73 (m, 1H), 1.69-1.61 (m, 3H), 1.55-1.38 (m, 3H), 1.51 (s, 3H), 1.20-1 .02 (m, 3H), 0.98-0.92 (m, 5H) ppm.

A mixture of two epimers of compound 11-5R and compound 11-5S from table. 1 (0.30 g, 0.58 mmol) was isolated by chiral HPLC (instrument: Gilson-281, column: OZ-H 20 * 250 mm, 10 μm (Dacel), using mobile phase: hexane (0.1% DEA) /ethanol (0.1% DEA) = 70/30 at a flow rate of 60 ml/min, determined at 214 nm The resulting solution was concentrated to separately obtain compound 11-5S (30 mg, 10% yield) and compound 11-5R ( 50 mg, 17% yield) as a white solid.The structures of compound 11-5S and compound 11-5R were determined by 2D-NOESY.

(1S,2S,4R,8S,9S,11S,12S,13R)-8-[2-(4-aminophenoxy)acetyl]-11-hydroxy-9,13-dimethyl-6-propyl5,7-dioxapentacyclo[10.8 .0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-5S): First peak on HPLC; Mass/charge at NMR: 522 (M+H) ⁺ . HPLC hold time (method A): 7.54 minutes; chiral SCFC(CC4): Exposure time 4.71 minutes, 99.5d.e.%;

1H NMR (400 MHz, CDCI3) δ 7.21 (d, J=10.1 Hz, 1H), 6.77 (d, J=8.8 Hz, 2H), 6.63 (d, J=8 .8 Hz, 2H), 6.24 (dd, J=10.1, 1.6 Hz, 1H), 6.02 (s, 1H), 5.20 (d, J=6.8 Hz, 1H ), 5.18 (t, J=4.8 Hz, 1H), 4.99 (d, J=-17.9 Hz, 1H), 4.61 (d, J=-17.9 Hz, 1H ), 4.43 (s, 1H), 3.46 (s, 2H), 2.57 (td, J=13.2, 4.4 Hz, 1H), 2.34 (dd, J=13, 4, 3.2 Hz, 1H), 2.16-2.01 (m, 4H), 1.85-1.68 (m, 3H), 1.59-1.49 (m, 3H), 1 .44 (s, 3H), 1.44-1.26 (m, 2H), 1.18-1.09 (2H), 1.00 (s, 3H), 0.91 (t, J=7 ,3 Hz, 3H) ppm, ¹³ C NMR (100 MHz, CDCl·,) δ 204.0, 186.7, 170.0, 156.3, 151.4, 141.0, 127.9, 122, 6, 116.5, 116.4, 108.4, 98.6, 83.2, 72.6, 69.8, 55.3, 53.0, 47.2, 44.2, 41.5, 37.3, 34.1, 33.0, 32.0, 31.1, 21.2, 17.9, 17.7, 14.1 ppm.

(1S,2S,4R,8S,9S,11S,12S,13R)-8-[2-(4-aminophenoxy)acetyl]-11-hydroxy-9,13-dimethyl-6-propyl5,7-dioxapentacyclo[10.8 .0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-5R): Second peak on HPLC; Mass/charge at NMR: 522 (M+H)+; HPLC hold time (method A): 7.58 minutes; chiral SCFC (CC4): Holding time 3.80 minutes, 98.1 de%;

1H NMR (400 MHz, CDC13) δ 7.23 (d, J=10.1 Hz, 1H), 6.79 (dd, J=8.8 Hz, 2H), 6.65 (d, J=8 .8 Hz, 2H), 6.27 (dd, J=10.1, 1.7 Hz, 1H), 6.04 (s, 1H), 4.94 (d, J=4.4 Hz, 1H ), 4.89 (d, J=-18.0 Hz, 1H), 4.65 (d, J=-18.0 Hz, 1H), 4.61 (t, J=4.4 Hz, 1H ), 4.48 (d, J=2.1 Hz, 1H), 3.51 (s, 2H), 2.58 (td, J=13.3, 4.9 Hz, 1H), 2.35 (dd, J=13.4, 2.8 Hz, 1H), 2.23-1.99 (m, 4H), 1.79-1.61 (m, 6H), 1.46-1.38 (m, 2H), 1.44 (s, 3H), 1.23-1.09 (m, 2H), 0.95 (s, 3H), 0.93 (t, J=7.3 Hz, 3H) ppm, ¹³ C NMR (101 MHz, CDCI3) δ 204.9, 186.6, 170.0, 156.2, 151.2, 141.0, 127.9, 122.5, 116.3, 116.3, 104.5, 97.6, 81.9, 72.6, 69.9, 55.1, 49.8, 45.7, 44.0, 41.1, 35.0, 34, 0, 33.3, 31.9, 30.3, 21.1, 17.5, 17.1, 14.0 ppm.

Example 13.

This example demonstrates the method for preparing compounds 11-5S and (11-5R) in table. 1. Given

- 137 045579 example refers to the connection numbered in FIG. 2. Compound 9R was prepared from (R)budesonide and compound 9S was prepared from (S)-budesonide, respectively, according to General Procedure A in Example 9. Using the method as described in Example 12, compound (11-5S) was prepared as a result reacting compound (9S) with compound (10-12), and compound (11-5R) was prepared from reacting compound (9R) with compound (10-9), respectively. A representative procedure is as follows. To a solution of compound (9R) or compound (9S (100 mg) in acetone (10 ml), compound 10-9 (2 eq.) and Cs ₂ CO ₃ (2 eq.) were simultaneously added. The mixture was refluxed for 2 hours and the crude product was worked up by removing the acetone in vacuo, extracting the crude material with ethyl acetate, washing the inorganic salts with water and purifying the resulting product by chromatography (050% ethyl acetate in petroleum ether) to give compound 11-5R or compound 11-5S (2560% yield ) as a pale yellow solid NMR Mass/Charge: 522 (M+H)+ Analy HPLC: 98% 2D-NOESY spectra of compound 11-5R and compound 11-5S are shown in Figures 7 and 8 .

Example 14

This example demonstrates the method for preparing compounds 11-6S and 11-6R from table. 1. This example refers to the connection numbered in FIG. 2. (1S,2S,4R,6S,8S,9S,11S,12S,13R)-8-[2-(4amino-3-fluorophenoxy)acetyl]-11-hydroxyl-9,13-dimethyl-6-propyl -5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-16-one (11-6S) and (1S,2S,4R,6R,8S, 9S,11S,12S,13R)8-[2-(4-amino-3-fluorophenoxy)acetyl]-11-hydroxyl-9,13-dimethyl-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 . 0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-6R).

Compounds of the racemic mixture 11-6R/S were prepared according to the method described in Example 12. The racemic products were separated by chiral GFC (see details in section 2.3) to give compound 11-6S (second peak) and compound 11-6R (first peak ) as a yellowish solid.

Compound 11-6S (30 mg, 7.9% yield). Mass/charge at NMR: 540.2 (M+H) ⁺ .

^1H NMR (500 MHz, DMSO _d6 ) δ 7.32 (d, J=10.1 Hz, 1H), 6.71-6.62 (m, 2H), 6.49 (dd, J=8, 5, 2.0 Hz, 1H), 6.19-6.16 (m, 1H), 5.93 (s, 1H), 5.21 (t, J=4.8 Hz, 1H), 5, 10 (d, J=7.3 Hz, 1H), 5.02 (d, J=18.1 Hz, 1H), 4.69 (dd, J=58.9, 28.6 Hz, 4H), 4.31 (s, 1H), 2.56-2.51 (m, 1H), 2.29 (d, J=10.6 Hz, 1H), 2.06-1.97 (m, 3H) , 1.89 (s, 2H), 1.79-1.72 (m, 1H), 1.30 (m, 10H), 0.88-0.85 (m, 6H) ppm. Holding time: 2.94 min, 98% with chiral SCPC (AD). Anal. HPLC: > 96.94%, Holding time: 7.94 minutes (method B).

Compound 11-6R (28 mg, 7.4% yield). Mass/charge at NMR: 540.3 (M+H) ⁺ .

1H NMR (500 MHz, DMSCOd6) δ 7.32 (d, J=10.1 Hz, 1H), 6.72-6.68 (m, 2H), 6.52 (dd, J=8.6, 2.1 Hz, 1H), 6.18 (d, J=10.1 Hz, 1H), 5.93 (s, 1H), 5.01 (d, J=18.3 Hz, 1H), 4 .77 (dd, J=12.9, 3.3 Hz, 2H), 4.71 (s, 2H), 4.65 (t, J=4.3 Hz, 1H), 4.32 (s, 1H), 3.17 (d, J=5.2 Hz, 1H), 2.57-2.51 (m, 1H), 2.30 (d, J=10.5 Hz, 1H), 2, 10 (d, J=7.2 Hz, 1H), 2.01-1.99 (m, 1H), 1.84 (s, 2H), 1.62-1.52 (m, 5H), 1 .39-1.33 (m, 5H), 1.23 (s, 1H), 1.02-0.95 (m, 2H), 0.87 (t, J=7.4 Hz, 3H), 0.83 (s, 3H) ppm. Holding time: 2.25 minutes, 100% with chiral SCPC (Ad). Anal. HPLC: > 98.50%, Holding time: 8.01 minutes (method B).

Example 15

This example demonstrates the method for preparing compound 11-7R in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12S,13R)-8-[2-(4-amino-3-fluorophenoxy)acetyl]-11-hydroxyl-9,13-dimethyl-6propyl-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-7S and 11-7R).

The racemic steroid mixture 11-7-22R/S was prepared according to the method described in Example 12. The racemic products were separated by chiral SCPC (see section 2.3 for details), yielding compound 11-7S (second peak) and compound 11-7R ( first peak).

Compounds 11-7R: NMR mass/charge: 540.2 (M+H) ⁺ .

1H NMR (500 MHz, CDCL) δ 7.25 (d, J=10.1 Hz, 1H), 6.87 (dt, J=15.5, 7.7 Hz, 1H), 6.47 (dd , J=12.8, 2.4 Hz, 1H), 6.37 (d, J=8.7 Hz, 1H), 6.29 (dd, J=9.9, 4.4 Hz, 1H) , 6.04 (s, 1H), 5.22-4.49 (m, 5H), 3.61 (s, 2H), 2.58 (td, J=13.5, 4.9 Hz, 1H ), 2.36 (d, J=10.3 Hz, 1H), 2.19-2.03 (m, 3H), 1.87-1.72 (m, 2H), 1.67-1, 55 (m, 3H), 1.511.33 (m, 7H), 1.21-1.11 (m, 2H), 1.00-0.90 (m, 6H), Anal. HPLC: > 62.24%, 36.49%, Hold time: 7.78, 7.86 minutes (method B).

Example 16.

This example demonstrates the method for preparing compound 11-8R in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxyl-9,13-dimethyl-8-{2-[4(methylamino)phenoxy]acetyl}-6-propyl-5 ,7-dioxapentacyclo-[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16one (11-8R).

Steroid 11-8 was prepared according to the method outlined in Example 13.

Compounds (11-8R) were obtained as a white solid (14 mg, 54% yield). Mass/charge at NMR: 525.3 (M+H) ⁺ .

1H NMR (500 MHz, MeOD _d4 ) δ 7.47 (d, J=10.1 Hz, 1H), 6.83-6.80 (m, 2H), 6.65-6.62 (m, 2H ), 6.28 (dd, J=10.1, 1.9 Hz, 1H), 6.04 (s, 1H), 4.99 (d, J=18.2 Hz, 1H), 4.90 (d, J=4.8 Hz, 1H), 4.74 (d, J=18.1 Hz, 1H), 4.66 (t, J=4.5 Hz, 1H), 4.46 (d , J=3.0 Hz, 1H), 2.75 (s, 3H), 2.67 (td, J=13.6, 5.2 Hz, 1H), 2.40 (dd, J=13, 5, 2.7 Hz,

- 138 045579

1H), 2.30-2.22 (m, 1H), 2.16-2.12 (m, 1H), 2.02 (dd, J=13.7, 3.3 Hz, 1H), 1 .85 (dd, J=13.7, 2.6 Hz, 1H), 1.76 (d, J=6.9 Hz, 1H), 1.67-1.63 (m, 4H), 1, 51 (s, 3H), 1.48-1.44 (m, 2H), 1.17-1.08 (m, 1H), 1.05 (dd, J=11.2, 3.5 Hz, 1H), 0.98-0.94 (m, 6H) ppm. Anal. HPLC: 100%, Hold time: 7.56 minutes (method A).

Example 17.

This example demonstrates the method for obtaining the compound (11-10R/S), in table. 1. This example refers to the connection numbered in FIG. 2. (1S,2S,4R,6R,8S,9S,11S,12S,13R)-8-[2-(4fluorophenoxy)acetyl]-11-hydroxy-9,13-dimethyl-6-propyl-5,7dioxapentacyclo [10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-10R/S). Steroids 11-10R/S were prepared according to the method outlined in Example 13. Compounds 11-10R/S were obtained as a white solid (14 mg, 54% yield). Mass/charge at NMR: 525.2 (M+H)+.

^1H NMR (400 MHz, MeOD _d4 ) δ 7.47 (d, J=10.1 Hz, 1H), 7.02 (t, J=8.7 Hz, 2H), 6.94-6.90 (m, 2H), 6.27 (dd, J=10.1, 1.8 Hz, 1H), 6.03 (s, 1H), 5.06 (d, J=18.1 Hz, 1H) , 4.90-4.88 (m, 1H), 4.82 (d, J=18.1 Hz, 1H), 4.69 (t, J=4.4 Hz, 1H), 4.46 ( d, J=2.8 Hz, 1H), 2.71-2.63 (m, 1H), 2.42-2.38 (m, 1H), 2.30-2.11 (m, 2H) , 2.05-2.01 (m, 1H), 1.89-1.84 (m, 1H), 1.77-1.63 (m, 5H), 1.51-1.41 (m, 5H), 1.18-1.02 (m, 2H), 0.97-0.93 (m, 6H) ppm. Anal. HPLC: 100%, Hold time: 9.94 minutes (method A).

Example 18.

This example demonstrates the method for preparing compound 11-11R/S in table. 1. This example refers to the connection numbered in FIG. 2.

N-(4-{2-[(1S,2S,4R,6R,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}phenyl)acetamide (11-11R/S).

Steroids 11-11R/S were prepared in accordance with the method outlined in example 13.

Compounds 11-11R/S were obtained as a white solid (25 mg, 46% yield).

Mass/charge at NMR: 564.3 (M+H) ⁺ .

1H NMR (500 MHz, MeOD _d4 ) δ 7.49-7.45 (m, 3H), 6.89 (d, J=9.0 Hz, 2H), 6.28 (d, J=10.2 Hz, 1H), 6.04 (s, 1H), 5.09 (d, J=18.1 Hz, 1H), 4.91-4.89 (m, 1H), 4.83 (d, J =18.1 Hz, 1H), 4.70 (t, J=4.3 Hz, 1H), 4.47 (d, J=3 Hz, 1H), 2.72-2.65 (m, 1H ), 2.43-2.39 (m, 1H), 2.30-2.22 (m, 1H), 2.18-2.12 (n, 4H), 2.06-2.03 (m , 1H), 1.901.86 (m, 1H), 1.77-1.65 (m, 5H), 1.48 (m, 5H), 1.18-1.09 (m, 1H), 1, 07-1.04 (m, 1H), 0.99-0.95 (m, 6H) ppm. Anal. HPLC: 100%, Holding time: 7.33 minutes (method B).

Example 19.

This example demonstrates the method for preparing compound 11-12R/S in table. 1. This example refers to the connection numbered in FIG. 2. (1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-[2-(4aminophenoxy)acetyl]-12,19-difluoro-11-hydroxy-9,13-dimethyl-6 -propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-12R/S).

Step 1: Compound (9B) was prepared according to the general procedure A in Example 9. To a solution of (6S,9R)2F-budesonide (80 mg, 0.17 mmol) in dichloromethane (1 ml) was added dropwise triethylamine (34 mg , 0.34 mmol) and methanesulfonyl chloride (30 mg, 0.26 mmol) at 0°C. The mixture was stirred at this temperature for half an hour until (6S,9R)2F-budesonide was consumed, which was monitored by TLC. The reaction mixture was then diluted with dichloromethane (100 ml) and quenched with saturated aqueous ammonium chloride (30 ml). The organic solution was washed with saturated aqueous ammonium chloride and brine, dried over sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography (0-2% MeOH in dichloromethane) to give the corresponding mesylate product (9B).

Step 2: Compound 9B was dissolved in acetone (0.5 ml). 4-Aminophenol (10-9.37 mg, 0.34 mmol) and cesium carbonate (0.11 g, 0.34 mmol) were added to the solution. The reaction mixture was refluxed for 1.5 hours or until completely consumed (9B) according to TLC and LCMS. The mixture was then diluted with ethyl acetate and filtered. The filtrate was concentrated in vacuo and the residue was purified by pre-HPLC (Method B) to give compounds 11-12R/S (6.0 mg, 6.3% yield from (6S,9R)2Fbudesonide) as a white solid. Mass/charge at NMR: 558 (M+H) ⁺ .

1H NMR (500 MHz, MeOD _d4 ) δ 7.34 (d, J=10.0 Hz, 1H), 6.78-6.71 (m, 4H), 6.37-6.33 (m, 2H ), 5.63-5.49 (m, 1H), 5.10-4.99 (m, 1H), 4.77-4.63 (m, 2H), 4.33 (d, J=9 ,1 Hz, 1H), 2.74-2.57 (m, 1H), 2.39-2.13 (m, 3H), 1.98-1.31 (m, 12H), 1.03- 0.93 (m, 6H) ppm. Anal. HPLC: purity 97.4%, Holding time: 7.55 minutes (method B).

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-[2-(4-aminophenoxy)acetyl]-12,19-difluoro-11-hydroxy9,13-dimethyl-6 -propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-12R).

Compound 9BR was prepared according to general procedure A in Example 9. Compound 9BR (0.90 g, 1.7 mmol) was reacted with 4-aminophenol (0.20 g, 1.8 mmol) and cesium carbonate (1, 1 g, 3.4 mmol) in acetonitrile (20 ml) gave (11-12R) (0.20 g, 54% yield) as a yellow oil after purification by silica gel column chromatography (50-80% ethyl acetate in petroleum ether) . Mass/charge at NMR: 558 (M/+H) ⁺ .

1H NMR (500 MHz, DMSO _d6 ) δ 7.26 (d,J=10.5 Hz, 1H), 6.64 (d,J=5.0 Hz, 2H), 6.50 (d,J= 5.0 Hz, 2H), 6.30 (dd, J=10 Hz, 2 Hz, 1H), 6.11 (s, 1H), 5.72-5.65 (m, 0.5H), 5 ,62-5.55 (m, 0.5H), 5.52-5.48 (m, 1H), 5.0 (s,

- 139 045579

0.5H), 4.95 (s, 0.5H), 4.80-4.78 (m, 1H), 4.75-4.65 (m, 1H), 4.24-4.16 ( m, 1H), 2.70-2.52 (m, 1H), 2.30-2.21 (m, 1H), 2.11-2.00 (m, 2H), 1.77 (d, J=13.0 Hz, 1H), 1.61-1.54 (m, 4H), 1.49 (s, 3H), 1.36 (q, J=7.5 Hz, 3H), 1, 23 (s, 1h), 0.87 (d, J=7.5 Hz, 3H), 0.83 (s, 3H) ppm. Anal. HPLC: 100%, dwell time: 8.44 minutes (method B).

Example 20

This example demonstrates the method for preparing compound 11-13R in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-[2-(3-aminophenoxy)acetyl]-12,19-difluoro-11-hydroxy9,13-dimethyl-6 -propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-13R).

Steroid 11-13R was prepared according to the method outlined in example 19.

Compound (11-13R) was obtained as a light orange liquid (9.0 mg, 44% yield) after purification by pre-HPLC (Method A). Mass/charge at NMR: 558 (M+H)+.

^1H NMR (500 MHz, MeOD _d4 ) δ 7.35 (dd, J=10.1, 1.3 Hz, 1H), 7.29 (t, J=8.1 Hz, 1H), 6.76 -6.70 (m, 3H), 6.40-6.29 (m, 2H), 5.66-5.48 (m, 1H), 5.14 (d, J=18.1 Hz, 1H ), 4.93-4.91 (m, 1H), 4.90-4.87 (m, 1H), 4.77 (t, J=4.3 Hz, 1H), 4.35 (d, J=9.3 Hz, 1H), 2.76-2.62 (m, 1H), 2.41-2.18 (m, 3H), 1.83-1.56 (m, 9H), 1 .50 (dt, J=15.4, 7.6 Hz, 2H), 0.99-0.96 (m, 6H) ppm. Anal. HPLC: 100%, Hold time: 7.77 minutes (method A).

Example 21.

This example demonstrates the method for preparing compound 11-14R/S in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-[2-(4-amino-3-fluorophenoxy)acetyl]-12,19-difluoro-11-hydroxy9,13-dimethyl -6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-diene-16-og (11-14R/S).

To a solution of (9B) (0.20 g, 0.37 mmol) in DMSO (3 ml) was added 4-amino-3-fluorophenol (10-14, 0.25 g, 2.0 mmol) and potassium hydroxide (0 .11 g, 2.0 mmol) at room temperature. The resulting mixture was stirred at 60°C for an hour under the protection of nitrogen until the reaction was complete, which was monitored by TLC and LCMS. After cooling to room temperature and membrane filtration, the reaction solution was directly purified by Prep-HPLC (Method A) to obtain compound 11-14R/S (40 mg, 19% yield) as a yellowish white solid. Mass/charge at NMR: 576 (M+H) ⁺ .

1H NMR (500 MHz, MeODd4) δ 7.40-7.31 (m, 1H), 7.20 (td, J=9.1, 1.9 Hz, 1H), 6.91-6.84 ( m, 1H), 6.806.76 (m, 1H), 6.40-6.30 (m, 2H), 5.57 (ddd,J=48.6, 9.7, 6.8 Hz, 1H) , 5.15 (d,J=18.1 Hz, 1H), 4.90-4.79 (m, 2H), 4.75 (t, J=4.3 Hz, 1H), 4.41- 4.28 (m, 1H), 2.78-2.57 (m, 1H), 2.40-2.12 (m, 3H), 1.98-1.39 (m, 11H), 1, 07-0.92 (m, 6H) ppm. Anal. HPLC: 100%, Holding time: 8.10 minutes (method A).

Example 22.

This example demonstrates the method for preparing compound 11-15R/S in table. 1. This example refers to the connection numbered in FIG. 2.

tert-Butyl N-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-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}phenyl)methyl]carbamate (N -Boc-11-15R/S).

Step 1: To a solution of 4-(aminomethyl)phenol (1.2 g, 10 mmol) in methanol (70 ml) and water (5 ml) add Boc2O (2.4 g, 11 mmol) dropwise using a syringe at room temperature temperature. The resulting mixture was stirred at room temperature for an hour until 4(aminomethyl)phenol was completely consumed, which was monitored by TLC and LCMS. Volatiles were removed in vacuo and the residue was dissolved in ethyl acetate (150 ml). The solution was washed with saturated aqueous citric acid (50 mLx2) and brine, dried over sodium sulfate and concentrated in vacuo to give N-Boc-4-aminomethylphenol (2.1 g, 94% yield) as a brown oil. Mass/charge at NMR: 246 (M+Na)+.

1H NMR (500 MHz, CDCl·,) δ 7.12 (d, J=7.8 Hz, 2H), 6.82-6.71 (m, 2H), 4.84 (s, 1H), 4 .23 (d, J=5.3 Hz, 2H), 1.46 (s, 9H) ppm.

Step 2: compound (N-Boc-11-15R/S) was prepared according to the method outlined in example 19. (1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-{2-[ 4-(aminomethyl)phenoxy]acetyl}-12,19-difluoro-11-hydroxy-9,13dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.8 ]icosa-14,17-dien-16-one (11-15R/S).

To a solution of (N-Boc-11-15R/S) (30 mg, 45 μmol) in DCM (2 ml), TFA (0.4 ml) was added dropwise using a syringe at 0°C. The resulting mixture was stirred at room temperature for an hour until Boc was completely removed, which was monitored by LCMS. Volatiles were removed in vacuo and the residue was purified by pre-HPLC (Method A) to give compound (11-15R/S) (15 mg, 49% yield) as a white solid. Mass/charge at NMR: 572 (M+H) ⁺ .

1H NMR (500 MHz, MeOD _d4 ) δ 7.45-7.32 (m, 3H), 7.01-6.96 (m, 2H), 6.41-6.30 (m, 2H), 5 .57 (ddd, J=18.2, 10.4, 7.3 Hz, 1H), 5.21 (dd, J=19.7 Hz, 1H), 4.93-4.91 (m, 1H ), 4.85 (d, J=18.0 Hz, 1H), 4.77 (t, J=4.3 Hz, 1H), 4.37-4.32 (m, 1H), 4.07 (s, 2H), 2.75-2.58 (m, 1H), 2.40-2.15 (m, 3H), 1.86-1.40 (m, 11H), 1.08-0 .92 (m, 6H) ppm. Anal. HPLC: 100%, dwell time: 7.47 minutes (method A).

Example 23.

This example demonstrates the method for preparing compound 11-16R/S in table. 1. This example

- 140 045579 refers to the connection numbered in FIG. 2. (1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-8[2-(4-hydroxyphenoxy)acetyl]-9,13-dimethyl-6-propyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa14,17-dien-16-one (11-16R/S).

Compounds 11-16R/S were prepared according to the method outlined in Example 12. Compounds 11-16R/S (20 mg, 38% yield) were obtained as a light brown solid after pre-HPLC purification (Method A). Mass/charge at NMR: 523.2 (M+H)+.

1H NMR (500 MHz, MeODd4) δ 7.47 (d, J=10.1 Hz, 1H), 6.82-6.77 (m, 2H), 6.75-6.70 (m, 2H) , 6.28 (dd, J=10.1, 1.8 Hz, 1H), 6.04 (s, 1H), 5.00 (d, J=18.1 Hz, 1H), 4.91- 4.89 (m, 1H), 4.75 (d, J=18.1 Hz, 1H), 4.67 (t, J=4.5 Hz, 1H), 4.46 (d, J=3 ,1 Hz, 1H), 2.68 (td, J=13.6, 5.8 Hz, 1H), 2.40 (dd, J=13.5, 2.8 Hz, 1H), 2.31 -2.21 (m, 1H), 2.17-2.13 (m, 1H), 2.02 (dd, J=13.7, 3.3 Hz, 1H), 1.86 (dd, J =13.7, 2.6 Hz, 1H), 1.80-1.58 (m, 5H), 1.531.40 (m, 5H), 1.18-0.93 (m, 8H) ppm. Anal. HPLC: 100%, Hold time: 8.92 minutes (method A).

Example 24.

This example demonstrates the method for preparing compound 11-17R/S in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-{2-[(6-Aminopyridin-2-yl)oxy]acetyl}-12,19-difluoro-11hydroxy-9, 13-dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (1117R/S).

Compounds 11-17R/S were prepared according to the method outlined in Example 19. Compounds (11-17R/S) (50 mg, 24% yield) were obtained as a white solid after purification by flash chromatography (1050% ethyl acetate in petroleum ether) . IER: 559 (M+H) ⁺ .

¹ H NMR (500 MHz, DMSOd6) δ 7.35-7.31 (m, 2H), 6.31 (d, J=11.5 Hz, 1H), 6.13 (s, 1H), 6, 03 (d, J=8.0 Hz, 1H), 5.98 (d, J=7.5 Hz, 1H), 5.84-5.82 (m, 1H), 5.68-5.56 (m, 3H), 5.25-4.72 (m, 4H), 4.29 (br s, 1H), 2.662.57 (m, 1H), 2.28-2.05 (m, 4H) , 1.63-1.58 (m, 4H), 1.50-1.30 (m, 6H), 0.95-0.87 (m, 6H) ppm. Anal. HPLC: 100%, Holding time: 8.65 minutes (method A).

Example 25.

This example demonstrates the method for preparing compound 11-19 in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-(2-Azidoacetyl)-12,19-difluoro-11-hydroxy-9,13-dimethyl-6propyl-5,7- dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (11-19).

Step 1: A suspension of compound 9B (1.0 g, 1.8 mmol), sodium azide (1.2 g, 18 mmol) in acetone (15 ml) was stirred at 50°C overnight until the reaction was complete according to LCMS . After cooling, the reaction mixture was poured into cold water (80 ml). The aqueous mixture was extracted with ethyl acetate (50 mlx3). The combined organic solution was washed with brine (30 mL), dried over sodium sulfate and concentrated in vacuo to give the crude azido precursor compound (1119R/S) (0.90 g, >99% yield) as a yellow solid, which used for the next step without further purification. Mass/charge at NMR: 492 (M+H) ⁺ .

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-(2-aminoacetyl)-12,19-difluoro-11-hydroxy-9,13-dimethyl-6propyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one; trifluoroacetic acid salt (11-19R/S).

Step 2: To a solution of the precursor of compounds 11-19R/S (0.85 g, 1.7 mmol) in THF (20 ml) was added an aqueous solution of hydrochloride (1 N, 10 ml). The mixture was stirred at 28-32°C until it became clear, then triphenylphosphine (0.68 g, 2.6 mmol) was added to it at this temperature. The resulting yellow transparent solution was stirred at 28-32°C for 18 hours until the reaction was complete according to TLC and LCMS. The mixture was concentrated in vacuo and the residue was purified by reverse phase flash chromatography (0-50% acetonitrile in aqueous TFA (0.05%)) to give compounds 11-19R/S (0.56 g, 57% yield, TFA salt) as a yellowish solid. Mass/charge at NMR: 466 (M+H) ⁺ .

1H NMR (400 MHz, MeOD _d4 ) δ 7.33 (d, J=9.9 Hz, 1H), 6.40-6.29 (m, 2H), 5.69-5.45 (m, 1H ), 4.93-4.92 (m, 1H), 4.71 (t, J=4.3 Hz, 1H), 4.35-4.27 (m, 2H), 3.90-3, 84 (m, 1H), 2.81-2.54 (m, 1H), 2.42-2.06 (m, 3H), 1.82-1.32 (m, 11H), 1.09- 0.87 (m, 6H) ppm. ^19F NMR (376 MHz, MeOD _d4 ) δ -77.01, -166.24, -166.92, -188.81, 188.83 ppm. Anal. HPLC: 100%, Hold time: 6.86 minutes (method A).

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-(2-aminoacetyl)-12,19-difluoro-11-hydroxy-9,13-dimethyl-6propyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one; salt of trifluoroacetic acid (11-19R).

Step 1: Using the same procedure as above, the azido precursor (1119R) (0.12 g, 87% yield) was obtained from compound (9BR) as a white solid after purification by flash chromatography (0-50% ethyl acetate in petroleum ether). Mass/charge at NMR: 492 (M+H) ⁺ .

1H NMR (500 MHz, CDCl ₃ ) δ 7.10 (dd, J=10.2, 1.3 Hz, 1H), 6.44 (s, 1H), 6.38 (dd, J=10.2 , 1.8 Hz, 1H), 5.48-5.31 (m, 1H), 4.92 (d, J=5.4 Hz, 1H), 4.62 (t, J=4.4 Hz , 1H), 4.43 (dd, J=5.6, 2.7 Hz, 1H), 4.22 (d, J=18.7 Hz, 1H), 3.94 (d, J=18, 7 Hz, 1H), 2.56-2.39 (m, 2H), 2.32-2.18 (m, 2H), 1.85-1.71 (m, 3H), 1.67-1 .54 (m, 7H), 1.46-1.37 (m, 2H), 0.97-0.90 (m, 6H) ppm.

- 141 045579

Step 2: Using the same procedure as above, compound 11-19R (30 mg, 66% yield) was obtained as a white solid after purification by pre-HPLC (Method A). Mass/charge at NMR: 466 (M+H)+.

1H NMR (500 MHz, MeOD _d4 ) δ 7.34 (d, J=10.0 Hz, 1H), 6.40-6.30 (m, 2H), 5.65-5.46 (m, 1H ), 4.94-4.91 (m, 1H), 4.72 (t, J=4.3 Hz, 1H), 4.34-4.28 (m, 2H), 3.88 (d, J=18.8 Hz, 1H), 2.78-2.60 (m, 1H), 2.39-2.34 (m, 1H), 2.33-2.18 (m, 2H), 1 .77-1.54 (m, 9H), 1.53-1.40 (m, 2H), 0.99-0.95 (m, 6H) ppm. Anal. HPLC: 100%, Hold time: 6.85 minutes (method A).

Example 26.

This example demonstrates the method for preparing compound 11-20R/S in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-8-(2-{[(4methoxyphenyl)methyl](methyl)amino}acetyl)- 9,13-dimethyl-6-propyl-5,7dioxapentacyclo[10.8.0.0 ^{2.9.0 4.8.0 13.18} ]icosa-14,17-dien-16-one; trifluoroacetic acid (11-20R/S).

To a solution of compound 9B (0.54 g, 1.0 mmol) in acetonitrile (10 ml), NPMB-methylamine (0.30 g, 2.0 mmol) and potassium carbonate (0.28 g, 2.0 mmol) were added sequentially ) at room temperature. The reaction mixture was stirred at 70°C overnight. After cooling, the mixture was diluted with DCM and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash chromatography (10-90% ethyl acetate in petroleum ether) to give the crude compound (11-20R/S) (0.20 g, 33% yield) as a white solid. The crude product (30 mg) was further purified by HPLC (Method A) to obtain the pure compound (11-20R/S) as a white solid (12 mg, 13% yield). Mass/charge at NMR: 600 (M+H) ⁺ .

^1H NMR (500 MHz, MeOD _d4 ) δ 7.50-7.43 (m, 2H), 7.34 (d, J=10.1 Hz, 1H), 7.07 (d, J=8, 5 Hz, 2H), 6.396.30 (m, 2H), 5.56 (δ, J=48.5, 10.7, 6.5 Hz, 1H), 5.24-5.21 (m, 1H ), 4.94-4.92 (m, 1H), 4.64-4.53 (m, 1H), 4.384.16 (m, 4H), 3.86 (s, 3H), 2.92- 2.91 (m, 3H), 2.76-2.56 (m, 1H), 2.39-2.31 (m, 1H), 2.28-2.09 (m, 2H), 1. 97 (td, J=13.2, 7.8 Hz, 1H), 1.78-1.23 (m, 10H), 1.08-0.88 (m, 6H) ppm. Anal. HPLC: 100%, Hold time: 7.81 minutes (method A).

Example 27.

This example demonstrates the method for preparing compound 11-21R/S in table. 1. This example refers to the connection numbered in FIG. 2.

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-8-[2(methylamino)acetyl]-6-propyl- 5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one; trifluoroacetic acid (11-21R/S).

To compounds 11-20R/S (30 mg, 0.053 mmol) in a 4 ml screw cap vial was added 1-chloroethylcarbonochloridate (1 drop) and chloroform (0.4 ml). The mixture was stirred at 70°C for 2 hours until the material was consumed according to TLC. After cooling to room temperature, methanol (1.5 ml) was added to the mixture. The mixture was stirred at 70°C for 1 hour until the reaction was complete, which was monitored by TLC and LCMS. Volatiles were removed in vacuo and the residue was purified by pre-HPLC (Method A) to give 11-21R/S (8.0 mg, 28% yield) as a white solid. Mass/charge at NMR: 480 (M+H) ⁺ .

1H NMR (400 MHz, MeOD _d4 ) δ 7.34 (d, J=10.1 Hz, 1H), 6.41-6.26 (m, 2H), 5.56 (ddd, J=48.7 , 10.0, 6.8 Hz, 1H), 5.28 (t, J=4.9 Hz, 1H), 5.23 (d, J=7.4 Hz, 1H), 4.47-4 .41 (m, 1H), 4.34-4.30 (m, 1H), 4.07-4.00 (m, 1H), 2.82-2.54 (m, 4H), 2.43 -2.09 (m, 3H), 1.96 (td, J=13.6, 7.9 Hz, 1H), 1.81-1.34 (m, 10H), 1.10-0.85 (m, 6H) ppm. ¹⁹ F NMR (376 MHz, MeOD _d4 ) δ -76.96, -166.28, -166.95, -188.80, -188.83 ppm. Anal. HPLC: 99%, Hold time: 6.97 minutes (method A).

Example 28.

This example demonstrates the method for preparing compound 14-2 in table. 1. This example refers to the connection numbered in FIG. 3.

(1R,2S,8S,10S,11S,13S,14R,15S,17S)-1,8-difluoro-17-hydroxy-2,13,15-trimethyl-14-[2(methylamino)acetyl]-5- Octotetracyclo[8.7.0.0 ^2.7 .0 ^11.15 ]heptadeca-3,6-dien-14-yl propanoate (14-2).

The synthesis of flumethasone mesylate (12) was published in Bioorg. Med. Chem. Lett., 2015, 25, 2837-2843, the entire contents of which are incorporated herein by reference for all purposes.

A solution of 12 (crude 82 mg) in methylamine (2M solution in THF, 1.5 ml, 3000 mmol) in a sealed tube was stirred at room temperature overnight and then heated at 60°C for 3 hours to complete the reaction. The solution was concentrated in vacuo and the residue was purified by pre-HPLC (0-80% acetonitrile in water with 10 mmol NH4HCO3) to give 14-2 (8 mg, 11% yield over two steps) as a white solid. Mass/charge at NMR: 480.2 (M+H).

1H NMR (DMSO-d6, 400 MHz) δ 7.27-7.25 (d, J=10.4 Hz, 1H), 6.30-6.27 (dd, J=10.4, 2.0 Hz, 1H), 6.10 (s, 1H), 5.73-5.56 (m, 1H), 5.43-5.32 (m, 2H), 4.62-4.42 (m , 1H), 4.25-4.18 (m, 1H), 4.15 (brs, 1H), 2.87 (s, 2H), 2.70 (s, 1H), 2.60-2, 56 (m, 1H), 2.36-1.90 (m, 7H), 1.49-1.35 (m, 5H), 1.10-0.91 (m, 10H).

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Example 29.

This example demonstrates a method for preparing compound 15-5 of Table 1. This example refers to the compound numbered in FIG. 3.

(1R,2S,8S,10S,11S,13R,14R,15S,17S)-14-[2-(4-aminophenoxy)acetyl]-1,8-difluoro-14,17-dihydroxy2,13,15-trimethyltetracyclo [8.7.0.0 ^2.7 .0 ^11.15 ]heptadeca-3,6-dien-5-one (15-5).

Step 1: mixture of compounds (12) (0.16 g, 0.33 mmol), 4-nitrophenol (10-5, 92 mg, 0.67 mmol) and potassium carbonate (92 mg, 0.67 mmol) in acetone (15 ml) was refluxed (60°C) for 18 hours. After cooling to room temperature, volatiles were removed in vacuo. The residue was purified by flash chromatography (0-1% ethyl acetate in petroleum ether) to give the nitro intermediate (0.14 g, 79% yield) as a white solid. Mass/charge at NMR: 532 (M+H)+.

¹ H NMR (CDCl ₃ , 400 MHz) δ 8.20 (d, J=9.0 Hz, 2H), 7.10 (d, J=10.5 Hz, 1H), 6.94 (d, J =9.0 Hz, 2H), 6.43 (br s, 1H), 6.39-6.37 (m, 1H), 5.45-5.32 (m, 1H), 5.26 (d , J=18.0 Hz, 1H), 4.85 (d, J=18.0 Hz, 1H), 4.43-4.40 (m, 1H), 3.21-3.16 (m, 1H), 2.60 (s, 1H), 2.52-2.40 (m, 2H), 2.30-2.20 (m, 2H), 2.06-1.99 (m, 1H) , 1.86-1.68 (m, 3H), 1.53-1.48 (m, 2H), 1.09 (s, 3H), 0.99 (d, J=7.0 Hz, 3H ) chnm.

Step 2: To a solution of the nitro intermediate (0.13 g, 0.25 mmol) in a combined solution of ethanol (20 ml) and water (2 ml), add iron powder (0.14 g, 2.5 mmol), then ammonium chloride (0.14 g, 2.5 mmol). After stirring at 80°C for 2 hours, the suspension was cooled to room temperature and filtered through celite to remove inorganic salts. The filtrate was concentrated in vacuo and the residue was purified by prep-HPLC (Method B) to give compound 15-5 (90 mg, 70% yield) as a white solid. Mass/charge at NMR: 502 (M+H) ⁺ .

1H NMR (DMSO _d6 , 500 MHz) δ 7.27 (d, J=10.0 Hz, 1H), 6.59 (d, J=8.5 Hz, 2H), 6.49 (d, J= 8.5 Hz, 2H), 6.31-6.28 (m, 1H), 6.11 (s, 1H), 5.77-5.57 (m, 1H), 5.42-5.39 (m, 1H), 5.22 (s, 1H), 5.07 (d, J=18.5 Hz, 1H), 4.63 (s, 1H), 4.59 (d, J=18, 5 Hz, 1H), 4.29-4.10 (m, 1H), 2.99-2.91 (m, 1H), 2.55-2.43 (m, 3H), 2.25-2 .19 (m, 3H), 1.71-1.64 (m, 1H), 1.56-1.43 (m, 5H), 1.15-1.10 (m, 1H), 0.88 (s, 3H), 0.83 (d, J=6.0 Hz, 3H) ppm.

Example 30.

This example demonstrates the method for preparing compound 16-5 in table. 1. This example refers to the connection numbered in FIG. 4.

(1R,2S,10S,11S,13R,14R,15S,17S)-14-[2-(4-aminophenoxy)acetyl]-1-fluoro-14,17-dihydroxy-2,13,15trimethyltetracyclo[8.7.0.0 ^2.7 .0 ^11.15 ]heptadeca-3,6-dien-5-one (16-5).

The synthesis of dexamethasone mesylate (13) was described in J. Pharmacol., 172, 1360 (2015), the entire contents of which are incorporated herein by reference for all purposes.

A mixture of dexamethasone mesylate (13.94 mg, 0.20 mmol), 4-nitrophenol (10-5.42 mg, 0.30 mmol) and potassium carbonate (55 mg, 0.40 mmol) in acetone (10 ml) was boiled with reflux condenser. (60°C) for 3 hours and then concentrated. The crude product was concentrated in vacuo and then directly purified by flash chromatography (0-50% ethyl acetate in petroleum ether) to give the nitro intermediate (0.10 g, 97% yield) as a white solid. Mass/charge at NMR: 514 (M+H) ⁺ .

1H NMR (MeOD _d4 , 400 MHz) δ 8.23 (d, J=9.0 Hz, 2H), 7.43 (d, J=10.5 Hz, 1H), 7.04 (d, J= 9.0 Hz, 2H), 6.31 (dd, J=10.0 Hz, 2.0 Hz, 1H), 6.11 (br s, 1H), 5.41 (d, J=18.0 Hz, 1H), 4.96 (d, J=18.0 Hz, 1H), 4.34-4.30 (m, 1H), 3.13-3.06 (m, 1H), 2.79 -2.72 (m, 1H), 2.57-2.41 (m, 3H), 2.32-2.26 (m, 1H), 1.94-1.90 (m, 1H), 1 .82-1.75 (m, 1H), 1.62 (s, 3H), 1.62-1.53 (m, 2H), 1.28-1.23 (m, 1H), 1.07 (s, 3H), 0.92 (d, J=7.0 Hz, 3H) ppm.

Iron powder ( ₆₇ mg, 1.2 mmol) and then ammonium chloride (64 mg, 1.2 mmol). After stirring at 80°C for 1.5 hours, the suspension was cooled to room temperature and filtered through celite to remove inorganic salts. The filtrate was concentrated in vacuo and the residue was purified by prep-HPLC (Method B) to give compound 16-5 (20 mg, 35% yield) as a white solid. Mass/charge at NMR: 484 (M+H) ⁺ .

1H NMR (MeOD _d4 , 500 MHz) δ 7.42 (d, J=10.5 Hz, 1H), 6.78-6.74 (m, 2H), 6.73-6.70 (m, 2H ), 6.31 (dd, J=10.0 Hz, 2.0 Hz, 1H), 6.10 (br s, 1H), 5.08 (d, J=18.0 Hz, 1H), 4 .71 (d, J=18.0 Hz, 1H), 4.30-4.27 (m, 1H), 3.14-3.09 (m, 1H), 2.78-2.71 (m , 1H), 2.54-2.37 (m, 3H), 2.30-2.24 (m, 1H), 1.94-1.89 (m, 1H), 1.81-1.74 (m, 1H), 1.62 (s, 3H), 1.59-1.52 (m, 2H), 1.26-1.21 (m, 1H), 1.06 (s, 3H), 0.91 (d, J=7.5 Hz, 3H) ppm.

Example 31.

This example demonstrates methods for separating stereoisomers of certain compounds disclosed herein.

SLC (supercritical liquid chromatography) technology has been used to purify low molecular weight compounds that are thermally labile, including chiral compounds. SCLC used supercritical liquid carbon dioxide as the mobile phase and a solid adsorbent based on organic polymers as the stationary phase. Based on the different partition coefficient of epimers in the two phases, mixed epimers can be separated

- 143 045579 pour, adjusting the density of the mobile phase. Instrument and column conditions are described as follows: Instrument: SKZHK - 80 (Thar, Waters), Column: AD 20 * 250 mm, 5 µm (Decial), Column temperature: 35°C, Mobile phase: CO ₂ /EtOH (1% Methanol Ammonia) = 65/35, flow rate: 80 g/min, back pressure: 100 bar, detection wavelength: 214 nm, cycle time: 4.5 min, test solution: 130 mg dissolved in 30 ml methanol, injection volume : 1.5 ml. Using a chiral AD-H column, 20 grams of 22R/S-budesonide were separated to give 8.9 grams of R-budesonide and 8.9 grams of S-budesonide with an overall recovery yield of 89%. Similarly, the two epimers of compound 11-5R/S were also separated by SCLC. Detailed separation conditions are described below in the table. 5.

Table 5

Conditions for the chiral separation of budesonide and compound (11-5) in table. 1

Compound Budesonide 11-5^/5 Device SKZHH-200 (Thar, Waters) SKZHH-200 (Thar, Waters) Column AD-H 20*250 mm, 5 µm (Dacel) SC 20*250 mm, 5 microns Column temperature 35°С 35°С Mobile phase CO ₂ /methanol (0.5% NH ₄ OH) = 70/30 CO ₂ /methanol (0.5% NH ₄ OH) = 60/40 Flow rate 120 g/min. 140 g/min Back pressure 100 bar 100 bar Detection wavelength 214 nm 214 nm Cycle time 4.0 minutes 5.0 minutes Test solution 20 g dissolved in 130 ml methanol 10 g dissolved in 130 ml methanol Injection volume 1.0 ml 0.5 ml

The structures of 22R/S-budesonide were determined stereospecifically using 2D-NOESY. Compared with proton NMR data for 22R/S-budesonide, the first compound from the chiral SCPC was identified as an R-epimer and the second as an S-epimer. The configuration at the C ²² position affects the magnetic resonances of neighboring protons. In the S spectrum, a double doublet was observed with J ₁₆ eH- ₁₅ eH = 5.0 Hz and J ₁₆ eH- _15a H = 2.5 Hz, which was a consequence of steric repulsion from the 22-propyl substituent, which shields the C ¹⁶ proton in S -epimere. This effect is not observed in the R epimer. The ^C22 proton in the S epimer also shifts downward compared to the R epimer, indicating decreased protection of the ^C22 proton in the S epimer due to steric repulsion between the 17β-ketol substituent and the 22β-propyl chain in the S epimer. Similarly, the ^C22 proton in the R epimer was protected by the anisotropy effect from the ^C20 carbonyl group in the 22R epimer. Details of the chemical shifts are described below in the table. 6.

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Table 6

Protons in pos. Chemical shifts (ppm) in D-chloroform _o ^{n N} /T o-^d λκ,λ ⁰ but 2о1 ' ¹ he nix ^{H Η} ΛΛ OTD HO 2θ1 \H * OH 1 Reported R- epimer 1st compound according to chiral SCFC data Reported S'- epimer 2nd compound according to chiral SCPC data S-1 7.26 (d.7i, ₂ = 10.1) 7.26 (d, 7i, ₂ = 10.1) 7.23 (d, 71.2= 10.1) 7.26 (d, 71.2= 10.0) S-2 6.27 (dd, 7i, ₂ = 10.1, 7 ₂ .4 = 1.8) 6.28 (dd, 71.2= 10.1, 7 ₂ .4 = 1.7) 6.27 (dd, 71.2= 10.1, 7 ₂ .4 = 1.8) 6.27 (dd, 71.2= 10.1, 7 ₂ .4 = 1.7) S-4 6.03(m) 6.03(s) 6.02(m) 6.02(s) S-11 4.4-4.6(m) 4.42-4.60 (m) 4.50(m) 4.50 (br s) S-16 4.90 (dd, 7ΐ6 _βΗ -ΐ5βΗ = 4.2) 4.90 (d, 7ιββΗ-ΐ5βΗ = 4.4) 5.16 (dd, 7ΐ6βΗ-ΐ5βΗ = 5.0, 7ΐ6βΗ-15αΗ = 2.5) 5.23-5.11(m) S-18 0.92(s) 0.92(s) 0.99(s) 0.99(s) S-19 1.45(s) 1.44(s) 1.45(s) 1.46(s) S-21 4.50 (dd), 4.25 (dd) (7 ₂ w,n' = -20.2, 7 ₂ ih-2ioh = 4.8) 4.50 (m), 4.26 (dd, 7 ₂ ih,h' = 20.1, 7 ₂ ih-2ioh = 4.3) 4.60 (dd), 4.20 (dd) (7 ₂ w,n' = -20,2,7 ₂ ih- 2YN = 4.8) 4.62 (d), 4.21 (d) (72Ш.Н’ = 19.9) S-22 4.55 (t, 7 ₂₂ , ₂ 3 = 4.2) 4.55 (t, 722.23 = 4.6) 5.16(t.7 _22.23 = 4.6) 5.23-5.11 (m) S-25 0.92 (t, 7 ₂₄ , ₂₅ = 6.7) 0.92 (t, 7 ₂₄ .25 = 7.3) 0.91(t.7 ₂ 4.25 = 7.3) 0.91 (t, 7 ₂₄ .25 = 7.3)

Example 32.

This example generally demonstrates methods for producing linkers and payload linkers.

In fig. Figure 9 shows three methods for producing payload linkers. In fig. 9, R' is a steroidal amine or aniline; R is an alkyne containing moiety, such as moiety A or B, or a malimide moiety, such as C; R1 means an amino acid residue; P is a protecting group such as Fmoc or Boc; n means an integer from 0 to 11; m means an integer from 2 to 4; p is an integer from 0 to 5. In approach I, the amide (23) is formed by a coupling reaction between the steroidal amine or aniline (21, Q = NH or NR) and the dipeptide (22), followed by N deprotection. The amine (23) is then coupled to an acid or its active ester (24), such as V-5, V7, V in FIG. 10, VI - 8 and VI in Fig. 11 and VII in FIG. 12 to generate a linker with payload (25). In approach II, amide (28) is formed by coupling the acid or its active ester (26) and VC-pAB (27) followed by N-protection. Compound 28 was then converted to its derivative PNP, which was further reacted with 21 to form a carbamate linker payload (29). In approach III, a carbamate (30) is formed from an N-protected dipeptide-pAB-PNP (19) and a steroidal amine or aniline (21) followed by N-protection; the amine moiety in 30 was then combined with the acid or its active ester (26) to form 29.

Example 33.

This example demonstrates methods for preparing the DIBAC-Suc-NHS (V) linker. The following example relates to FIG. 10.

See methods in J. Org. Chem., 2010, 75, 627-636, which is incorporated herein by reference in its entirety for all purposes.

Step 1: N-[tricyclo[9.4.0.03,8]nentadeca-1(11),3,5,7,9,12,14-heptaen-2-ylidene]hydroxylamine (V2): dibenzosuberenone mixture (V-1 ) (21 g, 0.10 mol) and hydroxylamine hydrochloride (9.3 g, 0.14 mol) in a combined solution of absolute ethanol (100 ml) and pyridine (200 ml) were stirred and refluxed for 15 hours. TLC showed that the starting material was consumed (TLC: 5% methanol in methylene chloride). After cooling to below 25°C, the reaction mixture was diluted with methylene chloride (500 ml) and the resulting solution was washed with aqueous HCl (1 N, 3x200 ml) and then brine (200 ml). The organic solution was dried over sodium sulfate and concentrated in vacuo to give crude V-2 (22 g, 98% crude yield) as a light brown solid. Mass/charge at NMR: 222.1 (M+H)+.

Step 2: 2-azatricyclo[10.4.0.04' ⁹ ]hexadeca-1(16),4(9),5,7,10,12,14-heptene (V-3): to oxime solution (V-2) (5.5 g, 25 mmol) in dry methylene chloride (here also dichloromethane or DCM) (150 ml) at -5°C, add DIBAL-H (1 mol in toluene, 250 ml) dropwise while maintaining the temperature below -5° WITH.

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The reaction mixture was then stirred at room temperature overnight and then quenched with a solution of solid sodium fluoride (38 g, 0.90 mol) in water (12 ml) at 0°C. The suspension was stirred at 0°C for another 30 minutes and filtered through celite. The celite was washed thoroughly with methylene chloride and the combined organic solution was concentrated in vacuo to give V-3 (4.6 g, 89% yield) as a yellow solid. Mass/charge at NMR: 222.1 (M+H)+.

Step 3: 4-[2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(16),4(9),5,7,10,12,14-heptaen-2-yl]-4-oxobutanoic acid (V-5): To a solution of (V-3) (5.0 g, 24 mmol) in methylene chloride (50 ml) was added DIPEA (3.1 g, 24 mmol), then succinic anhydride (V-4, 2, 9 g, 29 mmol). The mixture was then stirred at room temperature for 4 hours, quenched with aqueous sodium bisulfate (1N, 100 ml) and extracted with methylene chloride (3x100 ml). The combined organic solution was washed with water (100 ml), then brine (100 ml), dried over sodium sulfate and concentrated in vacuo to give (V-5) (7.7 g, 95% yield) as a white solid, which used without further purification. Mass/charge at NMR: 308.2 (M+H) ⁺ .

Step 4: 4-{10,11-dibromo-2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(16),4(9),5,7,12,14-hexaen-2-yl}- 4-oxobutanoic acid (V-6): A solution of (V-5) (15 g, 49 mmol) in methylene chloride (200 ml) was washed with nitrogen and cooled to 0°C. Liquid bromine (23 g, 0.14 mol) was added dropwise to the solution at 0°C through a syringe. The reaction mixture was stirred at the set temperature for 2 hours and TLC indicated that the reaction was complete (TLC: 10% methanol in methylene chloride). The reaction mixture was diluted with methylene chloride (50 ml) and allowed to warm to room temperature. The organic solution was washed with saturated (saturated) aqueous sodium sulfite (3x50 ml), water (50 ml), then brine (50 ml), dried over sodium sulfate and concentrated in vacuo to give (V-6) (13 g, crude yield product 99%) in the form of a yellowish solid. Mass/charge at NMR: 467.9 (M+H) ⁺ .

¹ H NMR (CDCl ₃ , 400 MHz): δ 7.71 (d, J=6.8 Hz, 1H), 7.25-7.01 (m, 6H), 6.94-6.88 (m , 1H), 5.90 (d, J=9.6 Hz, 1H), 5.84-5.79 (m, 1H), 5.25-5.25 (m, 1H), 4.24- 4.10 (m, 1H), 2.87-2.80 (m, 1H), 2.68-2.47 (m, 3H) ppm.

Step 5: 4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(16),4(9),5,7,12,14-hexaen-10-yn-2-yl}-4oxobutanoic acid (V-7): A solution of (V-6) (5.0 g, 11 mmol) in anhydrous THF (50 ml) was cooled to 40°C in a dry ice/acetonitrile bath, and a solution of potassium tert-butanolate in tetrahydrofuran. (1H, 37 ml, 37 mmol) dropwise under argon. The reaction mixture was stirred at this temperature for half an hour after addition. TLC showed that the reaction was complete (TLC: 10% methanol in methylene chloride). The reaction mixture was allowed to warm to room temperature and quenched with aqueous sodium bisulfate (1N) to pH 1. The mixture was extracted with methylene chloride (3x50 ml). The combined organic solution was washed with water (50 ml), then brine (50 ml), dried over sodium sulfate and concentrated in vacuo to give compound (V-7) (2.7 g, 95% yield) as a yellowish solid. Mass/charge at NMR: 306.1 (M+H) ⁺ .

1H NMR (DMSOd6, 500 MHz): δ 11.98 (s, 1H), 7.67-7.29 (m, 8H), 5.02 (d, J=13.5 Hz, 1H), 3, 61 (d, J=14.5 Hz, 1H), 2.61-2.56 (m, 1H), 2.32-2.27 (m, 1H), 2.21-2.16 (m, 1H), 1.80-1.76 (m, 1H) ppm.

Step 6: 4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-yn-2-yl}-4oxobutanoic acid (V): To a solution of acid (V-7) (50 mg, 0.16 mmol) in methylene chloride (10 ml), N-hydroxysuccinimide (HOSu, 28 mg, 0.24 mmol) and N-(3dimethylaminopropyl)- N-ethylcarbodiimide hydrochloride (EDCI, 47 mg, 0.24 mmol). After stirring at room temperature overnight, the mixture was washed with water, then brine, dried over sodium sulfate and concentrated in vacuo to give intermediate V, which was used directly for the next step. Mass/charge at NMR: 403.0 (M+H) ⁺ .

Example 34.

This example demonstrates methods for preparing the DIBAC-Suc-PEG4-acid/NHS (VI) linker. The following example relates to FIG. eleven.

Step 1: tert-Butyl-1-hydroxy-3,6,9,12-tetraoxapentadecane-15-oate (VI-3): to a solution of tetraethylene glycol (VI-1, 58 g, 0.30 mol) in dry THF ( 200 ml) sodium (0.12 g) was added, the mixture was stirred until sodium was consumed. Then, tert-butyl acrylate (VI-2, 13 g, 0.10 mol) in dry THF (50 ml) was added dropwise to the resulting solution, and the resulting mixture was stirred at room temperature overnight. The reaction was quenched first with acetic acid (0.1 ml), then with water (0.5 ml), the resulting mixture was stirred at room temperature for half an hour, then extracted with ethyl acetate (3x200 ml). The combined organic solution was washed with water (30 ml), then brine (3 x 100 ml), dried over sodium sulfate, filtered and concentrated to give the product (VI-3, 26 g, 81% yield) as a colorless oil. Mass/charge at NMR: 340 (M+18) ⁺ .

Step 2: tert-Butyl 1-(methanesulfonyloxy)-3,6,9,12-tetraoxapentadecane-15-oate (VI-4): to solution (VI-3) (26 g, 81 mmol), triethylamine (12 ml , 89 mmol) in methylene chloride (150 ml) in an ice bath, a solution of methanesulfonyl chloride (10 g, 89 mmol) in DCM (50 ml) was added dropwise. The mixture was stirred at room temperature for 14 hours and then concentrated in vacuo. The residue was mixed with water (30 ml), then extracted with ethyl acetate (3x200 ml). Combined organic layer

- 146 045579 washed with brine (3x100 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give the desired product (VI-4) (31 g, 95% yield) as a light yellow oil. Mass/charge at NMR: 418 (M+18)+.

Step 3: tert-Butyl 1-azido-3,6,9,12-tetraoxapentadecane-15-oate (VI-5): to a solution of (VI-4) (27 g, 67 mmol) in N,N-dimethylformamide ( 70 ml) sodium azide (6.6 g, 0.10 mol) was added, which was then stirred at 80°C for 4-16 hours. After cooling to room temperature, the mixture was diluted with ethyl acetate (3x150 ml). The combined solution was washed with water (30 ml), then brine (3x100 ml), dried over sodium sulfate, filtered and concentrated in vacuo. The crude product was purified by silica gel column chromatography (petroleum ether/ethyl acetate (1% to 2% methanol) = 4/1) to give (VI-5) (18 g, 67% yield) as a colorless oil. Mass/charge at NMR: 365 (M+18) ⁺ .

Step 4: tert-butyl 1-amino-3,6,9,12-tetraoxapentadecane-15-oate (VI-6): to solution (VI-5) (1.5 g, 4.3 mmol in ethyl acetate (20 ml ) wet Pd/C (10%, 0.15 g) was added under a nitrogen atmosphere. The mixture was then purged with hydrogen and stirred at room temperature under a hydrogen balloon overnight. The mixture was then filtered through celite. The celite was washed with ethyl acetate (10 ml). The combined filtrate was concentrated in vacuo to give crude (VI-6) (1.4 g) as a light yellow oil, which was used for the next step without further purification.NMR mass/charge: 322 (M+H) ⁺ .

Step 5: 1-Amino-3,6,9,12-tetraoxapentadecane-15-oic acid (VI-7): to a solution of (VI-6) above (1.4 g) in methylene chloride (10 ml) TFA (5 ml) was added. The mixture was stirred at room temperature for an hour. Volatiles were removed in vacuo to obtain the crude product (VI-7) as its TFA salt (1.6 g) as a yellow oil, which was used for the next step without further purification. Mass/charge at NMR: 266 (M+H) ⁺ .

Step 6: 1-(4-{2-azatricyclo[10.4.0.0 ^4-9 ]hexadeca-1(12),4(9),5,7,13,15-hexan-10-yn-2-yl} -4oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-oic acid (VI-8): mixture of 4-{2azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5 ,7,13,15-hexaen-10-yn-2-yl}-4-oxobutanoic acid (V in Fig. 11, 1.0 g, 2.5 mmol) and (VI-7) (0.91 g , 2.5 mmol) in N,N-dimethylformamide (10 ml) was added to triethylamine (0.50 g, 5.0 mmol). The mixture was stirred at room temperature overnight. The mixture was directly purified by reverse phase flash chromatography (0-100% acetonitrile in water (NH ₄ HCO ₃ , 10 mmol) to give (VI-8) (1.0 g, 74% yield in 3 steps from VI- 5) as a brown oil. Mass/charge at NMR: 553.3 (M+H) ⁺ .

^1H NMR (MeODd4, 400 MHz): δ 7.65 (d, J=7.2 Hz, 1H), 7.64-7.58 (m, 1H), 7.49-7.42 (m, 3H), 7.40-7.30 (m, 2H), 7.28-7.22 (m, 1H), 5.12 (d, J=13.6 Hz, 1H), 3.75-3 .68 (m, 3H), 3.63-3.50 (m, 12H), 3.50-3.39 (m, 2H), 3.25 (t, J=5.6 Hz, 2H), 2.76-2.66 (m, 1H), 2.52 (t, J=6.0 Hz, 2H), 2.41-2.30 (m, 1H), 2.21-2.14 ( m, 1H), 2.031.93 (m, 1H) ppm.

Step 7: 2,5-dioxopyrrolidin-1-yl 1-(4-{2-azatricyclo[10.4.0.0 ^4-9 ]hexadeca-1(12),4(9), 5,7,13,15hexane-10 -in-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-oate (VI): to a solution of (VI-8) (40 mg, 72 µmol) in methylene chloride (10 ml) HOSu (1-hydroxypyrrolidine-2,5dione, 12 mg, 0.11 mmol) and EDCI (21 mg, 0.11 mmol) were added sequentially. The mixture was stirred at room temperature overnight, then diluted with methylene chloride (50 ml). The organic solution was washed with water (50 ml), then brine (50 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give intermediate (VI), which was used for the next step without further purification. Mass/charge at NMR: 650 (M+H) ⁺ .

1H NMR ( _CDCl3 , 400 MHz): δ 7.70 (m, 1H), 7.66 (m, 1H), 7.55-7.47 (m, 3H), 7.38-7.24 ( m, 4H), 6.33 (br s, 1H), 5.13 (d, J=13.6 Hz, 1H), 3.83-3.78 (m, 1H), 3.66-3, 60 (m, 13H), 3.47-3.35 (m, 2H), 2.99-2.82 (m, 6H), 2.51-2.43 (m, 2H), 2.20- 1.89 (m, 4H) ppm.

Example 35.

This example demonstrates methods for the preparation of 1-((1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-yl)-3oxo-2,7,10,13,16-pentaoxa-4-azanonedecane -19-onic acid (BCN-PEG ₄ -acid, VII). The following example relates to FIG. 12.

To a solution of intermediate VII-1 (0.10 g, 0.33 mmol) in tetrahydrofuran (THF) (5 ml), diisopropylethylamine (0.17 g, 1.3 mmol), intermediate (VI-7) ( 89 mg, 0.33 mmol) and 1-hydroxybenzotriazole (HOBt, 43 mg, 0.33 mmol). The mixture was stirred at room temperature overnight. After filtration to remove insoluble solids and concentration in vacuo, the reaction mixture was directly purified by prep-HPLC (Method B) to obtain BCN-PEG4 acid (VII) (25 mg, 17% yield) as a colorless oil.

1H NMR (CDCl·, 400 MHz): δ 5.07 (br s, 1H), 4.14 (d, J=7.6 Hz, 2H), 3.77 (t, J=6.4 Hz , 2H), 3.70-3.55 (m, 14H), 3.40-3.31 (m, 2H), 2.58 (t, J=6.0 Hz, 2H), 2.30- 2.19 (m, 6H), 1.61-1.52 (m, 2H), 1.43-1.32 (m, 1H), 1.00.92 (m, 2H) ppm.

Example 36.

This example demonstrates methods for producing {4-[(2S)-2-[(2S)-2-[1-(4-{2

- 147 045579 azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7,13,15-hexaen-10-in-2-yl}-4-oxobutanamido)-3, 6,9,12tetraoxapentadecane-15-amido]-3-methylbutanamido]-5 -(carbamoylamino)pentanamido]phenyl} methyl 4nitrophenyl carbonate (DIBAC-Suc-PEG ₄ -VC-pAB-PNP, VIII). The following example relates to FIG. 13.

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

Step 1: HATU (309 mg, 0.81 mmol), then DIEA (140 mg, 1.08 mmol). The mixture was stirred at room temperature for 3 hours. After filtration to remove insoluble solids and concentration in vacuo, the reaction mixture was directly purified by reverse flash purification (NH ₄ HCO ₃ as buffer) to obtain a white solid (VIII-3) (300 mg, 60%). Mass/charge at NMR: 617(M+1).

{4-[(2s)-2-[(2S)-2-[1-(4-{2-azatricyclo[10.4.0.0 ^{4 9} ]hexadeca-1(12),4(9),5,7, 13,15-hexan-10-in-2-yl}4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5 (carbamoylamino)pentanamido]phenyl}methyl 4- nitrophenyl carbonate (VIII).

Step 2: To a solution of (VIII-3) (150 mg, 0.16 mmol) and (VIII-4) (150 mg, 0.49 mmol) in N,Ndimethylformamide (10 ml) was added DIEA (63 mg, 0. 49 mmol). The mixture was stirred at room temperature for 3 hours. After filtration to remove insoluble solids and concentration in vacuo, the reaction mixture was directly purified by reverse flash chromatography (NH4HCO3 as buffer) to give (VIII) as a yellow solid (50 mg, 28%). Mass/charge at NMR: 1079 (M+1).

Example 37.

This example demonstrates methods for producing a linker payload (LP1). The following example relates to FIG. 14.

tert-Butyl N-[(1S)-1-({4-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-8-(2-hydroxyacetyl)-9 ,13dimethyl-16-oxo-5,7-dioxapentacyclo[10.8.00 ^{2.9 .0 4.8} .0 ^13.18 ]icosa-14,17-dien-6yl]phenyl}carbamoyl)ethyl]carbamate (31) .

Step 1: mixture of Boc-Ala-OH (0.20 g, 0.42 mmol), DIPEA (0.12 g, 0.84 mmol) and HATU (0.24 g, 0.63 mmol) in N,N -dimethylformamide (5 ml) was stirred at 23°C for 30 minutes. Compound 7-1R (87 mg, 46 mmol) was then added to the solution. After stirring at 23°C for an additional 2 hours, the mixture was directly purified by pre-HPLC (Method B) to obtain compound 31 (0.11 g, 40% yield) as a white solid. Mass/charge at NMR: 651 (M+H) ⁺ .

(2S)-2-amino-N-{4-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-8-(2-hydroxyacetyl)-9,13dimethyl- 16-oxo-5,7-dioxapentacyclo[10.8.00 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-6-yl]phenyl}propanamide (32).

Step 2: TFA (0.3 mL) was added dropwise to a solution of compound 31 (0.10 g, 0.15 mmol) in methylene chloride (3 mL). The mixture was stirred at 23°C for one hour and volatiles were removed in vacuo to give crude (32) (83 mg) as an oil, which was used for the next step without further purification. Mass/charge at NMR: 551 (M+H) ⁺ .

tert-Butyl N-[(1S)-1-{[(1S)-1-({4-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-8 -(2hydroxyacetyl)-9,13-dimethyl-16-oxo-5,7-dioxapentacyclo[10.8.00 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-6yl]phenyl} carbamoyl)ethyl]carbamoyl}-2-methylpropyl]carbamate (33).

Step 3: mixture (32) (83 mg, 0.15 mmol), triethylamine (31 mg, 0.31 mmol) and Boc - Val-NHS (58 mg, 0.19 mmol) in N,N-dimethylformamide (5 mL) was stirred at 23°C for 4 hours, the reaction mixture was directly purified by pre-HPLC (Method B) to obtain (33) (52 mg, 20% yield in two steps) as a white solid. Mass/charge at NMR: 750 (M+H) ⁺ .

^1H NMR (500 MHz, DMSO _d6 ) δ 10.00 (s, 1H), 8.07 (d, J=7.0 Hz, 1H), 7.58 (d, J=8.5 Hz, 2H ), 7.40 (d, J=8.0 Hz, 2H), 7.31 (d, J=10.0 Hz, 1H), 6.72 (d, J=9.0 Hz, 1H), 6.16 (dd, J=1.5, 10.0 Hz, 1H), 5.91 (s, 1H), 5.38 (s, 1H), 5.08 (t, J=6.5 Hz , 1H), 4.92 (d,J=5.1 Hz, 1H), 4.78 (d, J=3.0 Hz, 1H), 4.55-4.46 (m, 1H), 4 .42 (t, J=7.0 Hz, 1H), 4.29 (s, 1H), 4.21-4.14 (m, 1H), 3.82 (t, J=8.5 Hz, 1H), 2.65-2.52 (m, 1H), 2.37-2.25 (m, 1H), 2.18-2.06 (m, 1H), 2.04-1.88 ( m, 2H), 1.85-1.57 (m, 5H), 1.40 (s, 3H), 1.37 (s, 9H), 1.29 (d, J=7.0 Hz, 3H ), 1.15-0.98 (m, 2H), 0.96-0.76 (m, 9H) ppm.

(2S)-2-amino-N-[(1S)-l-({4-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-8-(2 -hydroxyacetyl)9,13-dimethyl-16-oxo-5,7-dioxapentacyclo[10.8.00 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-6yl]phenyl} carbamoyl) ethyl]-3-methylbutanamide (34g).

Step 4: To a solution of compound 33 (50 mg, 67 μmol) in methylene chloride (3 ml) was added TFA (0.3 ml) dropwise, then the mixture was stirred at 23°C for an hour. Volatiles were removed in vacuo to obtain crude compound 34g (42 mg) as an oil, which was used for the next step without further purification. Mass/charge at NMR: 650 (M+H) ⁺ .

1-(4-{2-azatricyclo[10.4.0.0 ^4,9] hexadeca-1(12),4(9),5,7,13,15-hexan-10-in-2-yl}-4- oxobutanamido)N-(1S)-1-{[(1S)-l-({4-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-8-( 2-hydroxyacetyl)-9,13-dimethyl16-oxo-5,7-dioxapentacyclo[10.8.00 ^{2.9.0 4.8.0 13.18} ]icosa-14,17-dien-6-148 045579 yl] phenyl}carbamoyl)ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12-tetraoxapentadecane-15-amide (LP1).

Step 5: Solution of DIBAC-suc-PEG ₄ -OH (VI-8, 41 mg, 74 µmol), DIPEA (24 mg, 0.19 mmol) and HATU (47 mg, 0.12 mmol) in N,N- dimethylformamide (5 ml) was stirred at 23°C for 30 minutes, then (34g) was added (40 mg, 62 µmol). After stirring at 23°C for an additional 2 hours, the reaction mixture was directly purified by pre-HPLC (Method B) to obtain LP1 (33 mg, 44% yield in 2 steps) as a white solid. Mass/charge at NMR: 1185 (M+H)+.

^1H NMR (500 MHz, DMSOd6) δ 9.97 (s, 1H), 8.18 (d, J=6.5 Hz, 1H), 7.87 (d, J=8.5 Hz, 1H) , 7.75 (t, J=5.5 Hz, 1H), 7.67 (d, J=6.5 Hz, 1H), 7.63-7.56 (m, 3H), 7.53- 7.41 (m, 3H), 7.42 7.27 (m, 6H), 6.19-6.14 (m, 1H), 5.93 (s, 1H), 5.38 (s, 1H ), 5.08 (t, J=6.5 Hz, 1H), 5.03 (d, J=14.0 Hz, 1H), 4.92 (d, J=5.1 Hz, 1H), 4.78 (d, J=3.0 Hz, 1H), 4.55-4.46 (m, 1H), 4.42 (t, J=7.0 Hz, 1H), 4.29 (s , 1H), 4.21-4.14 (m, 2H), 3.63-3.55 (m, 3H), 3.50-3.40 (m, 12H), 3.32-3.26 (m, 2H), 3.10-3.05 (m, 2H), 2.65-2.52 (m, 2H), 2.48-2.48 (m, 2H), 2.40-2 .25 (m, 3H), 2.182.06 (m, 1H), 2.04-1.88 (m, 3H), 1.85-1.57 (m, 5H), 1.40 (s, 3H ), 1.28 (d, J=7.0 Hz, 3H), 1.15-0.98 (m, 2H), 0.96-0.84 (m, 6H), 0.84-0, 80 (d, J=7.0 Hz, 3H) ppm.

Example 38.

This example demonstrates a method for producing a payload linker (LP2).

The following example relates to FIG. 15.

tert-Butyl N-[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{2-[(1R,2S,8S,10S,11S,13R,14R,15S ,17S)-1,8difluoro-14,17-dihydroxy-2,13,15-trimethyl-5-octotetracyclo[8.7.0.0 ^2.7 .0 ^11.15 ]heptadeca-3,6-dien-14-yl] -2oxoethoxy}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamate (34e).

General Procedure C: To a solution of Boc-Val-Ala-OH or Boc-Val-Cit-OH (1.0 equiv.) in an organic solution (such as DCM or DMF) was added an alkali (such as DIPEA) (2.0 equiv. .) and HATU (1.2 equiv.) at 20-25°C. The mixture was stirred at 20-25°C for 30 minutes, after which alinine (1.1 equiv.) was added. The mixture was continued to stir for 16 hours until the peptide was consumed according to LCMS data. TFA (0.05 ml per 10 mg of peptide) was then added to the reaction mixture. The mixture was stirred at 20-25°C for another hour. Volatiles were removed under reduced pressure and the residue was purified directly by pre-HPLC (Method B).

Step 1: To a solution of Boc-VC (VC is Val-Cit) (67 mg, 0.18 mmol) in N,N-dimethylformamide (3 ml) add HATU (68 mg, 0.18 mmol) and NMM (30 mg , 0.30 mmol), the resulting solution was stirred at 23°C for 10 minutes. Compound 15-5 (75 mg, 0.15 mmol) was then added to the reaction mixture. After stirring at 23°C overnight, the reaction mixture was poured into ethyl acetate (80 ml), washed with brine, and then dried over anhydrous sodium sulfate. The combined organic solution was concentrated in vacuo and the residue was purified by flash chromatography (0-10% methanol in methylene chloride) to give (34e) (0.12 g, 89% yield) as a white solid. Mass/charge at NMR: 858 (M+H) ⁺ .

1H NMR (MeOD _d4 , 500 MHz) δ 7.54-7.47 (m, 2H), 7.36 (d, J=10.0 Hz, 1H), 6.90-6.87 (m, 2H ), 6.34 (dd, J=10.0, 1.5 Hz, 1H), 6.31 (s, 1H), 5.63-5.50 (m, 1H), 5.20 (d, J=18.0 Hz, 1H), 4.80 (d, J=18.0 Hz, 1H), 4.54-4.47 (m, 1H), 4.32-4.30 (m, 1H ), 3.92-3.81 (m, 1H), 3.23-3.11 (m, 3H), 2.65-2.52 (m, 1H), 2.43-2.32 (m , 3H), 2.111.99 (m, 1H), 1.79-1.58 (m, 9H), 1.46-1.24 (m, 11H), 1.06 (s, 3H), 1, 00-0.92 (m, 9H) ppm.

Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{2[( 1R,2S,8S,10S,11S,13R,14R,15S,17S)-1,8-difluoro-14,17-dihydroxy-2,13,15-trimethyl-5octotetracyclo[8.7.0.0 ^2.7 0 ^{11, 15} ]heptadeca-3,6-dien-14-yl]-2-oxoethoxy}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamoyl}-3,6,9,12-tetraoxatetradecan-1-yl)carbamate (LP2).

Step 2: To a solution of intermediate 34e (25 mg, 29 µmol) in methylene chloride (2 ml) was added TFA (1 ml), the resulting mixture was stirred at 23°C for an hour. Volatiles were removed in vacuo to leave a residue (25 mg, NMR Weight/Charge: 758.3 (M+H)+) as a brown oily residue.

To a solution of BCN-PEG ₄ -acid (VII in Fig. 12, 18 mg, 41 μmol) in N,N-dimethylformamide (2 ml) was added HATU (15 mg, 41 μmol) and NMM (6.9 mg, 41 μmol ), the resulting solution was stirred at 23°C for half an hour. Then, a solution of the brown oil residue obtained above in N,N-dimethylformamide (1 ml) was added to the reaction solution. After stirring at 23°C overnight, the mixture was isolated and purified directly by pre-HPLC (Method B) to obtain LP2 (15 mg, 37% yield) as a white solid. Mass/charge at NMR: 1181.4 (M+H) ⁺ .

1H NMR (DMSOd6, 400 MHz) (rotamer) δ 9.82 and 9.37 (s, 1H), 8.39 (d, J=8.0 Hz, 0.4H), 8.09 (d,J =7.2 Hz, 0.6H), 8.00 (d,J=8.0 Hz, 0.4H), 7.88 (d,J=8.8 Hz, 0.6H), 7.55 (d, J=8.8 Hz, 1H), 7.49 (d, J=8.8 Hz, 1H), 7.27 (d, J=10.0 Hz, 1H), 7.10 (br s, 1H), 6.80 (m, 2H), 6.29 (dd, J=10.0, 1.0 Hz, 1H), 6.11 (s, 1H), 5.99-5.94 (m, 1H), 5.72-5.56 (m, 1H), 5.43-5.41 (m, 3H), 5.31 (s, 1H), 5.22 (d, J=18 .0 Hz, 1H), 4.71 (d, J=18.0 Hz, 1H), 4.37-4.31 (m, 1H), 4.24-4.14 (m, 2H), 4 .04 (s, 1H), 4.02 (s, 1H), 3.62-3.56 (m, 2H), 3.50-3.45 (m, 12H), 3.40-3.37 (m, 2H), 3.13-3.08 (m, 2H), 3.00-2.92 (m, 3H), 2.54-2.33 (m, 2H), 2.25-2 .08 (m, 8H), 2.09-1.90 (m, 1H), 1.78-1.23 (m, 15H), 1.14-1.09 (m, 1H), 0.89 -0.82 (m, 14N) ppm.

Example 39.

This example demonstrates a method for producing a payload linker (LP3). Next

- 149 045579 example refers to FIG. 15.

(4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl} methyl N-(4{2-[(1R,2S,8S,10S ,11S,13R,14R,15S,17S)-1,8-difluoro-14,17-dihydroxy-2,13,15-trimethyl-5octotetracyclo[8.7.0.0 ^2.7 0 ^11.15 ]heptadeca-3,6 -dien-14-yl]-2-oxoethoxy}phenyl)carbamate (34f).

General procedure D.

Step 1: Fmoc-vcPAB-PNP (1.1 equiv), HOBt (1.5 equiv), and DIPEA (2.0 equiv) were added to a solution of aniline payload (1.0 equiv) in N,N-dimethylformamide .) at room temperature. The mixture was stirred at room temperature (18-30°C) until the starting material was consumed in accordance with LCMS data.

Step 2: Piperidine (0.03 ml per 10 mg payload) was added to the reaction mixture, the mixture was stirred at room temperature (18-30°C) for an hour until the Fmoc was removed, the process was monitored using LCMS. After membrane filtration, the reaction solution was directly purified by reverse phase flash chromatography or V-HPLC preparation to obtain vcPAB carbonate.

When N-Boc-vcPAB-PNP was used to replace Fmoc-vcPAB-PNP in the Step 1 reaction, NBoc vcPAB carbonate was obtained from Step 1. After purification, N-Boc vcPAB carbonate was redissolved in DCM, and treated with TFA (TFA concentration <25% ) at 0°C until Boc was removed, the process was monitored using LCMS. The reaction mixture was concentrated to remove volatiles, and the resulting residue was purified by chromatography or pre-HPLC to yield vcPAB carbonate.

To a solution of Fmoc-vcPAB-PNP (73 mg, 96 μmol) in N,N-dimethylformamide (1 ml), compound 15-5 (40 mg, 80 μmol), DMAP (20 mg, 0.16 mmol), HOBt was successively added (23 mg, 0.16 mmol) and DIPEA (55 mg, 0.40 mmol) at room temperature. The reaction mixture was stirred at room temperature for half an hour until completely consumed (15-5) in accordance with LCMS data. (ESI: 565.3 (M+H)+). Piperidine (34 mg, 0.40 mmol) was then added to the resulting mixture at room temperature. After stirring at room temperature for a further 30 minutes, which was monitored by LCMS, the resulting mixture was directly purified by reverse phase flash chromatography (0-30% acetonitrile in water) to (34f) (50 mg, 69% yield) as pale yellow solid. IER: 907 (M+N) ⁺ .

Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[( 4-{2[(1R,2S,8S,10S,11S,13R,14R,15S,17S)-1,8-difluoro-14,17-dihydroxy-2,13,15-trimethyl-5octotetracyclo[8.7.0.0 ^2.7 0 ^11.15 ]heptadeca-3,6-dien-14-yl]-2oxoethoxy}phenyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2methylpropyl]carbamoyl}-3.6.9 ,12-tetraoxatetradecane-1-yl)carbamate (LP3).

Step 3: HATU (27 mg, 70 µmol) and DIPEA (20 mg, 0.15 mmol) were successively added to a solution of BCN-PEG4 acid (60 mg, 67 µmol) in N,N-dimethylformamide (3.6 ml) at room temperature. The reaction mixture was stirred at room temperature for half an hour, after which compound (34f) (50 mg, 60 μmol) was added in portions. The reaction mixture was then stirred at room temperature for 2 hours until compound 34f was completely consumed according to LCMS data. The reaction mixture was then directly purified by pre-HPLC (Method B) to obtain LP3 (36 mg, 54% yield) as a white solid. ESI: 1330 (M+H) ⁺ .

1H NMR (400 MHz, DMSOd6) δ 10.02 (s, 1H), 9.56 (s, 1H), 8.14 (d, J=7.2 Hz, 1H), 7.89 (d, J =8.8 Hz, 1H), 7.62 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.4 Hz, 4H), 7.27 (d, J=10 .4 Hz, 1H), 7.11 (t, J=4.4 Hz, 1H), 6.78 (d, J=8.8 Hz, 2H), 6.33-6.26 (m, 1H ), 6.10 (s, 1H), 5.98 (t, J=5.4 Hz, 1H), 5.75-5.52 (m, 1H), 5.42 (s, 3H), 5 .30 (s, 1H), 5.20 (d, J=18.4 Hz, 1H), 5.05 (s, 2H), 4.70 (d, J=18.4 Hz, 1H), 4 .43-4.35 (m, 1H), 4.26-4.15 (m, 2H), 4.02 (d, J=7.6 Hz, 2H), 3.64-3.55 (m , 2H), 3.49 (s, 11H), 3.38 (t, J=6.0 Hz, 2H), 3.11 (dd, J=11.8, 5.9 Hz, 2H), 3 .05-2.88 (m, 3H), 2.44-2.31 (m, 2H), 2.28-2.08 (m, 9H), 2.02-1.90 (m, 1H) , 1.76-1.10 (m, 16H), 0.91-0.77 (m, 14H) ppm. HPLC purity: >99%, residence time: 7.03 minutes.

Example 40.

This example demonstrates a method for producing a payload linker (LP4). The following example relates to FIG. 16.

(2S)-2-amino-N-[(1S)-1-[(4-{2-[(1S,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-14,17-dien-8-yl]-2-oxoethoxy}phenyl )carbamoyl]ethyl]-3-methylbutanamide (34a).

General Procedure E: DIPEA (3.0 equiv) and HATU (1. 4 equiv.) at 20-25°C. The mixture was stirred at 20-25°C for 5 minutes after which alanine (1.0 equiv.) was added. The mixture was stirred for another 2 minutes until the peptide was completely consumed in accordance with LCMS data. Piperidine (5.0 equiv) was then added to the reaction mixture. The mixture was stirred at 20-25°C for 2 hours. After membrane filtration, the reaction solution was directly purified by reverse phase flash chromatography (0100% acetonitrile in aqueous ammonium bicarbonate (10 mmol)) or pre-HPLC (method B). Compound (34a) was prepared according to this General Procedure.

Alternatively, compound (34a) was prepared according to General Procedure C. To a solution of Boc-Val-AlaOH (0.29 g, 1.0 mmol) in methylene chloride (5 ml) was added DIPEA (0.26 g, 2.0 mmol) and HATU (0.46 g, 1.2

- 150045579 mmol), the mixture was stirred at 23°C for 30 minutes, then compound (11-5) (0.57 g, 1.1 mmol) was added to the reaction mixture. After stirring at 23°C for another 16 hours, TFA (1.5 ml) was added to the reaction mixture and the resulting mixture was stirred at 23°C for another hour. Volatiles were removed under reduced pressure and the residue was directly purified by pre-HPLC (Method B) to give 34a (0.17 g, 25% yield in 2 steps) as a white solid. Mass/charge at NMR: 692 (M+H)+.

^1H NMR (500 MHz, DMSOd6) δ 10.00 (s, 1H), 8.47 (d, J=6.5 Hz, 1H), 7.57-7.47 (m, 2H), 7, 33 (d, J=10 Hz, 1H), 6.87-6.82 (m, 2H), 6.18 (d, J=10 Hz, 1H), 5.93 (s, 3H), 5, 25-5.11 (m, 1H), 5.09 (d, J=6.5 Hz, 1H), 4.92-4.65 (m, 3H), 4.55-4.40 (m, 1H), 4.40-4.30 (m, 1H), 2.32-2.22 (m, 1H), 2.18-1.80 (m, 5H), 1.65-1.45 ( m, 5H), 1.451.25 (m, 9H), 1.25-0.98 (m, 2H), 0.96-0.76 (m, 13H) ppm.

Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1S)-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 ]icosa-14,17-dien-8yl]-2-oxoethoxy} phenyl)carbamoyl]ethyl]carbamoyl} -2-methylpropyl]carbamoyl} -3,6,9,12tetraoxatetradecane-1-yl)carbamate (LP4).

General Procedure F: HATU (1 equiv.) and DIPEA (2.5 equiv.) were added to a solution of BCN-PEG4 acid or its NHS ester in N,N-dimethylformamide. The mixture was stirred at 25°C for 30 minutes followed by the addition of the amine solution. After stirring at 25°C for 2 hours under LCSM control, the starting materials were consumed and the mixture was purified directly by pre-HPLC to obtain the desired amide.

To a solution of BCN-PEG4 acid (IX, 70 mg, 0.16 mmol) in N,N-dimethylformamide (8 ml), HATU (66 mg, 0.17 mmol) and DIPEA (56 mg, 0.43 mmol) were successively added ). The mixture was stirred at 25°C for 30 minutes, after which solution 34a (0.10 g, 0.15 mmol) was added. After stirring at 25°C for 2 hours, the mixture was purified directly by pre-HPLC (Method B) to obtain LP4 (25 mg, 16% yield) as a white solid. Mass/charge at NMR = 1116 (M+H) ⁺ .

Using chiral compound 11-5R as starting material, chiral (R)-LP4 was obtained as a white solid (24 mg, 31% yield) according to General Procedure F. NMR mass/charge: 1115 (M+H) ⁺ .

1H NMR (500 MHz, DMSOd6) (rotamers) δ 9.78 (s, 0.5H), 9.69 (s, 0.5H), 8.40 (d, J=7.5 Hz, 0.5H ), 8.15 (d, J=7.0 Hz, 0.5H), 8.01 (d, J=8.0 Hz, 0.5H), 7.89 (d, J=9.0 Hz , 0.5H), 7.57 (d, J=9.0 Hz, 1H), 7.51 (d, J=9.0 Hz, 1H), 7.32 (d, J=10.1 Hz , 1H), 7.09 (s, 1H), 6.85 (d, J=9.1 Hz, 2H), 6.18 (d, J=11.4 Hz, 1H), 5.93 (s , 1H), 5.10 (d, J=18.5 Hz, 1H), 4.86-4.67 (m, 4H), 4.45-4.36 (m, 1H), 4.33 ( s, 1H), 4.20 (t, J=7.5 Hz, 0.5H), 4.10 (t, J=7.8 Hz, 0.5H), 4.03 (d, J=8 .0 Hz, 2H), 3.59 (d, J=6.6 Hz, 2H), 3.49-3.45 (m, 11H), 3.39 (s, 2H), 3.30 (s , 2H), 3.11 (dd, J=11.4, 5.9 Hz, 2H), 2.47-2.43 (m, 1H), 2.38-2.12 (m, 8H), 2.03-1.83 (s, 5H), 1.62-1.51 (m, 6H), 1.42-1.24 (m, 10H), 1.02-0.94 (m, 2H ), 0.90-0.82 (m, 14H) ppm. Anal. HPLC: 100%, dwell time: 9.49 minutes (method A).

Example 41.

This example demonstrates a method for producing a payload linker (LP5).

The following example relates to FIG. 16.

(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)-N-(4-{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-diene-8 -yl]-2-oxoethoxy}phenyl)pentanamide (34c).

Compound 34c was prepared following general procedure C. A mixture of Boc-vc (0.26 g, 0.50 mmol), DIPEA (0.19 g, 0.60 mmol) and HATU (0.23 g, 0.60 mmol) in N,N-dimethylformamide (10 ml) was stirred at 23°C for 30 minutes, then mixture 11-5 (0.28 g, 0.55 mmol) was added.

After stirring at 23°C for 16 hours, the reaction mixture was directly purified using reverse phase flash chromatography (0-50% acetonitrile in water) to obtain the crude product (wt/charge at NMR 878 (M+H)+), which was dissolved in methylene chloride (8 ml) and treated with TFA (3 ml). The resulting mixture was stirred at 23°C for one hour. Volatiles were removed under reduced pressure and the residue was purified directly by pre-HPLC (Method B) to give 34c (0.12 g, 31% yield in 2 steps) as a white solid. Mass/charge at NMR: 778 (M+H) ⁺ .

1H NMR (500 MHz, DMSOd6) δ 9.97 (d,J=12.0 Hz, 1H), 8.10 (m, 1H), 7.51 (d,J=6.5 Hz, 2H), 7.32 (dd, J=10.1, 2.5 Hz, 1H), 6.83 (dd, J=15.9, 9.0 Hz, 2H), 6.17 (d, J=10, 0 Hz, 1H), 5.97 (t, J=5.0 Hz,1H), 5.93 (s, 1H), 5.40 (s, 2H), 5.22 (t, J=4, 8 Hz, 1H), 5.12 (d, J=6.0 Hz, 1H), 5.09 (d, J=6.5 Hz, 1H), 4.83-4.67 (m, 3H) , 4.47-4.37 (m, 1H), 4.35-4.29 (m, 1H), 3.05-2.90 (m, 3H), 2.57-2.51 (m, 1H), 2.30 (d, J=12.0 Hz, 1H), 2.13-1.74 (m, 7H), 1.70-1.46 (m, 7H), 1.45-1 .29 (m, 7H), 1.17-0.93 (m, 2H), 0.91-0.82 (m, 9H), 0.77 (dd, J=6.7, 2.7 Hz , 3H) ppm.

Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-[(4-{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]-2oxoethoxy}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamoyl}-3,6,9,12tetraoxatetradecane-1-yl)carbamate (LP5).

- 151 045579

LP5 was prepared following general procedure F. A solution of BCN-PEG4 acid (IX in FIG. 15, 0.28 g) in methylene chloride (6 ml) was added to a mixture of HATU (59 mg, 0.15 mmol) and DIPEA (50 mg, 0.39 mmol) in N,Ndimethylformamide (5 ml). The reaction mixture was stirred at 25°C for 30 minutes, and compound 34c (0.10 g, 0.13 mmol) was added thereto in one portion. The resulting mixture was stirred at 25°C overnight and directly purified by pre-HPLC (Method B) to obtain LP5 (35 mg, 23% yield) as a pale yellow solid. Mass/charge at NMR = 1202 (M+H)+.

^1H NMR (400 MHz, MeODd4) δ 7.61-7.43 (m, 3H), 6.87 (t, J=8.6 Hz, 2H), 6.26 (d, J=10.0 Hz, 1H), 6.02 (s, 1H), 5.29-5.02 (m, 2H), 4.84-4.65 (m, 2H), 4.51-4.44 (s, 2H), 4.22-4.05 (m, 3H), 3.80-3.68 (m, 2H), 3.67-3.45 (m, 14H), 3.22-3.08 ( m, 2H), 2.72-2.50 (m, 3H), 2.45-2.33 (m, 1H), 2.30-2.02 (m, 10H), 1.99-1, 82 (m, 2H), 1.81-1.32 (m, 17H), 1.26-0.85 (m, 17H) ppm.

Example 42.

This example demonstrates a method for producing a payload linker (LP6). The following example relates to FIG. 16.

(4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-(4{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 ]icos -14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamate (34d).

Compound 34d was prepared according to general procedure D.

Step 1: to a solution of compound (11-5) from the table. 1 (66 mg, 0.10 mmol) in N,N-dimethylformamide (3.5 ml), Boc-vcPAB-PNP (64 mg, 0.12 mmol), HOBt (14 mg, 0.10 mmol) and DIPEA ( 13.0 mg, 0.10 mmol). The reaction mixture was stirred at 13°C overnight and purified directly by pre-HPLC (Method B) to obtain intermediate Boc-34d (61 mg, 58% yield) as a white solid. Mass/charge at NMR: 1027.3 (M+H) ⁺ .

^1H NMR (MeOD _d4 , 400 MHz) δ 7.60 (d, J=8.4 Hz, 2H), 7.46 (d, J=10.4 Hz, 1H), 7.38-7.33 (m, 4H), 6.876.83 (m, 2H), 6.26 (dt, J=10.0, 2.0 Hz, 1H), 6.02 (s, 1H), 5.26-5, 03 (m, 4.2H), 4.82-4.67 (m, 1.8H), 4.54-4.51 (m, 1H), 4.48-4.43 (m, 1H), 3.91 (d, J=6.4 Hz, 1H), 3.31-3.18 (m, 1H), 3.14-3.08 (m, 1H), 2.70-2.63 ( m, 1H), 2.40-2.37 (m, 1H), 2.26-2.00 (m, 4H), 1.94-1.72 (m, 4H), 1.68-1, 35 (m, 20H), 1.22-0.92 (m, 14H) ppm.

Step 2: To a solution of Boc-34d (59 mg, 58 µmol) in DCM (2 ml) and MeOH (1 ml) was added dropwise HCl in dioxane (4 N, 1.5 ml) at 0°C. The mixture was then stirred at room temperature (14°C) for 4 hours. Volatiles were removed in vacuo to obtain 34d (60 mg, crude) as a brown oil, which was then used directly for the next step. Mass/charge at NMR: 927 (M+H) ⁺ .

Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1S)-4-(carbamoylamino)-1-{[4-({[( 4-{2[(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}phenyl)carbamoyl]oxy}methyl)phenyl]carbamoyl}butyl]carbamoyl}-2- methylpropyl]carbamoyl}-3,6,9,12-tetraoxatetradecane-1yl)carbamate (LP6). LP6 was obtained as a white solid (24 mg, 31% yield) following general procedure F. NMR mass/charge: 1350.5 (M+H) ⁺ .

1H NMR (DMSO _d6 , 400 MHz) δ 10.02 (s, 1H), 9.58 (s, 1H), 8.13 (d, J=7.6 Hz, 1H), 7.88 (d, J=8.4 Hz, 1H), 7.61 (d, J=8.4 Hz, 2H), 7.36-7.30 (m, 5H), 7.11 (t, J=4.8 Hz, 1H), 6.84-6.78 (m, 2H), 6.19-6.16 (m, 1H), 5.98 (t, J=5.2 Hz, 1H), 5.93 (s, 1H), 5.42 (s, 2H), 5.23-5.06 (m, 4H), 4.80-4.67 (m, 3H), 4.39-4.31 (m , 2H), 4.23 (t, J =7.2 Hz, 1H), 4.02 (d, J = 8.0 Hz, 2H), 3.64-3.55 (m, 2H), 3 .49 (m, 12H), 3.42-3.27 (m, 3H), 3.13-2.89 (m, 4H), 2.41-2.12 (m, 9H), 2.03 -1.95 (m, 2H), 1.91-1.82 (m, 2H), 1.75-1.68 (m, 1H), 1.61-1.20 (m, 16H), 1 .15-0.95 (m, 2H), 0.92-0.81 (m, 15H) ppm. Anal. HPLC: 69% + 31% = 100%, holding time: 8.86 minutes and 8.92 minutes (method B).

Example 43.

This example demonstrates a method for producing a linker with an LP7 payload. The following example relates to FIG. 16.

(Bicyclo[6.1.0]non-4-yn-9-ylmethyl N-(14-{[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S, 4R,

6R,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]-2oxoethoxy} phenyl)carbamoyl]ethyl]carbamoyl} -2-methylpropyl]carbamoyl} -3,6,9,12tetraoxatetradecane-1-yl)carbamate (LP7). LP7 (24 mg, 31% yield in 3 steps from 34a) was obtained as a white solid following general procedure F. NMR mass/charge: 1115 (M+H) ⁺ .

1H NMR (500 MHz, DMSOd6) (rotamers) δ 9.78 (s, 0.5H), 9.69 (s, 0.5H), 8.40 (d, J=7.5 Hz, 0.5H ), 8.15 (d, J=7.0 Hz, 0.5H), 8.01 (d, J=8.0 Hz, 0.5H), 7.89 (d, J=9.0 Hz , 0.5H), 7.57 (d, J=9.0 Hz, 1H), 7.51 (d, J=9.0 Hz, 1H), 7.32 (d, J=10.1 Hz , 1H), 7.09 (s, 1H), 6.85 (d, J=9.1 Hz, 2H), 6.18 (d, J=11.4 Hz, 1H), 5.93 (s , 1H), 5.10 (d, J=18.5 Hz, 1H), 4.86-4.67 (m, 4H), 4.45-4.36 (m, 1H), 4.33 ( s, 1H), 4.20 (t, J=7.5 Hz, 0.5H), 4.10 (t, J=7.8 Hz, 0.5H), 4.03 (d, J=8 ,0 Hz, 2H), 3.59 (d, J=6.6 Hz, 2H), 3.49-3.45 (m, 11H), 3.39 (s, 2H), 3.30 (s , 2H), 3.11 (dd, J=11.4, 5.9 Hz, 2H), 2.47-2.43 (m, 1H), 2.38-2.12 (m, 8H), 2.03-1.83 (s, 5H), 1.62-1.51 (m, 6H), 1.42-1.24 (m, 10H), 1.02-0.94 (m, 2H ), 0.90-0.82 (m, 14H) ppm. Anal. HPLC: 100%, dwell time: 9.47 minutes (method A).

- 152 045579

Example 44.

This example also demonstrates the payload acquisition method (LP7). The following example relates to FIG. 26.

The following reaction conditions were used.

Amine mg (µmol) Acid mg (µmol) Step 1 mg % exit ms mass/charge HATU mg (µmol) DIPEA mg (µmol) DMF (ml) Pace. (°C) Time (h) 26b thirty (43) VI-8 48 (87) 40(105) 17(132) 1 25 16 30 56% 1227.6 (M+H) ⁺

To a solution of acid (VI-8) (1.0-2.5 equiv.) in N,N-dimethylformamide (or DCM/DMF), DIPEA (1.5-10 equiv.) and HATU (2.5- 4.0 equiv.) at room temperature. The resulting mixture was stirred at this temperature for 0.5-1 hour, then amine (26b) (1.0 equiv.) was added. The reaction mixture was stirred at room temperature for 2-16 hours until the amine was completely consumed under LCMS control. The reaction mixture was membrane filtered, the filtrate was concentrated, then separated by pre-HPLC (Method B) to give the approximate compound LP7 (20-69% yield) as a white solid.

-(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-{[(1S)-1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12S,13R)-11-hydroxy-9 ,13-dimethyl16-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}phenyl )carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12-tetraoxapentadecan-15-amide (LP7).

Mass/charge at NMR: 1227.6 (M+H)+.

¹ H NMR (500 MHz, DMSOd6) (rotamers) δ 9.79 (s, 0.5H), 9.70 (s, 0.5H), 8.41 (d, J=7.5 Hz, 0. 5H), 8.17 (d, J=7.0 Hz, 0.5H), 8.02 (d, J=8.0 Hz, 0.5H), 7.89 (d, J=8.6 Hz, 0.5H), 7.77 (t, J=4.8 Hz, 1H), 7.68 (d, J=7.3 Hz, 1H), 7.62 (d, J=7.3 Hz, 1H), 7.58 (d, J=9.0 Hz, 1H), 7.53-7.43 (m, 4H), 7.40-7.28 (m, 4H), 6.88 -6.82 (m, 2H), 6.18 (d, J=9.1 Hz, 1H), 5.93 (s, 1H), 5.10 (d, J=18.4 Hz, 1H) , 5.03 (d, J=14.0 Hz, 1H), 4.83-4.67 (m, 4H), 4.454.29 (m, 2H), 4.23-4.17 (m, 0 ,5H), 4.11 (t, J=7.7 Hz, 0.5H), 3.64-3.40 (m, 15H), 3.31-3.26 (m, 2H), 3, 13-3.03 (m, 2H), 2.65-2.52 (m, 2H), 2.47-1.26 (m, 24H), 1.06-0.93 (m, 2H), 0.90-0.80 (m, 12H) ppm.

Anal. HPLC: 99%, dwell time: 8.55 minutes (method B).

Solubility: <0.1 mg/ml water; 0.06 mg/ml 20% DMSO in water; 0.07 mg/ml 30% DMSO in water.

Example 45.

This example demonstrates a method for producing a payload linker (LP15). The following example relates to FIG. 27-28. Note that in FIG. 27, connection 11b is identical to connection 11-5 in FIG. 2.

Step 1: obtaining connection (13b), with reference to FIG. 27.

HATU (1.0-2.8 equiv.) and TEA (2.0-5.0 equiv.) were added to a solution of acid Fmoc-Val-Ala-OH (12b) in N,N-dimethylformamide at 25°C. After stirring the mixture at 25°C for 30 minutes, a solution of amine (11b, i.e., payload, 1.0 equiv.) in N,Ndimethylformamide (1 ml) was added using a syringe. The resulting mixture was stirred at 25°C for 2-24 hours until most of the amine was consumed in accordance with LCMS data. Piperidine or diethylamine (excess) was then added to the mixture and the mixture was stirred at 25°C for 1-16 hours until complete removal of Fmoc under LCMS control. The reaction mixture was membrane filtered, the filtrate was concentrated and purified directly by pre-HPLC (Method B) or reverse phase flash chromatography to obtain 13b (23-64% yield) as a white solid.

- 153 045579

In particular, the following conditions applied.

Amine mg (mmol) Acid mg (mmol) Step 1 Step 2 Cleansing * % Output D mg Mass/charge (M+1) ⁺ HAT U mg (mmol) DIPE A mg (mmol) DM F (ml) Time (h) Et ₂ N H (ml) Be me (h) 11 b 85 (0.0 76) 12b 69 (0.08 8) 69 (0.18) TEA 18 (0.18) 3 2 0.5 16 R.P. 17.43% 832.2 (M/2+N) ⁺

Step 2: Preparation of compound (17a), with reference to FIG. 27.

HATU (1.0-2.8 equiv.) and DIPEA or TEA (2.0-5.0 equiv.) were added to a solution of compound 13b in N,N-dimethylformamide at 25°C. After stirring the mixture at 25°C for 30 minutes, a solution of Fmoc-Lys-(PEG)4-COT (13c, 1.0 equiv.) in N,N-dimethylformamide (1 ml) was added using a syringe. The resulting mixture was stirred at 25°C for 2-24 hours until more of the amine (13b) was consumed, according to LCMS data. Piperidine or diethylamine (excess) was then added to the mixture and the mixture was stirred at 25°C for 1-16 hours until complete removal of Fmoc under LCMS control. The reaction mixture was membrane filtered, the filtrate was concentrated and purified directly by pre-HPLC (Method B) or reverse phase flash chromatography to give compound (17a) (23-64% yield) as a white solid.

Step 3: Preparation of compound (27b), with reference to FIG. 27.

αcyclodextrin azide (16a) was added to a solution of alkyne (17а) (1.0 equiv.) in N,N-dimethylformamide or DMSO (See Synth. Sotp., 2002, 32(21), 3367-3372; J. Am Chem. Soc., 2012, 134(46), 19108-19117; J. Med. Chem., 1997, 40(17), 2755-2761; J. Am. Chem. Soc., 1993, full content of each of of these publications are incorporated herein by reference for all purposes, 115(12), 50355040) (1.5-3.0 equiv.). The resulting mixture was then stirred at 20-30°C for 16 hours-3 days until compound 16a was completely consumed and the desired mass of the intermediate was obtained, under LCMS control. After filtration, the resulting mixture was directly purified by pre-HPLC (or used directly) to give 27b (25-58% yield) as a white solid (with triazole regioisomers).

In particular, the following conditions applied.

Alkyne mg (mmol) 16a mg (mmol) Solution (ml) Pace. (°C) Time (h) Cleansing Exit Mass/charge 50 (0.030) 60 (0.06) DMSO (2) 25 48 RP-B 46 mg, 58% 887.9 (M/3+N) ⁺ .

Step 4: Receive connection (LP15), with reference to FIG. 28. The following reaction conditions were used.

Amine mg (µmol) Acid mg (µmol) mg % yield ms mass/charge HATU mg (µmol) DIPEA mg (µmol) DMF (ml) Pace. (°C) Time (h) 27b 13 (6.0) VI8 20 (36) 15 (39) 4.0(31) 2 25 2 6.0 36% 1259.1 (M/2+N) ⁺

To a solution of acid (VI-8) (1.0-2.5 equiv.) in N,N-dimethylformamide (or DCM/DMF), DIPEA (1.5-10 equiv.) and HATU (2.5- 4.0 equiv.) at room temperature. The resulting mixture was stirred at this temperature for 0.5-1 hour, then amine (27b) (1.0 equiv.) was added. The reaction mixture was stirred at room temperature for 2-16 hours until amine (27b) was completely consumed, n under LCMS control. The reaction mixture was membrane filtered, the filtrate was concentrated, then separated by pre-HPLC (Method B) to give the approximate compound (20-69% yield) as a white solid.

-(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-[(1R)-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20, 25,30pentakis(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 ]dotetracotan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9Hcycloocta[d] [1,2,3]triazol-4-yl)oxy] acetamido }-1-{[(1S)-1-{[(1S)-1 -[(4-{2[(1S,2S,4R,8S,9S,ΠS,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 ]]icos-14,17-dien-8-yl] -2-oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}-2methylpropyl]carbamoyl}pentyl]-3,6,9,12-tetraoxapentadecane-15-amide (LP15)

- 154 045579

Mass/charge at NMR: 1259.1 (M/2+H)+.

1H NMR (500 MHz, DMSOd ₆ ) (rotamers) δ 9.84 (s, 1H), 8.34 (s, 0.5H), 8.15 (d, J=7.3 Hz, 1H), 8 .04 (d, J=6.6 Hz, 1H), 7.90-7.84 (m, 1H), 7.81-7.74 (m, 1.5H), 7.72-7.56 (m, 4H), 7.56-7.27 (m, 11H), 6.89-6.79 (m, 2H), 6.17 (d, J=10.0 Hz, 1H), 5, 93 (s, 1H), 5.64-5.44 (m, 12H), 5.24-5.00 (m, 5H), 4.86-4.51 (m, 16H), 4.404.16 ( m, 5H), 4.05-3.96 (m, 1H), 3.86-3.73 (m, 10H), 3.67-2.88 (m, 35H), 2.80-2, 69 (m, 1H), 2.62-2.55 (m, 1H), 2.41-2.20 (m, 6H), 2.10-1.71 (m, 10H), 1.66- 1.07 (m, 26H), 1.05-0.79 (m, 17H) ppm. Anal. HPLC: 97%, Holding time: 6.62 and 6.67 minutes (method B). The exposure time is given for triazole regioisomers.

Example 46.

This example demonstrates a method for producing a payload linker (LP16). The following example relates to FIG. 27-28. The method for producing LP16 was the same as the method for producing LP15 in Example 45 herein, except that a different payload was used, as shown in FIG. 27-28.

The following reaction conditions were used.

Amine Acid Step 1 mg MG (µmol) MG (µmol) HATU mg (µmol) DIPEA mg (µmol) DMF (ml) Pace. (°C) Time (h) Cleansing % YOU- PROGRESS MS mass/charge 27b 30 (15) VI8 10 (18) 8.0 (21) 6.0 (47) 1 15-20 16 IN 18 47% 1259.1 (M/2+N) ⁺

To a solution of acid VI-8 (1.0-2.5 equiv.) in N,N-dimethylformamide (or DCM/DMF), DIPEA (1.5-10 equiv.) and HATU (2.5-4. 0 equiv.) at room temperature. The resulting mixture was stirred at this temperature for 0.5-1 hour, then amine (27b) (1.0 equiv.) was added. The reaction mixture was stirred at room temperature for 2-16 hours until the amine (27b) was completely consumed, under control LCMS. The reaction mixture was membrane filtered, the filtrate was concentrated, then separated by pre-HPLC (Method B) to give the approximate compound (20-69% yield) as a white solid.

-(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-[(1R)-5-{2-[(1-{[31,32,33,34,35,36,37,38,39,40,41,42-dodecahydroxy-10,15,20, 25,30pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29-dodecaoxaheptacyclo[26.2.2.2 ³ ' ⁶ .2 ^8D1 .2 ^13D6 .2 ¹⁸ ' ²¹ .2 ²³ ' ²⁶ ]dotetracotan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9Hcycloocta[d] [1,2,3]triazol-4-yl)oxy]acetamido}-1 -{[(1S)-1-{[(1S)-1 -[(4- {2[(1S,2S,4R,6R,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 ^13D8 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl) carbamoyl]ethyl]carbamoyl}-2-methylpropyl]carbamoyl}pentyl]-3,6,9,12-tetraoxapentadecane-15-amide (LP16)

- 155 045579

Mass/charge at NMR: 839.5 (M/3+H)+, 1259.1 (M/2+H)+ (60%).

1H NMR (500 MHz, DMSO _d6 ) (with triazole regioisomer) δ 9.77-9.42 (m, 1H), 8.27-8.20 (m, 0.5H), 8.17-8.01 (m, 2H), 7.86-7.74 (m, 2.5H), 7.70-7.60 (m, 4H), 7.57-7.43 (m, 7H), 7.39 -7.28 (m, 6H), 6.88-6.81 (m, 2H), 6.21-6.14 (m, 1H), 5.93 (s, 1H), 5.61-5 .42 (m, 10H), 5.16-4.97 (m, 4H), 4.89-4.48 (m, 17H), 4.40-4.28 (m, 4H), 4.16 -4.10 (m, 1H), 4.04-3.94 (m, 1H), 3.83-3.74 (m, 7H), 3.65-3.56 (m, 9H), 3 .48-3.21 (m, 23H), 3.15-3.06 (m, 4H), 2.97-2.89 (m, 1H), 2.81-2.69 (m, 1H) , 2.61-2.53 (m, 2H), 2.40-2.20 (m, 6H), 2.14-2.06 (m, 2H), 2.031.95 (m, 4H), 1 .91-1.70 (m, 5H), 1.64-1.52 (m, 9H), 1.49-1.25 (m, 14H), 1.13-0.81 (m, 19H) chnm. Anal. HPLC: 98%, dwell time: 6.61 (59%) and 6.73 (39%) minutes (method B). The holding times are given for the two triazole regioisomers. Solubility: 0.1 mg/ml 10% DMSO in water.

Example 47.

This example demonstrates a method for producing a payload linker (LP8).

The following example relates to FIG. 16.

(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)-N-(4-{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-diene-8 -yl]-2-oxoethoxy}phenyl)pentanamide (34h).

Compound (34h) was obtained as a white solid according to General Procedure C after purification by pre-HPLC (Method B). Mass/charge at NMR: 778 (M+H) ⁺ .

^1H NMR (500 MHz, DMSOd6) δ 9.97 (d, J=12.0 Hz, 1H), 8.10 (m, 1H), 7.51 (d, J=6.5 Hz, 2H) , 7.32 (dd, J=10.1, 2.5 Hz, 1H), 6.83 (dd, J=15.9, 9.0 Hz, 2H), 6.17 (d, J=10 .0 Hz, 1H), 5.97 (t, J=5.0 Hz, 1H), 5.93 (s, 1H), 5.40 (s, 2H), 5.22 (t, J=4 .8 Hz, 1H), 5.12 (d, J=6.0 Hz, 1H), 5.09 (d, J=6.5 Hz, 1H), 4.83-4.67 (m, 3H ), 4.47-4.37 (m, 1H), 4.35-4.29 (m, 1H), 3.05-2.90 (m, 3H), 2.57-2.51(m , 1H), 2.30 (d, J=12.0 Hz, 1H), 2.13-1.74 (m, 7H), 1.70- 1.46 (m, 7H), 1.45- 1.29 (m, 7H), 1.17-0.93 (m, 2H), 0.91-0.82 (m, 9H), 0.77 (dd, J=6.7, 2.7 Hz, 3H) ppm.

-(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-{[(1S)-1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-12,19 -difluoro-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}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12-tetraoxapentadecan-15amide (LP8).

Compound LP8 (25 mg, 20% yield) was obtained as a white solid following general procedure F. NMR mass/charge: 1263 (M/+H) ⁺ .

1H NMR (400 MHz, DMSO _d6 ) δ 9.79 (s, 0.7H), 9.69 (s, 0.3H), 8.41 (d,J=8.0 Hz, 0.3H), 8.16 (d, J=8.0 Hz, 0.7H), 8.01 (d, J=7.6 Hz, 0.3H), 7.89 (d, J=7.6 Hz, 0 ,7H), 7.77 (t, J=5.2 Hz, 1H), 7.70-7.66 (m, 1H), 7.64-7.60 (m, 1H), 7.60- 7.54 (m, 1H), 7.54-7.44 (m, 4H), 7.40-7.24 (m, 4H), 6.90-6.82 (m, 2H), 6. 30 (dd, J=10 Hz, 1.2 Hz, 1H), 6.11 (s, 1H), 5.72-5.55 (m, 1H), 5.52-5.48 (m, 1H ), 5.16-5.08 (m, 1H), 5.06-5.00 (m, 1H), 4.88-4.80 (m, 1H), 4.80-4.76 (m , 1H), 4.74 (t, J=4.0 Hz, 1H), 4.42-4.33 (m, 1H), 4.26-4.06 (m, 2H), 3.64- 3.54 (m, 3H), 3.503.40 (m, 12H), 3.12-3.02 (m, 2H), 2.70-2.55 (m, 2H), 2.40-2, 20 (m, 4H), 2.12-1.90 (m, 4H), 1.86-1.70 (m, 2H), 1.64-1.54 (m, 4H), 1.49 ( s, 4H), 1.46-1.34 (m, 3H), 1.29 (d, J=6.8 Hz, 3H), 0.90-0.80 (m, 13H) ppm. Anal. HPLC: 100%, dwell time: 8.26 minutes (method B).

Example 48.

This example demonstrates a method for producing a payload linker (LP9). Next

- 156 045579 example refers to FIG. 16.

{4-[(28)-2-[(28)-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- (4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro11-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-dien8-yl]-2-oxoethoxy} phenyl)carbamate (LP9).

Compound (34i) was obtained as a white solid according to general procedure D.

Compound LP9 (20 mg, 22% yield) was prepared following general procedure F. NMR mass/charge: 1499 (M+H)+.

^1H NMR (400 MHz, DMSO _d6 ) δ 10.02 (s, 1H), 9.59 (s, 1H), 8.14 (d, J=7.6 Hz, 1H), 7.88 (d , J=8.8 Hz, 1H), 7.80-7.75 (m, 1H), 7.70-7.66 (m, 1H), 7.65-7.60 (m, 3H), 7.53-7.45 (m, 3H), 7.40-7.28 (m, 7H), 6.84 (d, J=9.2 Hz, 2H), 6.30 (dd, J= 10.4 Hz, J=1.6 Hz, 1H), 6.11 (s, 1H), 6.10-6.0 (m, 1H), 5.72-5.55 (m, 1H), 5.52 (s, 1H), 5.43 (s, 2H), 5.16-5.05 (m, 4H), 4.88-4.70 (m, 3H), 4.43-4, 33 (m, 1H), 4.25-4.20 (m, 2H), 3.65-3.55 (m, 3H), 3.50-3.40 (m, 12H), 3.30- 3.25 (m, 2H), 3.12-2.90 (m, 4H), 2.70-2.55 (m, 2H), 2.48-2.43 (m, 1H), 2, 40-2.35 (m, 1H), 2.30-2.20 (m, 2H), 2.15-1.95 (m, 4H), 1.86-1.75 (m, 2H), 1.64-1.54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 4h), 1.23 (s, 2H), 0.90-0, 80 (m, 12H) ppm. Anal. HPLC: 100%, dwell time: 7.83 minutes (method B).

Example 49.

This example demonstrates a method for producing a payload linker (LP10). The following example relates to FIG. 16.

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-(2-aminoacetyl)-12,19-difluoro-11-hydroxy-9,13-dimethyl-6propyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (34j). Compound 34j (80 mg, 64% yield) was prepared from compound 1-19 following general procedure D. NMR mass/charge: 871 (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-tetraoxapentadecane-15-amido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methyl N- (4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S-12,19-difluoro-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} phenyl)carbamate (LP10).

Following general procedure F, compound (LP10) (20 mg, 22% yield) was obtained by reaction of 34j (43 mg, 50 μmol) with DIBAC-suc-PEG ₄ -NHS ester (VI), after purification by pre-prep-HPLC ( method B). Mass/charge at NMR: 1406 (M+H) ⁺ .

^1H NMR (DMSOd6, 500 MHz) δ 9.99 (s, 1H), 8.11 (d, J=7.5 Hz, 1H), 7.88 (d, J=8.5 Hz, 1H) , 7.80-7.75 (m, 1H), 7.70-7.66 (m, 1H), 7.65-7.60 (m, 3H), 7.53-7.33 (m, 6H), 7.33-7.28 (m, 3H), 6.30 (dd, J=10.0 Hz and 1.5 Hz, 1H), 6.11 (s, 1H), 6.10- 6.00 (m, 1H), 5.72-5.55 (m, 2H), 5.41 (s, 2H), 5.05-5.01 (m, 1H), 4.97 (s, 2H), 4.80-4.72 (m, 1H), 4.60-4.58 (m, 1H), 4.43-4.33 (m, 1H), 4.25-4.10 ( m, 3H), 3.88-3.80 (m, 1H), 3.65-3.55 (m, 3H), 3.503.40 (m, 12H), 3.30-3.25 (m, 2H), 3.12-2.90 (m, 4H), 2.70-2.55 (m, 2H), 2.48-2.35 (m, 2H), 2.30-2.20 ( m, 2H), 2.15-1.95 (m, 4H), 1.86-1.65 (m, 3H), 1.64-1.54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 5H), 0.90-0.80 (m, 12H) ppm. Anal. HPLC: 100%, holding time: 7.40 minutes (method B).

Example 50.

This example demonstrates a method for producing a linker with an LP11 payload. The following example relates to FIG. 16.

{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- [(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-12,19difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6- propyl 5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosas14,17-dien-8-yl]-2-oxoethoxy}phenyl)methyl]carbamate (LP11).

Compound 34k (80 mg, 64% yield) was prepared from (11-19) following general procedure D.

Following General Procedure C, Compound (LP11) (18 mg, 31% yield) was obtained as a white solid by reacting compound (34k). Mass/charge at NMR: 756.5 (M/2+H) ⁺ .

¹ H NMR (500 MHz, DMSOd ₆ ) δ 10.02 (s, 1H), 8.14 (d, J=8.0 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H ), 7.76(t, J=5.5 Hz, 1H), 7.72(t, J=5.5 Hz, 1H), 7.70-7.66 (m, 1H), 7.65 -7.60 (m, 3H), 7.53-7.45 (m, 3H), 7.40-7.31 (m, 2H), 7.31-7.25 (m, 4H), 7 ,20-7.15 (m, 2H), 6.86-6.80 (m, 2H), 6.30 (dd, J=10.4 Hz, 1.6 Hz, 1H), 6.11 ( s, 1H), 6.106.00 (m, 1H), 5.72-5.55 (m, 1H), 5.52 (s, 1H), 5.43 (s, 2H), 5.16-5 .10 (m, 1H), 5.06-5.00 (m, 1H), 5.00-4.93 (m, 2H), 4.90-4.76 (m, 2H), 4.75 (t, J=4.0 Hz, 1H), 4.43-4.33 (m, 1H), 4.25-4.20 (m, 2H), 4.12 (d, J=6.0 Hz, 2H), 3.65-3.55 (m, 3H), 3.50-3.40 (m, 12H), 3.30-3.25 (m, 2H), 3.12-2, 90 (m, 4H), 2.70-2.55 (m, 2H), 2.48-2.43 (m, 1H), 2.40-2.35 (m, 1H), 2.30- 2.20 (m, 2H), 2.15-1.95 (m, 4H), 1.86-1.70 (m, 3H), 1.64-1.54 (m, 5H), 1, 49 (s, 4h), 1.46-1.34 (m, 4H), 0.90-0.80 (m, 12h) ppm. Anal. HPLC: 99%, dwell time: 7.89 minutes (method B).

Example 51.

This example demonstrates a method for producing a linker with an LP12 payload. The following example relates to FIG. 17.

- 157 045579

[(2R,3R,4S,5R,6S)-3,4,5-tri(acetyloxy)-6-[4-formyl-3-(prop-2-yn-1-yloxy)phenoxy]oxan-2yl] methyl acetate (45).

Step 1: The synthesis of [(2R,3R,4S,5R,6S)-3,4,5-tris(acetyloxy)-6-(4-formyl-3-hydroxyphenoxy)oxan2-yl]methyl acetate (43) has been reported in Carbohydrate Research, 1986, 146, 241-249, the entire contents of which are incorporated herein by reference. To a solution of intermediate 43 (2.8 g, 6.0 mmol) in acetone (40 ml), sodium carbonate (1.7 g, 12 mmol) and 3-bromoprop-1-yne (44, 3.5 g) were simultaneously added , 30 mmol), the resulting mixture was refluxed overnight. The mixture was then concentrated in vacuo and the residue was purified by flash chromatography (033% ethyl acetate in petroleum ether) to give compound 45 (1.9 g, 63% yield) as a brown solid. Mass/charge at NMR: 507 (M+H)+.

^1H NMR (MeOD _d4 , 500 MHz) δ 10.26 (s, 1H), 7.78 (d, J=8.5 Hz, 1H), 6.87 (d, J=2.0 Hz, 1H ), 6.77 (dd, J=8.5, 2.0 Hz, 1H), 5.51 (d, J=8.0 Hz, 1H), 5.41 (t, J=9.5 Hz , 1H), 4.93 (t, J=2.5 Hz, 2H), 5.23-5.19 (m, 1H), 5.14 (t, J=9.5 Hz, 1H), 4 .34-4.30 (m, 1H), 4.22-4.15 (m, 2H), 3.11 (t, J=2.0 Hz, 1H), 2.05-1.99 (m , 12H) ppm.

[(2R,3R,4S,5R,6S)-3,4,5-tri(acetyloxy)-6-[4-(hydroxymethyl)-3-(prop-2-yn-1yloxy)phenoxy]oxane-2- yl]methyl acetate (46).

Step 2: Sodium borohydride (31 mg, 0.82 mmol) was added to a solution of compound 45 (0.83 g, 1.6 mmol) in isopropanol (50 mL). The mixture was stirred at 23°C for 2 hours, then concentrated in vacuo. The residue was diluted with ethyl acetate and washed with brine. The organic solution was dried over sodium sulfate and concentrated to give 46 (0.70 g, 84% yield) as a brown oil. Mass/charge at NMR: 526.1 (M+H ₂ O)+.

^1H NMR (MeOD _d4 , 500 MHz) δ 7.32 (d, J=8.0 Hz, 1H), 6.78 (d, J=2.0 Hz, 1H), 6.67 (dd, J =8.0, 2.0 Hz, 1H), 5.40 (t, J=9.0 Hz, 1H), 5.33 (dd, J=7.5 Hz, 1H), 5.20-5 .11 (m, 2H), 4.78 (t, J=2.5 Hz, 2H), 4.59 (s, 2H), 4.32 (d, J=12.5, 5.0 Hz, 1H), 4.21 (dd, J=12.5, 2.5 Hz, 1H), 4.12-4.08 (m, 1H), 3.02 (t, J=2.0 Hz, 1H ), 2.07-2.0.

[(2R,3R,4S,5R,6S)-3,4,5-tri(acetyloxy)-6-(4-{[(4-nitrophenoxycarbonyl)oxy]methyl}-3-(prop-2yn-1- yloxy)phenoxy)oxan-2-yl]methyl acetate (48).

Step 3: To a solution of compound 46 (0.40 g, 0.79 mmol) in methylene chloride (30 ml) was added 4-nitrophenyl chloroformate (47, 0.24 g, 1.2 mmol), 4-dimethylaminopyridine (0.19 g , 1.6 mmol) and diisopropylethylamine (0.20 g, 1.6 mmol). The mixture was stirred at 23°C overnight and diluted with methylene chloride (50 ml). The organic solution was washed with saturated aqueous ammonium chloride (50 ml), then brine (50 ml), dried over sodium sulfate and concentrated. The residue was purified by flash chromatography (0-33% ethyl acetate in petroleum ether) to give 48 (0.30 g, 57% yield) as a yellowish solid. Mass/charge at NMR: 691.0 (M+H ₂ O)+.

1H NMR (CDCl ₃ , 500 MHz) δ 8.27 (d, J=9.0 Hz, 2H), 7.38 (d,J=9.0 Hz, 2H), 7.35 (d, J= 8.5 Hz, 1H), 6.75 (d, J=2.5 Hz, 1H), 6.64 (dd, J=9.0, 2.5 Hz, 1H), 5.33-5, 26 (m, 4H), 5.21-5.17 (m, 1H), 4.76 (t, J=2.0 Hz, 2H), 4.28 (dd, J=12.5, 5, 0 Hz, 1H), 4.20 (dd, J=12.5, 2.5 Hz, 1H), 3.89-3.88 (m, 1H), 2.56 (t, J=7.0 Hz, 1H), 2.08-2.04 (m, 12H) ppm.

[(2R,3R,4S,5R,6S)-3,4,5-tri(acetyloxy)-6-[4-({[(4-{2-[(1S,2S,4R,8S,9S, 11S,12S,13R)-11-hydroxy9,13-dimethyl-16-oxo-6-propyl-5,7-dioxapentacyclo[10.8.002,9.04,8.013,18]icos-14,17-dien-8-yl ]-2oxoethoxy}phenyl)carbamoyl]oxy}methyl)-3-(prop-2-yn-1-yloxy)phenoxy]oxan-2-yl]methyl acetate (49).

Step 4: To a solution of compound 48 (0.15 g, 0.22 mmol) in K,K-dimethylformamide (5 ml), 11-5 (0.14 g, 0.26 mmol), HOBt (59 mg, 0.44 mmol) and diisopropylethylamine (57 mg, 0.44 mmol). The mixture was stirred at 23°C overnight, then purified by pre-HPLC (Method B) to give compound 49 (0.14 g, 62% yield) as a white solid. Mass/charge at NMR: 1056.3 (M+H) ⁺ .

1H NMR (MeOD _d4 , 400 MHz) δ 7.46 (d, J=10.4 Hz, 1H), 7.35-7.26 (m, 3H), 6.87-6.80 (m, 3H ), 6.67 (dd, J=8.0, 2.4 Hz, 1H), 6.26 (dt, J=10.0, 2.4 Hz, 1H), 6.03 (br s, 1H ), 5.42-5.34 (m, 2.5H), 5.26-5.03 (m, 5.5H), 4.884.64 (m, 4H), 4.46-4.43 (m , 1H), 4.34-4.30 (m, 1H), 4.21-4.18 (m, 1H), 4.12-4.08 (m, 1H), 3.03 (t, J =2.0 Hz, 1H), 2.71-2.62 (m, 1H), 2.41-2.38 (m, 1H), 2.28-2.15 (m, 2H), 2, 06-2.04 (m, 12H), 1.90-1.39 (m, 12H), 1,200.89 (m, 8H) ppm.

[2-(prop-2-yn-1-yloxy)-4-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2yl]oxy} phenyl]methyl N-(4-{2-[(1S,2S,4S,8R,9S,11S,12S,13R)-11-hydroxy-4,9,13-trimethyl-16-oxo-6propyl-7- Oxapentacyclo[10.8.002.9.04.8.013.18]icos-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamate (LP12).

Step 5: To a solution of compound 49 (35 mg, 33 µmol) in methanol (3 ml) was added another solution of LiOH in H ₂ O (14 mg, 0.33 mmol) in water (1 ml). The mixture was stirred at 23°C for 1.5 hours and quenched with HOAc (20 mg). The mixture was concentrated in vacuo and the residue was purified by pre-HPLC (Method B) to obtain linker payload LP12 (26 mg, yield) 88% as a white solid. Mass/charge at NMR: 888 (M+H) ⁺ .

1H NMR (MeOD _d4 , 400 MHz) δ 7.46 (d, J=10.0 Hz, 1H), 7.35-7.30 (m, 3H), 6.91 (d, J=2.0 Hz, 1H), 6.876.83 (m, 2H), 6.74 (dd, J=8.0, 2.0 Hz, 1H), 6.27 (dt, J=10.0, 2.0 Hz , 1H), 6.03 (s, 1H), 5.25 (t, J=4.8 Hz, 0.5H),

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5.19 (d, J=7.2 Hz, 0.5H), 5.13-5.03 (m, 3H), 4.94-4.91 (m, 1H), 4.82-4, 75 (m, 3H), 4.71-4.67 (m, 1H), 4.46-4.43 (m, 1H), 3.91 (dd, J=12.0, 2.0 Hz, 1H), 3.70 (dd, J=12.0, 5.2 Hz, 1H), 3.48-3.36 (m, 4H), 2.99 (t, J=2.4 Hz, 1H ), 2.71-2.62 (m, 1H), 2.40-2.37 (m, 1H), 2.26-2.12 (m, 2H), 2.07-2.00 (m , 1H), 1.88-1.61 (m, 5H), 1.56-1.35 (m, 6H), 1.20-0.92 (m, 8H) ppm.

Example 25

This example demonstrates a method for producing a linker with an LP13 payload. The following example relates to FIG. 18.

2-[(1S,2S,4S,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)-5(carbamoylamino)-2-[ (2S)-2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido]-3methylbutanamido]pentanamido]phenyl}methoxy)carbonyl] (methyl)amino}ethyl) -N-methylcarbamate (LP13).

To a solution of Budesonide-DME carbonate (20 mg, 0.037 mmol) in DMF (1 ml), MC-VC-PAB-PNP (22 mg, 0.03 mmol), DIPEA (12 mg, 0.09 mmol) and HOBt were successively added (6 mg, 0.05 mmol). The mixture was stirred at room temperature for 12 hours, then preparative HPLC was performed to obtain two epimers: epimer 1: 3.3 mg (yield 10%) and epimer 2: 4.1 mg (yield 12%).

Epimer 1: Mass/charge at NMR: 1143.4 (M+1).

^1H NMR (400 MHz, MeOD) δ 7.63-7.62 (m, 2H), 7.50-7.49 (m, 1H), 7.37-7.35 (m, 2H), 6 .81 (s, 1H), 6.296.27 (m, 1H), 6.03 (brs, 1H), 5.37-5.08 (m, 5H), 4.83-4.79 (m, 3H ), 4.53-4.46 (m, 2H), 4.18-4.15 (m, 1H), 3.693.37 (m, 6H), 3.25-3.13 (m, 3H), 3.12-2.96 (m, 5H), 2.90-2.86 (m, 2H), 2.68-2.64 (m, 1H), 2.41-2.40 (m, 1H ), 2.31-2.28 (m, 2H), 2.26-1.95 (m, 7H), 1.93-1.77 (m, 11H), 1.51 (s, 3H), 1.42-1.30 (m, 10H), 1.25-0.89 (m, 15H).

Epimer 2: Mass/charge at NMR: 1143.4 (M+1).

1H NMR (400 MHz, MeOD) δ 7.62-7.34 (m, 5H), 6.81 (brs, 1H), 6.29-6.23 (m, 1H), 6.05-6, 00 (m, 1H), 5.27-5.17 (m, 4H), 4.92-4.79 (m, 2H), 3.75-3.37 (m, 7H), 3.03- 2.86 (m, 5H), 2.72-2.63 (m, 1H), 2.41-2.28 (m, 3H), 2.23-2.04 (m, 7H), 1. 91-1.32 (m, 31H), 1.19-0.90 (m, 14H).

Example 53.

This example demonstrates a method for producing a linker with an LP14 payload. The following example relates to FIG. 18.

N-(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-12,19- difluoro-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}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-1-{2-[4-(2,5-dioxo-2,5-dihydro-1Hpyrrol-1 -yl)phenyl]acetamido }-3,6,9,12-tetraoxapentadecane-15-amide (LP14).

Compound 34h-2 (0.18 g, 74% yield in 2 steps) was prepared following general procedure F. NMR mass/charge: 728 (M+H) ⁺ .

Compound LP14 (20 mg, 14% yield in three steps from 34h) was obtained as a white solid. ESI: 1189 (M+H) ⁺ .

1H NMR (500 MHz, DMSO _d6 ) δ 9.81-9.67 (m, 1H), 8.43-8.13 (m, 2H), 8.03-7.84 (m, 1H), 7 ,61-7.47 (m, 2H), 7.35 (d, J=8.4 Hz, 2H), 7.29-7.21 (m, 3H), 7.17 (s, 2H), 6.88-6.81 (m, 2H), 6.33-6.28 (dd, J=10.1, 1.8 Hz, 1H), 6.11 (s, 1H), 5.71- 5.56 (m, 1H), 5.51 (s, 1H), 5.12 (d, J=18.5 Hz, 1H), 4.84 (d, J=18.5 Hz, 1H), 4.79-4.76 (m, 1H), 4.74 (t, J=4.3 Hz, 2H), 4.38-4.33 (m, 1H), 4.25-4.17 ( m, 2H), 3.63-3.55 (m, 2H), 3.52-3.44 (m, 14H), 3.42 (t, J=5.8 Hz, 2H), 3.21 (q,J=5.7 Hz, 1H), 2.69-2.55 (m, 1H), 2.47-2.41 (m, 1H), 2.41-2.34 (m, 1H ), 2.292.23 (m, 1H), 2.14-2.02 (m, 2H), 1.99-1.90 (m, 1H), 1.82 (d, J=13.0 Hz, 1H), 1.65-1.53 (m, 4H), 1.49 (s, 3H), 1.47-1.41 (m, 1H), 1.40-1.33 (m, 2H) , 1.29 (d, J=7.1 Hz, 3H), 0.90-0.80 (m, 12H) ppm. Anal. HPLC: 100%, dwell time: 8.45 minutes (method A). In table 7 below are some physical properties of LP1LP16.

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Table 7

Physical properties of some payload linkers

No. LP Molecular formula Molecular mass Purity (%) MS mass/charge (100%) Largest mass/charge value HPLC room temp. (min.) LP1 C ₆ 6H ₈ 1N ₅ O15 1184.4 98 593 (M/2+N) 1185 (M+N, 20%) 6.53(V) LP2 Sb1N ₈ bR ₂ N6O15 1181.4 100 1181.4 (M+H) 1181.4 (M+H) 7.83(V) LP3 C70H ₉₄ F ₂ N ₆ O17 1329.5 100 1330.4 (M+N) 1330.4 (M+N) 7.03 (V) LP4 Sb1EY9 ₄ O15 1115.4 100 1115 [M+N] 1115 [M+N] 8.17 (A) 8.24(V) LP5 _{C64H92N6O16 _ _} 1201.5 100 1201 [M+N] 1201 [M+N] 7.34 (A) 7.44(V) LP6 C+HggNvOlS 1350.6 100 1350.5 (M+N) 1350.5 (M+N) 8.87(A) LP7 C61H86N4O15 1226.5 100 1227.8 (M+H) 1227.8 (M+H) 9.47 (A) LP8 C ₆₉ H ₈₅ F ₂ N ₅ Oi ₅ 1262.4 100 1262.4 (M+H) 1262.4 (M+H) 8.26(V) LP9 C ₈ qH ₉₈ F ₂ N ₈ O1 ₈ 1497.7 100 749.5 (M/2+N) 1497.7 (M+H) 7.99(V) LP10 C74H ₉ 4F ₂ N8O17 1405.6 100 703.5 (M/2+N) 1405.7 (M+N) (5%) 7.40(V) LP11 C81H100F2N8O18 1511.7 99.3 756.5 (M/2+N) 756.5 (M/2+N) 7.89(V) LP12 C48H57NO15 888.0 100 566.2(Mglucose-PAB) 889.1 (M+H, 25%) 8.02 (A) 8.08(V) LP13 C ₇ 2H ₉₉ N ₇ O18 1350.6 100 1350.5 (M+N) 1350.5 (M+N) 8.87(A) LP14 C62H7 ₉ F2N5O16 1188.31 100 594.8 (M/2+N) 1188.5 (M+N, 40%) 7.99(V) LP15 C121H170N10O47 2516.71 97 1259 (M/2 +N) 1259.1 (M/2+N) ⁺ , 6.62 and 6.67 (IN) LP16 C121H170N10O47 2516.71 98 1258 (M/2+N) 1259.1 (M/2+N,60%) 6.61(59%) and 6.73 (39%) (B)

Example 54.

This example generally demonstrates a method for site-specific conjugation of a payload and an antibody or antigen-binding fragment thereof. This example refers to FIG. 19.

In one example, site-specific conjugates were prepared using a microbial transglutaminase (MTG EC 2.3.2.13, Zedira, Darmstadt, Germany) (hereinafter MTG-mediated) two-step conjugation of a mutated antibody N297Q or N297D. In the first step, the mutated antibody was functionalized with azido-PEG ₃ -amine through an MTG-mediated enzymatic reaction. See, for example, PCT International Patent Application No. PCT/US17/19537, filed February 24, 2017, entitled Optimized Site-Specific Transaminase-Antibody Conjugation, which is incorporated herein by reference in its entirety for all purposes. In the second step, an alkyne-functionalized payload linker was attached to the azido-functionalized antibody via a [2+3] 1,3-dipolar cycloaddition reaction (see, for example, FIG. 19, which shows a DIBAC-functionalized payload linker conjugated to an azido-functionalized antibody prepared by [2+3] cyclization.This process produced site-specific and stoichiometric conjugates in practical yields of approximately 50-80%.

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Example 55.

This example generally demonstrates a method for site-specific conjugation of a payload and an antibody or antigen-binding fragment thereof.

This example relates to the connections shown in FIG. 29.

In this example, site-specific conjugates were prepared in two steps. The first step is the microbial transglutaminase (MTG)-mediated enzymatic addition of a small molecule, such as the PEG3-amine azide (above), to an antibody bearing a Q tag (Qtag references) (hereinafter MTG-mediated conjugation). The second step involved attaching the payload linker to the azido-functionalized antibody using a [2+3] cycloaddition, such as a 1,3-dipolar cycloaddition between azides and cyclooctynes (so-called copper-free click chemistry). See, 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 entire contents of which are incorporated herein by reference for all purposes. In fig. 28 shows an example of a payload linker having a DIBAC moiety conjugated to an azido-functionalized antibody via a [2+3] cycloaddition. This process produced site-specific and stoichiometric conjugates in practical yields of approximately 50-80%.

ALS conjugation via [2+3] click reaction.

Step 1: Preparation of azido-functionalized antibody.

Aglycosylated human IgG antibody (IgG1, IgG4, etc.) or human IgG1 isotype with N297Q mutation in PBS (pH 6.5-8.0) was mixed with >200 molar equivalents of azido-dPEG ₃ amine (MW=218 .26 g/mol). The resulting solution was mixed with MTG (EC 2.3.2.13 manufactured by Zedira, Darmstadt, Germany, or Modernist Pantry [L#210115A] -ACTIVA TI contains maltodextrin manufactured by Ajinomoto, Japan) (25 units/ml; 5 units of MTG per 1 mg of antibody), which gave a final antibody concentration of 0.5-5 mg/ml solution, then the solution was incubated at 37°C for 4-24 hours with gentle shaking. The reaction was monitored using ESI-MS. After completion of the reaction, excess amine and MTG were removed by size exclusion chromatography or Protein A column chromatography to obtain the azido-functionalized antibody. This product was characterized by DNS-PAGE and ESI-MS. Azido-dPEG ₃ -amine added to two antibody sites results in an increase of 204 Da for the 2DAR antibody-PEG ₃ -azide conjugate.

In a specific experiment, Q-tagged N-terminal antibody (24 mg) in 7 ml potassium-free PBS buffer (pH 7.3) was incubated with >200 molar equivalent of azido-PEG ₃ -amine (MW 218.26) in the presence of MTG (0.350 ml, 35 units, mTGase, Zedira, Darmstadt, Germany). The reaction mixture was incubated at 37°C overnight with gentle stirring. Excess azido-PEG ₃ -amine and mTGase were removed by size exclusion chromatography (SEC, Superdex 200 PG, GE Healthcare).

Step 2: Preparation of site-specific drug-antibody conjugates using chemical click reactions.

Conjugates of site-specific human antibodies of the IgG class (IgG1, IgG4, etc.) in table. 10 was prepared using a [2+3] click reaction between azido-functionalized antibodies and an alkyne-containing payload linker. The detailed conjugation procedure is given below. A site-specific antibody linker payload (LP) conjugate was prepared by incubating mAb-PEG ₃ -N ₃ (1-3 mg/ml) in an aqueous medium (e.g. PBS, PBS with 5% glycerol, GEPES) with >6 molar equivalents of payload linker dissolved in a suitable organic solvent such as dimethyl sulfoxide, N,N-dimethylformamide or DMA (that is, the reaction mixture contains 520% organic solvent, v/v) at a temperature of 24 to 37°C for more than 6 hours. The progress of the reaction was monitored using ESI-MS; the absence of mAb-PEG ₃ -N ₃ indicated the completion of conjugation. Excess payload linker and organic solvent were removed by size exclusion chromatography eluting with PBS or by protein A column chromatography eluting with acid buffer followed by neutralization with hydroxymethylaminomethane (pH 8.0).

In a specific example, azido-functionalized antibody (1 mg) in 0.800 ml PBSg (PBS, 5% glycerol, pH 7.4) was treated with six molar equivalents of DIBAC-PEG4-DLys(COT-^-CD)-VC-PABC payload (concentration 10 mg/ml in dimethyl sulfoxide) for 6-12 hours at room temperature, excess payload (LP) linker was removed using size exclusion chromatography (SEC, Superdex 200 HR, GE Healthcare).

The final product was concentrated by ultracentrifugation and characterized by UV, size exclusion chromatography, DNS-PAGE and ESI-MS.

Example 56.

This example demonstrates a method for preparing a conjugate with azido-functionalized antibodies.

An aglycosylated human IgG1 isotype antibody in BupH™ (pH 7.6-7.8) was mixed with >200 molar equivalents of azido-dPEG ₃ -amine (molecular weight 218.26 g/mol). The resulting

- 161 045579 the solution was mixed with transglutaminase (25 units/ml; 5 units of MTG per 1 mg of antibody, Zedra, Darmstadt, Germany), which gave a final antibody concentration of 0.5-3 mg/ml, then the solution was incubated at 37° C for 4-24 hours with gentle shaking. The reaction was monitored using ESI-MS. After completion of the reaction, excess amine and MTG were removed by size exclusion chromatography (see FIG. 21) to obtain the azido-functionalized antibody. This product was characterized by DNS-PAGE (see FIG. 20) and ESI-MS (see FIG. 22). Azido-dPEG ₃ -amine added to two antibody sites results in an increase of 804 Da for the 4DAR aglycosylated antibody-PEG ₃ -azide conjugate. Conjugation sites were identified and confirmed on the EEQ ^Linker YQ ^Linker STYR for the azido-functionalized 4DAR antibody by mapping the trypsin-digested heavy chain peptide sequence.

Example 57.

This example demonstrates the preparation of site-specific drug-antibody conjugates using chemical click reactions.

Conjugates of site-specific aglycosylated antibodies with human IgG1 containing the N297Q mutation in Table. 8, described below, was prepared by a [2+3] click reaction between azido-functionalized antibodies with an alkyne-containing payload linker. As shown in table. 8, Her2-PEG3-N3 antibodies were conjugated to compounds LP1, LP2, LP3, LP4, LP5, LP6, LP7, LP8, LP9, LP10 and LP11. As shown in table. 8, anti-PRLR-PEG3-N3 antibodies were conjugated to LP1 LP2, LP3, LP4, LP5, LP6, LP7, LP8, LP9, LP10, LP11, LP15 and LP16. As shown in table. 8, anti-IL2Rg-PEG ₃ -N ₃ antibodies were conjugated to LP4 and LP7. As shown in table. 8, anti-Fel d 1-PEG3-N3 antibody was conjugated to LP4.

For conjugation, an azido-functionalized aglycosylated human IgG1 antibody conjugate (mAb-PEG ₃ -N ₃ ) and linker payload (LP) were prepared by incubating mAbPEG3-N3 (1-3 mg/ml) in an aqueous medium (e.g. PBS, PBS containing 5% glycerol, GEPES) with >6 molar equivalents of payload linker dissolved in a suitable organic solvent such as dimethyl sulfoxide, N,N-dimethylformamide or DMA (reaction mixture contains 5-20% organic solvent, v/v ) at temperatures between 24°C and 37°C for more than 6 hours. The progress of the reaction was monitored using ESI-MS; the absence of mAb-PEG ₃ -N ₃ indicated the completion of conjugation. Excess payload linker and organic solvent were removed by size exclusion chromatography by eluting with PBS. Purified conjugates were analyzed using size exclusion chromatography, DNS-PAGE and ESI-MS. In table Figure 8 shows a list of non-toxic steroidal antibody conjugates (ncADCs) from the corresponding payload linkers, their molecular weights and DAR values according to ESI. In table 8 Ab refers to an antibody, Ab-N3 refers to an azide-functionalized antibody, and ncADC refers to a noncytotoxic antibody-drug conjugate.

Table 8

Ab, Ab-Nj, or ncADC Mass/charge at MS (ncADC) LP№ Mol. weight (LP) DAR (IER-MS) 1 Antibody Her2 mAb 145126 2 Antibody Neg2-PEOs-Xs 145930 NH2-PEG3-N3 218.3 4 3 Antibody Her2-LPl 150683 LP1 1184.4 4 4 Her2-LP2 antibody 150671 LP2 1181.4 4

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5 Antibody Her2-LP3 151274 LP3 1330.5 4 6 Antibody Her2-LP4 150406 LP4 1115.4 4 7 Antibody Her2-LP5 150726 LP5 1201.5 4 8 Antibody Her2-LP6 151358 LP6 1350.6 4 9 Antibody Her2-LP7 151297 LP7 1226.5 4 10 Antibody Her2-LP8 152239 LP8 1262.4 4 eleven Antibody Her2-LP9 151868 LP9 1497.7 4 12 Antibody Her2-LP10 152293 LP10 1405.6 4 13 Antibody Her2-LPl 1 145430 LP11 1511.7 4 14 PRLR mAb 144579 15 Antibody PRLR-PEG3-N3 145373 NH2-PEG3-N3 218.3 4 16 PRLR-LP1 antibody 150110 LP1 1184.4 4 17 PRLR-LP2 antibody 150101 LP2 1181.4 4 18 PRLR-LP3 antibody 150721 LP3 1330.5 4 19 PRLR-LP4 antibody 149836 LP4 1115.4 4 20 PRLR-LP5 antibody 150181 LP5 1201.5 4 21 PRLR-LP6 antibody 150795 LP6 1350.6 4 22 Antibody PRLR-LP7 150311 LP7 1226.5 4 23 Antibody PRLR-LP8 150444 LP8 1262.4 4 24 PRLR-LP9 antibody 151386 LP9 1497.7 4 25 Antibody PRLR-LP10 151015 LP10 1405.6 4 26 Antibody PRLR-LP11 151447 LP11 1511.7 4 27 AHTHTeno-IL2Rg mAb 144960 28 AHTHTeno-IL2Rg-PEG3-N3 145768 NH2-PEG3-N3 218.26 4 29A Antibody-1E2E§-EP4 150237 LP4 1115.37 4 29V AHTHTeno-IL2Rg-LP7 150670 LP7 1226.5 4 thirty Antibody-Fel d 1 mAb 145430 31 Antibody-Fel d I-PEG3-N3 146235 NH2-PEG3-N3 218.26 4 32 Antibody Fei d 1-LP4 150705 LP4 1115.37 4 33 Antibody-PRLR-LP 15 155460 LP15 2515.1 4 34 Antibody-PRLR-LP 16 155486 LP16 2515.1 4

Example 58.

This example demonstrates a method for performing non-site-specific drug-antibody conjugation using a thiol-maleimide reaction.

Conjugation via antibodies to cysteines was carried out in two stages using methods described similar to those described in the Mol Pharm source. 2015 Jun 1; 12(6): 1863-71.

Monoclonal antibody (mAb, 10 mg/ml in 50 mmol GEPES, 150 mmol NaCl) at pH 7.5 was reconstituted with 1 mmol dithiothreitol (0.006 mg per 1 mg antibody) or TCEP (2.5 molar equivalent of antibody) at 37°C C for 30 minutes. After gel filtration (G-25, pH 4.5 sodium acetate), compound LP13 in dimethyl sulfoxide (10 mg/ml) was added to the reduced antibody, the mixture was adjusted to pH 7.0 with 1 mol HEPES (pH 7.4). The reaction was allowed to proceed for 3-14 hours. The resulting conjugate was purified by size exclusion chromatography. DAR values were determined using measured ncADC absorbance and antibody and LP13 extinction coefficients.

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Example 59.

This example demonstrates a method for characterizing antibodies and noncytotoxic antibody-drug conjugates (ncADCs).

The antibody and ncADC were characterized by DNS-PAGE, size exclusion chromatography and MS (ESI). Anti-PRLR-LP4 conjugate in table. 8, derived from an anti-prolactin receptor antibody through its azido-functionalized antibody (anti-PRLR-PEG ₃ -N ₃ ), was characterized by DNSPAG performed under non-reducing and reducing conditions (Fig. 20), size exclusion chromatography (Fig. 21) and ESI-MS (Fig. 22), and demonstrated the completion of the formation of the ncADC conjugate.

DNS-PAGE was used to analyze the integrity and purity of ALS conjugates. In one method, DNS-PAGE conditions included unreduced and reduced samples (2-4 μg), which were loaded along with a prestained BenchMark protein ladder (Invitrogen, cat. no. 10748-010; L no. 1671922.) fragment by fragment into a 20% triglycerol gel Novex 4 (1.0 mm x 10 wells) and treated at 180 M, 300 mA for 80 minutes. The analytical sample was prepared using Novex triglycerol SDS buffer (2X) (Invitrogen, cat. no. LC2676); reducing samples were prepared with SDS buffer (2X) containing 10% 2-mecaptoethanol.

In fig. Figure 20 shows the molecular weights of antibodies and ncADC conjugates according to DNS-PAGE performed under non-reducing and reducing conditions. Under non-reducing conditions, mass shifts were not evident due to the relatively small percentage of mass changes. However, the heavy chain masses increased from unprotected antibodies to azido-functionalized antibodies and further to the ncADC conjugate. There was no detectable cross-linked material.

As shown in FIG. 20, DNS-PAGE fragments included the following types based on the following fragments in table. 9.

Table 9

Fragment Sample 1 Standards (10 µl template) 2 Anti-prolactin receptor antibody 3 Anti-prolactin receptor antibody -NH-PEG ₃ -N ₃ 4 Anti-prolactin receptor antibody -LP4 8 Anti-prolactin receptor antibody (reconstituted) 9 Anti-prolactin receptor antibody NH-PEG ₃ -N ₃ (reduced) 10 Anti-prolactin receptor antibody -LP4 (reduced) ~2 µg unreduced/reduced sample/fragment.

ALS conjugates were analyzed for purity by size exclusion chromatography (SEC).

Size exclusion chromatography was performed to determine the purity of the antibody-drug conjugates. Analytical size exclusion chromatography experiments were carried out using a Waters 600 instrument on an HR Superdex 200 column (1.0 x 30 cm) at a flow rate of 0.80 ml/min, using PBS pH 7.4 and monitored at 280 nm using a Waters PDA 2998. The analytical sample consisted of 200 µl PBS (pH 7.4) with 30-100 µl of test sample. Preparative purification in the process of size exclusion chromatography was carried out using an ACT instrument manufactured by GE Healthcare on a Superdex 200 PG column (2.6x60 cm) with elution at a flow rate of 2 ml/min, with PBSg at pH 7.4 and control at a wavelength of X280 nm . Size exclusion chromatography results in Fig. 21 show typical residence times for monomeric mAb and its conjugates, with no detectable aggregation or degradation.

The antibody and ALS conjugate were analyzed using intact mass analysis by LC-IERMS.

Measurement of the intact mass of ncADC conjugate samples by LC-ESI-MS was performed to determine the drug distribution profile and to calculate the average DAR of intact forms of the ALS conjugate. Each test sample (20-50 ng, 5 µl) was loaded onto an Acquity UPLC Protein BEH C4 column (10 kPa, 300 A, 1.7 µm, 75 µm x 100 mm; catalog number 186003810). After 3 minutes of desalting, the protein was eluted and mass spectra were obtained using a Waters Synapt G2-Si mass spectrometer (Waters).

As shown in FIG. 22, the deconvoluted mass spectra show a predominant peak for the alycosylated anti-prolactin receptor antibody with a molecular weight of 144579.0 Da and a predominant peak for its azido-functionalized anti-prolactin receptor antibody with a molecular mass of 145373.0 Da, indicating an increase by 794.0 Yes compared to its aglyco

- 164 045579 fused parent antibody (corresponding to the conjugation of 4 amino-PEG ₃ -azide with each aglycosylated antibody). Additionally, the predominant peak for the anti-PRLR-LP4 conjugate had a molecular weight of 149836.0 Da, indicating an increase of 4463 Da compared to its aglycosylated parent antibody (corresponding to 4 LP4 conjugating to each aglycosylated antibody). As shown in table. 8, most of the site-specific ALS conjugates in this document have a 4DAR ratio.

For non-site-specific ALS conjugates, DAR values were determined based on ESI Q-TOF mass analysis. ESI Q - TOF mass spectra were deconvoluted to zero charge mass spectra using the maximum entropy algorithm (MassLunx). The resulting mass spectra demonstrated the distribution of each antibody conjugated to the drug(s). The peak area percentage represents the relative distribution of specific drug-loaded antibody species. The average DAR ratio was calculated using the percentage peak area information and the number of drug loads per antibody.

Example 60.

This example demonstrates, using the LanthaScreen TR-FRET GR competitive binding assay, that the steroids described herein bind to the glucocorticoid receptor (GR) in the amounts described herein.

To evaluate the ability of novel steroids to bind to the glucocorticoid receptor (GR), a cell-free binding assay was performed using the LanthaScreen TR-FRET GR Competitive Binding Assay Kit (Life Technologies, Cat. No. A15901). The analysis was carried out in accordance with the manufacturer's instructions. Budesonide is a commercial glucocorticoid receptor steroid and was used as a control in the binding assay and other cell-based assays described herein below. Briefly, a threefold serial dilution of budesonide and the derivative compound noted below was prepared in 100% dimethyl sulfoxide, starting at 100 nmol (100X the final value). Serial dilutions were further diluted 50-fold in nuclear receptor F buffer with 5 mM dithiothreitol and 0.1 mM stabilizing peptide and transferred to a 384-well assay plate. Fluormone GS1 Green, GR-LBD (GST), and anti-GST Tb antibody were then added sequentially to a 384-well assay plate. The plate was then incubated at room temperature for 2.5 hours while protected from light. The plate was analyzed on an Envision multilayer reader (PerkinElmer) with excitation set at 340 nm and emission filters at 520 nm and 486 nm. The FRET ratio was calculated as 520 nm/486 nm. IC ₅₀ values were determined using a four-parameter logistic equation on a 12-point response curve (GraphPad Prism).

As shown in table. 10, budesonide competed for GS1 Green fluormone binding in a glucocorticoid receptor assay with an IC ₅₀ value of 10 to 100 nmol. N-analogues of budesonide similarly competed for binding with IC ₅₀ values ranging from less than 10 nmol to more than 100 nmol. The novel steroids tested here performed comparable or better (lower IC ₅₀ values) in this assay and a similar bias for the GR ligand compared to budesonide. 22R isomers are generally more effective than 22S isomers or at least identical to 22S isomers.

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Table 10

Cell-free binding and cell functional activity

Connection numbers ECso of HEK293/9xUASLuc2P/pBind-GR/PRLR-HA cells (nmol) IC50 by GR competitive binding assay, (nmol) 1 Budesonide +++ Full activation ++ 2 Diflorazon +++ Full activation ++ 3 7-1 S + Partial activation +++ 4 7-1 R +++ Full activation ++ 5 7-2S/R +++ No activation +++ 6 7-4 S/R + No activation +++ 7 8-1 R ++ Full activation NT 8 8-2 S/R + No activation +++ 9 8-3 S/R + No activation +++ 10 11-1 S/R +++ Full activation ++ AND 11-2 S/R + Full activation ++ 12 11-3 S/R ++ Partial activation +++ 13 11-5 S/R +++ Full activation ++ 14 11-5S +++ Full activation ++ 15 11-5 R +++ Full activation ++ 16 11-6 S +++ Full activation ++ 17 11-6 S/R ++ Full activation ++ 18 11-7R ++ Full activation ++ 19 11-8R +++ Full activation ++ 20 11-10 S/R + No activation +++ 21 11-11 S/R + No activation +++ 22 11-12 S/R +++ Full activation ++ 23 11-13 R +++ Full activation +++ 24 11-14 S/R +++ Full activation +++ 25 11-15 S/R ++ Partial activation +++ 26 11-17 S/R ++ Full activation +++ 28 11-19 S/R +++ Full activation +++ 29 11-20 S/R + No activation +++ thirty 11-21 S/R ++ Partial activation +++ 31 14-2 + No activation +++ 32 15-5 ++ Full activation ++ 33 16-5 ++ Full activation ++

+++: < 10 nmol; ++: <100 nmol > 10 nmol; +: > 100 nmol;

NT: not tested.

Full activation: >75% of budesonide-induced fold activation. Partial activation: (20%, 75%) fold activation caused by budesonide. No activation: <20% fold activation induced by budesonide. A cell-free assay is used to evaluate the direct binding of compounds to the recombinant GR LBD, regardless of their permeability. Cell-based assays are used to measure how compounds activate intracellular GR-mediated transcription following

- 166 045579 passage through the plasma membrane, thus connection across the membrane permeability is a necessary condition for activity.

Example 61.

This example demonstrates that the PRLR-ncADC conjugate is internalized into HEK293/PRLR cells.

Internalization of anti-prolactin receptor antibody and isotype control antibodies was assessed in HEK293 cells engineered to express full-length human prolactin receptor (PRLR) (amino acids 1 to 622 of accession number NP000940.1 with K2E mutation; HEK293/PRLR). Parental HEK293 cells were also evaluated as a negative control. Cells were seeded at 20,000 cells per well in complete medium and incubated overnight at 37°C. The next day, the wells were washed with PBS and placed on ice. Serial dilutions of antibodies from 0.1 to 100 nmol were added to the appropriate wells in 2% FBS in PBS and incubated on ice for 30 minutes. Cells were washed twice with PBS and then incubated for 30 minutes on ice with Alexa 488-conjugated goat anti-hIgG Fab fragment (Jackson Immunoresearch, cat. no. 109-547-003). Cells were washed twice with PBS and then either fixed in 3.7% formaldehyde in PBS (4°C control conditions) or incubated at 37°C for 3 hours to allow internalization. After a 3-hour incubation, cells were fixed in 3.7% formaldehyde in PBS for 15 minutes, washed with PBS, and imaged on ImagExpress MicroXL molecular devices.

Anti-PRLR-ncADC and parental prolactin receptor antibody were internalized into HEK293/PRLR cells, whereas isotype control ncADC antibodies and parental isotype control were not internalized because they do not bind to the protein found in the cell lines tested. In parental HEK293 cells, no internalization was observed in any samples tested.

Example 62.

The bioassays described here were used to evaluate the efficacy of free steroids and anti-PRLR-ncADCs. In one example, a bioassay assessed steroid activity following internalization of a site-specific anti-PRLR-GC steroid conjugate ALS into cells for binding to pBIND-GR and subsequent activation of a luciferase reporter. For this analysis, the 293 cell line was developed to express the full-length human prolactin receptor (PRLR). This stable cell line was then transfected with a chimeric receptor consisting of the glucocorticoid receptor ligand binding domain fused to the yeast Gal4 DNA binding domain (pBind-GR, Promega cat. no. E1581) and a Gal4 activating sequence (9xGal4UAS-Luc2P) that drives gene expression luciferase. This assay format provides high sensitivity and low cross-reactivity with other nuclear receptors. Since the two vectors together are generally used to monitor glucocorticoid receptor ligand binding and transactivation, the resulting stable cell line is referred to here as 293/PRLR/GRE-Luc for simplicity (see Improved Dual-Luciferase Reporter Assays for Nuclear Receptors, Current Chem Genomics , 2010; 4: 43-49; Aileen Paguio, Pete Stecha, Keith V Wood, and Frank Fan).

In the second example, both the efficacy of free steroids and any non-specific anti-PRLR-ncADC activity were assessed by bioassay. For this analysis, cell line 293 was transfected with the pGL4.36 vector [Luc2P/MMTV/Hygro] (cat. no. E1360, Promega). The resulting cell line is referred to here as 293/MMTV-Luc.

Example 63.

A glucocorticoid receptor (GR) luciferase reporter cell co-activator assay was used to analyze the time-dependent activation of the GR by budesonide and the steroids described here.

Steroid activity in 293/PRLR/GRE-Luc cells was studied after 72 hours of incubation. For this assay, 20,000 cells were seeded in 96-well plates in medium containing Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% FBS and pencillin/streptomycin (complete medium) and grown overnight at 37°C. in 5% CO ₂ . For dose response curves of free drug or ncADC conjugate, serially diluted reagents ranging from 100 nmol to 5.1 pmol were added to the cells and incubated for 72 hours at 37°C. Luciferase activity was determined by adding One-Glo™ reagent (Promega, cat. no. E6130), and relative light units (RLU) were measured on a Victor luminometer (Perkin Elmer). EC ₅₀ values were determined from a four-parameter logistic equation using a 10-point response curve using GraphPad Prism. Steroid delivery will result in activation of the Luc reporter in 293/PRLR/GRE-Luc cells.

As shown in table. 11, at the 72 hour time point, budesonide activated 293/PRLR/GRE-Luc cells with an IC ₅₀ value of 10 to 100 nmol. N-analogs of budesonide-activated 293/PRLR/GRE-Luc cells with similar fold activation and IC ₅₀ values ranged from less than 10 nmol to more than 100 nmol.

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Example 64.

Selective activation of glucocorticoid receptors using ALS conjugates in targeted cell lines.

Activity of steroids and ncADC steroid conjugates after internalization into the 293/PRLR/GRE-Luc cell line, as well as into 293/MMTV-Luc cells that do not express the prolactin receptor, as described in example 61, and 293/PRLR cells that do not express The luciferase reporter as described in Example 62 was assayed at concentrations from 100 nmol to 5.1 pmol using the assay procedures described in Example 63 with 72 hours of incubation.

The PRLR-LP4 conjugate (in Table 8) and its isotype control conjugate, as well as free payloads and unconjugated antibodies, were studied in two types of cell lines. The PRLR-LP4 conjugate (anti-PRLR-LP4 in Table 8) demonstrated selective activation of the 293/PRLR/GRE-Luc cell line (Fig. 23A) and lack of glucocotic receptor activation (Fig. 23B) in 293/MMTV-Luc cells. not expressing the prolactin receptor.

As shown in FIG. 23A, in 293/PRLR/GRE-Luc cells, anti-prolactin receptor antibodies site-specifically conjugated to LP4 (anti-PRLR-LP4 in Table 8) induced full activation of GRE-Luc with an EC ₅₀ value of <10 nmol. An isotype control antibody conjugated to LP4 (Anti-Her2-LP4 in Table 8) did not significantly activate GRE-Luc. The unconjugated isotype control antibody did not significantly activate GRE-Luc. Payload 11-5 in table. 1 (LP4 payload) caused full activation of GRE-Luc with an EC ₅₀ <10 nmol. The reference, budesonide, induced full activation of GRE-Luc with an EC ₅₀ of <10 nmol. As shown in FIG. 23B, in 293/MMTV-Luc cells only free payload 11-5 in table. 1 (LP4 payload) and the reference, budesonide, induced GRE-Luc activation: payload 11-5 in Table. 1 (LP4 payload) induced full GRE-Luc activation with an EC ₅₀ value of 10 to 100 nmol, and budesonide induced full GRE-Luc activation with an EC ₅₀ value of 10 to 100 nmol.

The examples given here demonstrate that anti-PRLR-LP4 in table. 8 specifically activates 293/PRLR/GRE-Luc cells that express both the target prolactin receptor and the steroid-induced GRE-luciferase reporter, but does not affect steroid-sensitive 293-MMTV-Luc cell lines or target cell lines expressing 293 -PRLR.

Example 65.

This example examined the importance of the linker and payload for glucocorticoid receptor activation of ncADCs conjugates.

The activity of free steroids and their corresponding ncADC conjugates after internalization into the 293/PRLR/GRE-Luc cell line was studied at concentrations ranging from 100 nmol to 5.1 pmol using the assay procedures described in Example 63 with 72 hours of incubation.

As shown in table. 11 and fig. 24, in 293/PRLR/GRE-Luc cells, antiprolactin receptor antibodies site-specifically conjugated to LP4 (anti-PRLR-LP4 in Table 8) induced full activation of GRE-Luc with an EC50 value of <10 nmol. An isotype control antibody conjugated to LP4 (anti-Her2-LP4 in Table 8) did not significantly activate GRE-Luc. Free payload 11-5 in table. 1 (LP4 interval) caused full activation of GRE-Luc with an EC50 of <10 nmol.

Anti-prolactin receptor antibodies site-specifically conjugated to LP2 (anti-PRLRLP2 in Table 8) induced full activation of GRE-Luc with an EC ₅₀ value of <10 nmol. An isotype control antibody conjugated to LP2 (anti-Her2-LP2 in Table 8) did not significantly activate GRE-Luc. Free payload 16-5 in table. 1 (LP2 payload) caused full activation of GRE-Luc with an EC ₅₀ <10 nmol. Finally, anti-prolactin receptor antibodies site-specifically conjugated to LP1 (anti-PRLR-LP1 in Table 8) induced full activation of GRE-Luc with an EC ₅₀ value in the range of 10-100 nmol. An isotype control antibody conjugated to LP1 (anti-Her2-LP1 in Table 8) did not significantly activate GRE-Luc. Free payload 7-1R in table. 1 (LP1 payload) caused full activation of GRE-Luc with an EC ₅₀ in the range of 10-100 nmol.

This example demonstrates that with the same antibody and linker, the payload efficiency is 11-5 in the table. 1 (payload LP4) is higher than payload 16-5 in table. 1 (payload LP2), which is higher than payload 7-1R in Table. 1 (payload LP1). Anti-PRLR-LP4 payload in Table. 8 was more effective than anti-PRLR-LP2 in Table. 8, which had higher efficiency than anti-PRLR-LP1 in table. 8.

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Table 11

The importance of the linker with the payload for the activation of glucocorticoid receptors of ncadcs conjugates, studied using the example of 293/prlr/gre-luc cells

Test sample Activation degree ECso (nmol) Ahth-PRLR-LPI Full ++ Ahth-PRLR-LP2 Full +++ Ahth-PRLR-LP4 Full +++ Anti-Her2-bp 1 N.A. N.A. Anti-Her2-bp2 N.A. N.A. Anti-Neg2-EP4 N.A. N.A. 7-2 (LP1 payload) Full ++ 16-5 (LP2 payload) Full +++ 11-5 (LP4 payload) Full +++ Budesonide Full +++

NA=not applicable; +++: < 10 nmol; ++: 10 - 100 nmol, +: > 100 nmol.

Example 66.

Biological testing of IL2RY-ncADC using HEK293/MMTV-luc/IL2Ry/IL7R cells.

Common cytokine receptor γ-chain, also known as IL2R/ and CD132, is a type I cytokine receptor that is common to the signaling pathways for interleukin-2 (IL-2), IL4, IL-7, IL-9, IL- 15, and IL-21 and plays an important role in the formation and regulation of the immune system (Rochman et al. 2009). IL2Ry is expressed primarily on immune cells and therefore may be a useful target for delivering immunosuppressive drugs such as steroids via a noncytotoxic antibody-drug conjugate (ncADC) and suppress immune cell activity while avoiding the off-target side effects associated with systemic administration steroids.

The cell-based assay described here was used to detect transcriptional activation of the glucocorticoid receptor (GR) by ncADCs with the long terminal repeat region of mouse mammary tumor virus (LTR MMTV), which has been used to study glucocorticoid receptor activation (Deroo et al. 2001). First, the HEK293 cell line was established to stably express the luciferase reporter pGL4.36 [luc2P/MMTV/Hygro] (Promega, no. E136A), here called HEK293/MMTV-luc, which was maintained in DMEM containing 10% FBS, nonessential amino acids, penicillin / streptomycin /L - glutamine and 100 μg/ml hygromycin (complete medium). The parental HEK293/MMTV-luc stable cell line was then transfected with a plasmid encoding full-length human IL2Ry (expressing amino acids 1-369 with accession number NP_000197.1) and transduced with a plasmid encoding full-length IL7Ra (expressing amino acids 1-459 with accession number number NP_002176.2) and sorted by the degree of expression of IL2Ry and IL7Ra using flow cytometry.

The resulting cell line, designated here as HEK293/MMTV-luc/IL2R//IL7R, was maintained in complete medium supplemented with 1 μg/ml puromycin and 500 μg/ml G418 sulfate.

To perform the bioassay, HEK293/MMTV-luc or HEK293/MMTV-luc/IL2Ry/IL7R cells were seeded into 96-well assay plates at 10,000 cells per well in complete medium and incubated at 37°C in 5% CO ₂ overnight. The next morning, to test glucocorticoid receptor activation, budesonide, compound 11-5 in Table. 1 (payload LP4) and connection 16-5 in table. 1 (payload LP2), conjugatanti-IL2Rg-LP4 (in Table 8), isotype control antibody (in Table 8) and unprotected antibodies were serially diluted in a 1:3 ratio from 200 nmol-1 μmol to 0.0020.01 nmol and added to the cells. Concentrations were adjusted according to the drug-antibody ratio for ncADC and other methods known to those skilled in the art. One well without test sample was also included as a control.

Luciferase activity was measured after 6, 24, 48 and 72 hours of incubation at 37°C with 5% CO2 on a Victor X instrument (Perkin Elmer). The results were analyzed using nonlinear regression (four-parameter logistic model) using Prism 6 software (GraphPad) to obtain EC ₅₀ values. The degree of activation was calculated by determining the ratio of the luciferase activity of each sample to the luciferase activity observed without the addition of any test sample.

As shown in table. 12, after 6, 24 and 48 hours of incubation, budesonide showed the greatest activation of the glucocorticoid receptor with complete activation; compounds 16-5 in table. 1 (payload LP2) and 11-5 in table. 1 (LP4 payload) showed partial activation. With more

- 169 045579 long incubation periods equal to 72 hours, 16-5 in table. 1 (payload LP2) and 11-5 in table. 1 (LP4 payload) showed a level of glucocorticoid receptor activation similar to that of budesonide when fully activated. These results demonstrate that budesonide, compounds 16-5 (LP2 payload) and 11-5 (LP4 payload) in Table. 1 activate the glucocorticoid receptor with EC ₅₀ values in the range of 10-100 nmol.

Table 12

Activation of the glucocorticoid receptor in HEK293/MMTV-LUC/IL-2Ry/L7R cells by budesonide, payload 11-5 in table. 1 or payload 16-5 in table. 1 at 6, 24, 48 or 72 hours

Moment of time 6 hours 24 hours 48 hours 72 hours Medicine Activation degree ec ₅₀ [nmol] Activation degree ec ₅₀ [nmol] Activation degree ec ₅₀ [nmol] Activation degree ec ₅₀ [nmol] Budesonide Full activation ++ Full activation ++ Full activation ++ Full activation ++ Compound 11-5 in Table 1 Partial activation + Partial activation + Partial activation ++ Full activation ++ Compound 16-5 in Table 1 Partial activation + Partial activation + Partial activation ++ Full activation ++

+++: < 10 nmol; ++: 10-100 nmol, +: > 100 nmol.

Budesonide, linker with payload LP4 (Payload 11-5) and linker with payload LP7 (Payload R-11-5), anti-IL211γ, anti-IL2Rγ mAbs-ncADC with payloads LP4 and LP7 (referred to as anti -IL2Rγ-LP4 and anti-IL2Rγ-LP7), as well as control mAb-LP7 and unconjugated anti-IL2Rγ mAb, were added to HEK293/MMTV-Luc/IL2Ry/IL7R cells and incubated for 24 hours (A), for 48 hours (B), for 72 hours (C), or for HEK293/MMTV-Luc cells for 72 hours (D) with a maximum concentration at 200 nmol (RLU, relative light unit) in Fig. 25 and in table. 13.1.

As shown in table. 13.1 and Fig. 25, after 24 hours of incubation, budesonide showed the highest maximum degree of activation in HEK293/MMTV-Luc/IL2Ry/IL7R cells, and payloads 11-5 and R-11-5 showed relatively lower levels of activation compared to budesonide (Fig. 25A ). At longer incubation times of 48 and 72 hours, 11-5 and R-11-5 showed a similar level of activation to budesonide (Figure 25B and Figure 25C).

Ahtu-IL2Ry-LP4 and anti-IL2Rγ-LP7 showed little or no activation in HEK293/MMTV-Luc/IL2Ry/IL7R cells after 24 hours of incubation (Figure 25A), but higher levels of activation with a longer incubation period of 48 hours and 72 hours (Figs. 25B and 25C). Ahtu-IL2RyncADCs, anti-IL2Rγ-LP4 and anti-IL2Rγ-LP7, did not show any activation in HEK293/MMTV-Luc cells (Figure 25D), indicating that steroid delivery by the ncADC conjugate is dependent on binding to the IL2Ry antigen on cell surface and subsequent internalization. In contrast, unconjugated anti-IL2Rγ antibody and unconjugated and conjugated isotype control antibodies did not show any significant activation under any conditions. Budesonide, payloads 11-5 and R-11-5 showed activation in HEK293/MMTV-Luc cells after 72 hours of incubation, indicating activation of glucocorticoid receptors by free drugs (Figure 25D).

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Table 13.1

Activation of the glucocorticoid receptor in HEK293/MMTV-LUC/IL-2Ry/IL7R cells by steroid payloads and anti-IL2Rγ-steroid ALS conjugates and control ALS conjugate

Treatment Maximum degree of activation 24 hours 48 hours 72 hours Ahth-IL2Ry-LP4 N.A. + ++ AhthIL2Ry-LP7 N.A. + ++ Payload LP4 (11-5) + ++ +++ Payload LP7 (R-11-5) + ++ +++ Budesonide +++ +++ +++ Ab-LP7 control N.A. N.A. N.A.

NA=not applicable +++: < 10 nmol; ++: 10-100 nmol; +: > 100 nmol.

Example 67.

This example demonstrates the bioactivity of cytotoxic ALS conjugates with and without cyclodextrin linkers (Fig. 30).

To evaluate the comparability of CD and CD-free conjugates containing cytotoxic payloads, a cytotoxicity assay was performed using SKBR3 cells. SKBR3 cells have been commonly used to evaluate anti-Her2 ADC activity. Anti-PRLR ALS conjugates were used as a control ALS mAb conjugate in the SKBR3 cell cytotoxicity assay. For the assay, the in vitro cytotoxicity of the anti-PRLR ALS conjugate was assessed using the CellTiter-Glo assay kit (Promega, cat. no. G7573), in which the amount of ATP present is used to determine the number of viable cells in culture. For analysis, SKBR3 cells were seeded at 6000 cells per well onto white Nunclon 96-well plates in complete growth medium and grown overnight at 37°C in 5% CO ₂ . To generate cell viability curves, serially diluted 1:4 ALS conjugates or free payload were added to cells at concentrations starting at 100 nmol, including a no-treatment control, and then incubated for 5 days. After 5 days of incubation, cells were incubated at room temperature with 100 μl CellTiter-Glo reagents for 5 minutes. Relative luminescence units (RLU) were determined on a Victor plate reader (PerkinElmer). IC ₅₀ values were determined from a four-parameter logistic equation using a 10-point response curve (GraphPad Prism). All curves and EC ₅₀ values were adjusted to include equivalents. All IC ₅₀ values are expressed in nmol concentration, and the percentage of cells killed (%kill) is given for the highest concentration tested.

The bioactivity of ALS steroid conjugates with and without cyclodextrin linkers is shown in FIG. thirty.

To evaluate the comparability of conjugates with and without CD containing steroid payloads, their activity in 293/PRLR/GRE-Luc cells was studied after 72 hours of incubation. For this assay, 20,000 cells were seeded in 96-well plates in medium containing DMEM supplemented with 10% FBS and pencillin/streptomycin (complete medium) and grown overnight at 37°C in 5% CO ₂ . To generate dose-response curves for free drug or ALS conjugate, serially diluted reagents ranging from 100 nmol to 5.1 pmol were added to the cells and incubated for 72 hours at 37°C. Luciferase activity was determined by adding One-Glo™ reagent (Promega, cat. no. E6130), and relative light units (RLU) were measured on a Victor luminometer (Perkin Elmer). EC values were determined from a four-parameter logistic equation using a 10-point response curve using GraphPad Prism. Steroid delivery will result in activation of the Luc reporter in 293/PRLR/GRE-Luc cells. Full activation in this analysis is defined as activation that is between 90% and 100% of the maximum activation measured by the free payload. Partial activation in this analysis is defined as activation that is between 10% and 90% of the maximum activation measured by the free payload. Minimum activation in this analysis is defined as less than 10% of the maximum activation measured by the free payload.

As shown in table. 13.2 and in fig. 30, ALS anti-PRLR Ab conjugates containing CD (anti-PRLR Ab-Ex46) have potency and specific activity in activating the GRE-Luc reporter in 293/PRLR/GRE-Luc cells similar to those of ALS anti-PRLR conjugates Abs that do not contain CD (anti-PRLR Ab-Ex44). In this analysis

Isotype control ALS conjugates, whether containing CD or not, as well as unconjugated antibody, did not show any significant effects.

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Molecules studied Max, activation ec ₅₀ (nmol) Anti-PRLR AB- Ex46 Partial 8.6 AhthPRLR Ab- Ex45 Partial 9.5 Free payload (Connection 1c) Full 8.4

Table 13.2

Activation of glucocorticoid receptors by ALS steroid conjugates with and without cyclodextrin linkers in 293/PRLR/GRE-LUC cells

Molecules studied Max, activation ec ₅₀ (nmol) Isotype control AB-Ex46 Minimum N.A. Isotype control AB-Ex45 Minimum N.A. AhthPRLR Ab Minimum N.A.

NA=not applicable

Table 14

Chemical and physical properties of linker steroids

Enter Molecular formula Mol. weight cLogP HPLC Purity (%) HPLC at room, temp, (min.) ms (mass/charge) 100% Highest peak mass/charge LP101 Error 1163.35 4.04 100 7.07(V) 1163.6 (M+H) 1163.6 (M+H) LP102 C55H75F2N5O11 1020.21 5.41 >99 8.44 (A) 8.47(V) 510.8 (M/2+N) 1020.3 (M+H) (33%) LP103 SbbN88r2M ₈ O14 1255.45 5.9 >99 8.46 (A) 1255.5 (M+N) 1255.5 (M+H) LP104 C126H177F2N13O49 2695.81 -5.09 >99 6.23 (V) 899.2 (M/Z+N) 1348.6 (M/2+N) (40%) LP105 C118H ₁₇₆ F2N12O49 2584.71 -5.21 >99 7.37 (A) 7.41(V) 1293.6 (M/2+N) 1293.6 (M/2+N) LP108 C121H168F2N10O47 2552.66 -3.72 >99 7.76(V) 851.4 (M/Z+N) 1276.8 (M/2+N) (83%) LP110 C132H181F2N13O50 2787.9 -3.23 >99 7.94(A) 8.02(V) 930.3 (M/Z+N) 1394.2 (M/2+N) (33%) LP112 C74H ₉ 4F ₂ N ₈ O17 1405.60 4.39 >99 7.40(V) 703.5 (M/2+N) 703.5 (M/2+N) LP113 C54H74N4O15 1019.18 3.46 100 7.46(V) 510.3 (M/2+N) 1041.3 (M+Na) (10%) LP114 C70H ₈₇ F2N ₅ O16 1292.46 5.61 99 9.18 (A) 9.22(V) 1292.1 (M+H) 1292.1 (M+H) LP115 C69H ₈₄ F3N ₅ O15 1280.42 5.91 100 8.34 (V) 1279.6 (M+H) 1279.6 (M+H) LP116 C8oH ₉ 8F2N ₈ 018 1497.70 6.25 99 7.99(V) 1498.7 (M/2+N) 1498.7 (M+H) (10%)

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Table 15

Physical properties of steroid linkers

LP№ Structure cLogP Mol. weight Payload Linker Structure L3 split fragment Hydrophilic fragment L2 Conjugate linker LI LP112 F N I F T G n MDMKA 1 DT^yun Before vcPAB / Lk-DIBAC 4.39 1405.58 LP101 vcPAB / LkCCK 4.04 1163.35 LP104 vcPAB aCDCCK Lk-DIBAC -5.09 2695.81 LP105 vcPAB aCDCCK Lk-BCN -5.21 2584.71 LP102 F n G F| | n oХ 1 ⁿ 1 ¹ L /dg-^on ⁰ y-o 0 γΝΗ V.A. / Lk-CCK 5.41 1020.2 LP108 V.A. aCDCCK Lk-DIBAC -3.72 2552.66 LP116 vcPAB / Lk-DIBAC 6.25 1497.67 LP103 vcPAB / LkCCK 5.90 1255.5 LP110 vcPAB aCDCCK Lk-DIBAC -3.23 2787.9 LP113 D Tj o-\1 JV M-LM oβ γΝΗ V.A. / Lk-MAL 3.46 1019.18 LP114 F n GfT g n /MfMKA oDHD 1 /SON J ⁰ L° O V.A. / Lk-DIBAC 5.61 1292.46 LP115 F Ach^^gch^O Η Г F 1 I n o\L ⁿ L T dt^on J ° L° O L ^NH F V.A. / Lk-DIBAC 5.91 1280.42

- 173 045579

Table 16

List of site-specific steroid-antibody linkers

LP Ab, Ab-N3, or Ab-steroid conjugates EX MS mass/charge Name Mol.mass (Da) DAR ratio Anti-PRLR AB 144602 4.0 PEG3-N3 218.3 AHTH-PRLR-Ab-N ₃ 145385 3.9 LP112 1405.6 Anti-PRLR Ab-LP12z 151015 4.0 LP104 2695.9 Anti-PRLR Ab-LP4z 156198 4.0 LP116 1497.7 Anti-PRLR Ab-LP16z 151015 4.0 Anti-Fei DI Ab PEG3-N3 218.3 Anti-Fel DI Ab-N3z 146251 4 LP112 1405.6 Anti-Fel DI Ab-LP12z 151871 3.9 LP116 1497.7 Anti-Fel DI Ab-LP16z 152235 3.9

Example 68.

This example applies to the compounds in table. 2 and fig. 31.

Commercial steroids used as starting materials included fluocinolone acetonide (1a), dexamethasone (1c), flumethasone (1d), triamcinolone (1e), methylprednisolone (1f) and triamcinolone acetonide (1g). Compound 1b was prepared from 1a by ketal exchange with butyraldehyde in the presence of perchloric acid, and its two chiral isomers were obtained from chiral supercritical fluid chromatography (SCFC) separation. Using the same approach, compound 1h was obtained from 1g. Compounds 1b-f and 1h were converted to the corresponding mesylate derivatives (2b-f, 2h) followed by replacement of the mesylate group with an azide moiety to form compounds 3b-f and 3h, which were then reduced to amines (4b-f, 4h). The mesylate moiety in 2b was also replaced with anilines to give 5-Iz, replaced with an alkylamine to give 5-II, and replaced with phenols to give 6-I to 6-III. Compound 6-VI was obtained by replacing budesonide mesylate with 4-amino-phenol, 6-VII was obtained by replacing the mesylate in compound 2f with 4-amino-phenol.

Example 69.

Synthesis of compound 1b, R-1b, S-1b, and 1h.

This example applies to the compounds in table. 2 and fig. 31.

(1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6propyl-5,7 -dioxapentacyclo[10.8.0.0 ^{2.9.0 4.8.0 13.18} ]icosa-14,17-dien-16-one (1 b)

Butyraldehyde (0.27 ml, 3.0 mmol) was added to a mixture of fluocinolone acetonide (1a, 0.90 g, 2.0 mmol) and silica gel (18 g) in heptanes (90 ml) at 10°C, the suspension was stirred at 10-20°C for 10 minutes. Perchloric acid (70%, 0.68 mL, 8.3 mmol) was added dropwise to the mixture at 0°C. The reaction mixture was then stirred at 10-20°C overnight. Most of compound 1a was consumed according to TLC and LCMS data. The reaction mixture was diluted with petroleum ether and quenched with a saturated aqueous sodium carbonate solution. The suspension was filtered and the solid was washed with DCM/methanol (v/v = 1). The combined filtrate was concentrated in vacuo. The residue was purified by flash chromatography (0-100% ethyl acetate in petroleum ether) to give 1b (0.15 g, 16% yield) as a white solid. Mass/charge at NMR: 467.1 (M+H) ⁺ . Compound 1b was further purified by pre-HPLC (Method B) to obtain R-1b (40 mg, 39% yield) and S-1b (10 mg, 9% yield) as a white solid. Mass/charge at NMR: 467 (M+H) ⁺ .

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-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 (R-1b).

Certain methods and/or intermediates in EP0262108A1 were used, the entire contents of which are incorporated herein by reference for all purposes:

Compound R-1b means

- 174 045579

1H NMR (400 MHz, MeOD _d4 ) δ 7.34 (dd, J=10.1, 1.3 Hz, 1H), 6.37-6.32 (m, 2H), 5.65-5.48 (m, 1H), 4.63 (t, J=4.3 Hz, 1H), 4.55 (d, J=19.4 Hz, 1H), 4.33-4.28 (m, 2H) , 2.74-2.59 (m, 1H), 2.38-2.32 (m, 1H), 2.26-2.16 (m, 2H), 1.70-1.41 (m, 12H), 0.97-0.93 (m, 6H) ppm. Anal. HPLC: >99.9%, dwell time: 8.05 minutes (method A).

(1 S,2S,4R,8S,9S, 11S,12R, 13S)-12-fluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-propyl5,7-dioxapentacyclo [10.8 .0.0 ^2.9 .0 ^4.8 .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-16-one (1h)

Following the procedure for 1b, 1g (1.3 g, 3.0 mmol) was converted to 1h (1.1 g, 85% yield) as a white solid. Mass/charge at NMR: 449 (M+1)+.

Example 70.

General procedure A for the synthesis of methylates (Ms) 2 in Fig. 31:

This example applies to the compounds in table. 2 and fig. 31.

To a solution of compound 1 (1c, 1d, 1e, 1f, or 1h, 1 equiv.) in pyridine (10 ml per gram of compound 1), 4-dimethylaminopyridine (2 equiv.) and methanesulfonyl chloride (1.5 equiv.) were added dropwise .) at 0°C. After stirring at room temperature for 2 hours and monitoring the reaction by LCMS until compound 1 (1c, 1d, 1e, 1f, or 1h, 1 equiv.) was completely consumed, the resulting mixture was poured into ethyl acetate (100 ml). The mixture was washed with diluted hydrochloride (1N) to pH=7 and brine, dried over sodium sulfate and concentrated. The crude product was purified by flash chromatography (0-2% MeOH in DCM) to give compound 2 (2c, 2d, 2e, 2f, or 2h, 1 equiv.).

Example 71.

This example applies to the compounds in table. 2 and fig. 31.

2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-nronul-5,7dioxapentacyclo [10.8.0.0 ² ' ⁹ .0 ^4.8 .0 ¹³ ' ¹⁸ ]icosa-14,17-dien-8-yl]-2-oxoethyl methanesulfonate (2b).

This example applies to the compounds in table. 2 and fig. 31.

No° 9 -X . j it\° n I I f I n

F

To a solution of compound 1b (0.28 g, 0.65 mmol) in DCM (3 ml) was added triethylamine (0.13 g, 1.3 mmol) and methanesulfonyl chloride (89 mg, 0.78 mmol). After stirring at 0°C for half an hour until compound 1b was completely consumed by TLC, the reaction mixture was concentrated in vacuo. The silica gel residue was purified by silica gel column chromatography (0-50% ethyl acetate in petroleum ether) to give 2b (0.26 g, >99% yield) as a white solid. Mass/charge at NMR: 545 (M+H) ⁺ .

Example 72.

This example applies to the compounds in table. 2 and fig. 31.

2-[(1R,2S,10S,11S,13R,14R,15S,17S)-1-fluoro-14,17-dihydroxy-2,13,15-trimethyl-5octotetracyclo[8.7.0.0 ² ' ⁷ .0 ^{11 ,15} ]heptadeca-3,6-dien-14-yl]-2-oxoethyl methanesulfonate (2c).

Certain methods and/or intermediates were used in WO2015/71657, the full contents of which are incorporated herein by reference for all purposes:

Following general procedure A, compound 2c (0.32 g, 50% yield) was obtained as a white solid from dexamethasone (1c, 0.53 g, 1.4 mmol). Mass/charge at NMR: 471 (M+H) ⁺ .

1H NMR (MeOD _d4 , 500 MHz) δ 7.42 (d, J=10.0 Hz, 1H), 6.31 (dd, J=10.0, 2.0 Hz, 1H), 6.10 ( s, 1H), 5.27

- 175 045579 (d, J=18.0 Hz, 1H), 5.04 (d, J=18.0 Hz, 1H), 4.30-4.27 (m, 1H), 3.21 (s , 3H), 3.10-3.05 (m, 1H), 2.78-2.71 (m, 1H), 2.55-2.40 (m, 1H), 2.36-2.32 (m, 1H), 2.27-2.21 (m, 1H), 1.93-1.88 (m, 1H), 1.82-1.74 (m, 1H), 1.61 (s , 3H), 1.58-1.51 (m, 2H), 1.25-1.20 (m, 1H), 1.06 (s, 3H), 0.89 (d, J=7.5 Hz, 3H) ppm.

Example 73.

This example applies to the compounds in table. 2 and fig. 31.

2-[(1R,2S,8S,10S,11S,13R,14R,15S,17S)-1,8-difluoro-14,17-dihydroxy-2,13,15-trimethyl-5octotetracyclo[8.7.0.0 ^{2, 7} .0 ^11.15 ]heptadeca-3,6-dien-14-yl]-2-oxoethyl methanesulfonate (2d)

Specific methods and/or intermediates in the Bioorg source were used. Med. Chem. Lett., 2015, 25, 2837-2843, the entire contents of which are incorporated herein by reference for all purposes:

Following general procedure A, compound 2d (0.17 g, 71% yield) was obtained as a white solid from flumethasone (1d, 0.20 g, 0.49 mmol). Mass/charge at NMR: 489 (M+H)+.

Example 74.

This example applies to the compounds in table. 2 and fig. 31.

2-((8S,9R,10S,11S,13S,14S,16R,17S)-9-fluoro-11,16,17-trihydroxy-10,13-dimethyl-3-oxo6,7,8,9,10 ,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]p phenanthrene-17-yl)-2-oxoethyl methanesulfonate (2e)

Following general procedure A, 2e (0.38 g, 81% yield) was obtained as a white solid from triamcinolone (1e, 0.39 g, 1.0 mmol). Mass/charge at NMR: 473 (M+H) ⁺ .

Example 75.

This example applies to the compounds in table. 2 and fig. 31.

2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo6,7,8,9,10,11,12 ,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-yl)-2-oxoethyl methanesulfonate (2f)

Following general procedure A, 2f (0.16 g, 35% yield) was obtained as a white solid from methylprednisolone (1f, 0.38 g, 1.0 mmol). Mass/charge at NMR: 453 (M+H) ⁺ .

Example 76.

This example applies to the compounds in table. 2 and fig. 31.

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

This example applies to the compounds in table. 2 and fig. 31.

Following general procedure A, compound 2h (0.45 g, 85% yield) was obtained as a white solid from methylprednisolone (1h, 0.39 g, 1.0 mmol). Mass/charge at NMR: 528 (M+H) ⁺ .

Example 77.

This example applies to the compounds in table. 2 and fig. 31. Synthesis of steroid payload 4b.

(1 S,2S,4R,8S,9S, 11S,12R, 13S, 19S)-8-(2-azidoacetyl)-12,19-difluoro-11-hydroxy-9,13-dimethyl-6propyl-5,7 -dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (3b)

- 176 045579

- _n =n ⁺ -n P ⁴ —\ o^xX

NO./DN 1

I I H l\°

XzMX h

I I F I H qX^/

F

A suspension of compound 2b (1.0 g, 1.8 mmol) and sodium azide (1.2 g, 18 mmol) in acetone (15 ml) was stirred at 50°C overnight, during which time the reaction was completed according to the data obtained using the LCMS method. After cooling the suspension, the reaction mixture was poured into cold water (80 ml). The aqueous mixture was extracted with ethyl acetate (50 mlx3). The combined organic solution was washed with brine (30 mL), dried over sodium sulfate and concentrated in vacuo to give 3b (0.90 g, >99% yield) as a yellow solid, which was used for the next step without further purification. Mass/charge at NMR: 492 (M+H) ⁺ .

(1S,2S,4R,6R,8S,9S,11S,12R,13S,19S)-8-(2-aminoacetyl)-12,19-difluoro-11-hydroxy-9,13-dimethyl-6propyl-5, 7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one; trifluoroacetic acid salt (4b)

H ₂ N Р οχΧ . 1 ⁿ TV ⁰ HIMH n II f I n

F

Compound 3b (0.85 g, 1.7 mmol) was added to a 100 mL round bottom flask, followed by THF (20 mL) and aqueous hydrochloride (1 N, 10 mL). The mixture was stirred at 28-32°C until it became clear, then triphenylphosphine (0.68 g, 2.6 mmol) was added to it at this temperature. The resulting yellow transparent solution was stirred at 28-32°C for 18 hours, during which time the reaction was complete according to TLC and LCMS. The mixture was concentrated in vacuo and the residue was purified by reverse phase flash chromatography (0-50% acetonitrile in aqueous TFA (0.05%)) to give 4b (0.56 g, 57% yield, trifluoroacetic acid salt) in as a yellowish solid. Mass/charge at NMR: 466 (M+H) ⁺ .

^1H NMR (400 MHz, MeODd4) δ 7.33 (d,J=9.9 Hz, 1H), 6.40-6.29 (m, 2H), 5.69-5.45 (m, 1H ), 4.93-4.92 (m, 1H), 4.71 (t, J=4.3 Hz, 1H), 4.35-4.27 (m, 2H), 3.90-3, 84 (m, 1H), 2.81-2.54 (m, 1H), 2.42-2.06 (m, 3H), 1.82-1.32 (m, 11H), 1.09- 0.87 (m, 6H) ppm. ¹⁹ F NMR (376 MHz, MeODd4) δ -77.01, -166.24, -166.92, -188.81, 188.83 ppm. Anal. HPLC: 100%, dwell time: 6.86 minutes (method A).

Example 78.

This example applies to the compounds in table. 2 and fig. 31.

(1R,2S,10S,11S,13R,14R,15S,17S)-14-(2-aminoacetyl)-1-fluoro-14,17-dihydroxy-2,13,15trimethyltetracyclo[8.7.0.0 ^2.7 .0 ^11.15 ]heptadeca-3,6-dien-5-one trifluoroacetate (4c)

Following the procedure described in Example 77 for compound 4b, except replacing compound 2c with compound 2d, compound 4c was obtained as trifluoroacetic acid salt (0.50 g, 53% yield in 2 steps) as a white solid. Mass/charge at NMR: 392 (M+H)+.

1H NMR (400 MHz, DMSOd6) δ 8.22 (s, 3H), 7.35 (d, J=10.0 Hz, 1H), 6.19 (d, J=10.0 Hz, 1H), 5.98 (s, 1H), 5.60 (d, J=4.0 Hz, 1H), 5.38 (s, 1H), 4.30-4.10 (m, 2H), 3.62 (d, J=18.8 Hz, 1H), 2.98-2.83 (m, 1H), 2.65-2.50 (m, 1H), 2.50-2.22 (m, 2H ), 2.20-2.01 (m, 2H), 1.80-1.72 (m, 1H), 1.72-1.58 (m, 1H), 1.46 (s, 3H), 1.46-1.25 (m, 2H), 1.13-1.01 (m, 1H), 0.89 (s, 3H), 0.78 (d, J=6.8 Hz, 3H) ppm, ¹⁹ F NMR (376 MHz, DMSOd6) δ -73.79, 164.32 ppm. Anal. HPLC: >99%, dwell time: 6.34 minutes (method A).

Example 79.

This example applies to the compounds in table. 2 and fig. 31.

(1R,2S,8S,10S,11S,13R,14R,15S,17S)-14-(2-aminoacetyl)-1,8-difluoro-14,17-dihydroxy-2,13,15trimethyltetracyclo[8.7.0.0 ^{2 .7} .0 ^11.15 ]heptadeca-3,6-dien-5-one trifluoroacetate (4d)

- 177 045579

Following the procedure described in Example 77 for the preparation of compound 4b, except replacing compound 2d with compound 2b, compound 4d was obtained as trifluoroacetic acid salt (0.18 g, 21% yield in 2 steps), as a white solid. Mass/charge at NMR: 410 (M+H) ⁺ .

Ή NMR (400 MHz, DMSO _d6 ) δ 8.17 (s, 3H), 7.36 (d, J=10.3 Hz, 1H), 6.29 (dd, J=10.2, 1.7 Hz, 1H), 6.11 (s, 1H), 5.74-5.54 (m, 2H), 5.42 (s, 1H), 4.28-4.10 (m, 2H), 3 .70-3.59 (m, 1H), 3.02-2.89 (m, 1H), 2.58-2.40 (m, 1H), 2.31-2.12 (m, 3H) , 2.08 (s, 1H), 1.77-1.64 (m, 1H), 1.51-1.44 (m, 4H), 1.16-1.06 (m, 1H), 0 .91 (s, 3H), 0.82 (d, J=7.2 Hz, 3H), ppm, ¹⁹ F NMR (376 MHz, DMSO _d6 ) δ -73.65, -163.75, -186, 04 ppm. Anal. HPLC: > 99%, Hold time: 6.36 minutes (method A).

Example 80.

This example applies to the compounds in table. 2 and and in Fig. 31.

(8S,9R,10S,11S,13S,14S,16R,17S)-17-(2-aminoacetyl)-9-fluoro-11,16,17-trihydroxy-10,13-dimethyl7,8,11,12, 13,15,16,17-octahydro-6H-cyclopenta[a]phenanthrene-3(9H, 10H, 14H)-one trifluoroacetate (4e)

Following the procedure described in Example 77 for the preparation of compound 4b, except replacing compound 2e with compound 2b, compound 4e was obtained as trifluoroacetic acid salt (28 g, 21% yield in 2 steps), as a white solid.

Mass/charge at NMR: 394 (M+H)+.

^1H NMR (500 MHz, DMSO _d6 ) δ 8.04 (s, 3H), 7.33 (d, J=10 Hz, 1H), 6.24 (dd, J=10 Hz, 1.0 Hz, 1H), 6.02 (s, 1H), 5.53 (d, J=5.5 Hz, 1H), 5.50-5.45 (m, 1h), 5.04 (s, 1H), 4.76-4.70 (m, 1H), 4.20-4.12 (m, 2H), 3.68 (d, J=20 Hz, 1H), 2.66-2.57 (m, 1H), 2.40-2.20 (m, 3H), 2.20-2.10 (m, 1H), 1.90-1.70 (m, 2H), 1.50-1.20 ( m, 6H), 0.89 (s, 3H) ppm. Anal. HPLC: >99%, dwell time: 5.79 minutes (method A).

Example 81.

This example applies to the compounds in table. 2 and fig. 31.

(6S,8S,9S,10R,11S,13S,14S,17R)-17-(2-aminoacetyl)-11,17-dihydroxy-6,10,13-trimethyl6,7,8,9,10,11, 12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3-one (4f)

Following the procedure described in Example 77 to prepare compound 4b, except replacing compound 2b with compound 2f and stirring at 60°C rather than 28~32°C in step 2, compound 4f (10 mg, 14% yield in 2 steps) as a yellow solid after purification by pre-HPLC (Method B). Mass/charge at NMR: 466 (M+H) ⁺ .

^1H NMR (400 MHz, MeOD _d4 ) δ 8.50 (s, 1H), 7.50 (d, J=10.0 Hz, 1H), 6.27 (dd, J=1.6 Hz, 10 .0 Hz, 1H), 6.02 (s, 1H), 4.43-4.42 (m, 1H), 4.32-4.27 (m, 1H), 3.80-3.76 ( m, 1H), 2.79-2.73 (m, 2H), 2.29-2.15 (m, 3H), 1.83-1.50 (m, 7H), 1.10-0, 80 (m, 8H) ppm.

Example 82.

This example applies to the compounds in table. 2 and fig. 31.

(1S,2S,4R,8S,9S,11S,12R,13S)-8-(2-aminoacetyl)-12-fluoro-11-hydroxy-9,13-dimethyl-6-propyl-5,7dioxapentacyclo[10.8. 0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (4h) with 22R/S isomers (ratio 2:1)

Following the procedure described in Example 77 for compound 4b, except replacing compound 2h (0.26 g, 0.5 mmol) with compound 2b, compound 4h (5 mg, 6% yield in 2 steps) was obtained as a solid yellow after double purification by pre-HPLC. Mass/charge

- 178 045579 at NMR: 448 (M+H)+.

1H NMR (500 MHz, DMSOd6) δ 8.04 (s, 3H), 7.95-7.70 (m, 1H), 7.32 (d, J=10 Hz, 1H), 6.24 (d , J=9.0 Hz, 1H), 6.02 (s, 1H), 5.65-5.55 (m, 1H), 5.18 (t, J=4 Hz, 0.24H), 5 .12 (d, J=5 Hz, 0.24Н), 4.77 (d, J=5.0 Hz, 0.76Н), 4.66 (t, J=4 Hz, 0.76Н), 4 .25-4.10 (m, 2H), 3.80-3.70 (m, 1H), 2.65-2.55 (m, 1H), 2.36-2.30 (m, 1H) , 2.05-1.95 (m, 2H), 1.85-1.75 (m, 1H), 1.70-1.55 (m, 4H), 1.48 (s, 3H), 1 .40-1.30 (m, 3H), 1.25-1.20 (m, 1H), 0.90-0.80 (m, 6H) ppm. ^19F NMR (376 MHz, DMSO _d6 ) δ -73.51 (3F), -164.50 (0.3F), -165.27 (0.7F) ppm.

Example 83.

This example applies to the compounds in table. 2 and fig. 31.

(1S,2S,4R,8S,9S,11S,12R,13S,19S)-8-{2-[(4-aminophenyl)amino]acetyl}-12,19-difluoro-11-hydroxy9,13-dimethyl- 6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one trifluoroacetate (5-I)

To compound 2b (0.10 g, 0.18 mmol) in N,N-dimethylformamide (2 ml) in a screw cap test tube was added 4-hydroxyalinine (0.10 mg, 0.92 mmol), triethylamine (0.20 g, 2. 0 mmol) and sodium iodite (0.10 g, 0.67 mmol). The mixture was stirred at 70°C for 5 hours under LCMS control. The reaction mixture was purified directly twice by pre-prep-HPLC (Method A) to obtain compound 5-I (10 mg, 8%) as a white solid. Mass/charge at NMR: 557 (M+H) ⁺ .

1H NMR (500 MHz, DMSO _d6 ) δ 9.59 (br s, 3H), 7.50-5.96 (m, 8H), 5.76-3.81 (m, 7H), 2.73- 2.55 (m, 1H), 2.28 (s, 1H), 2.20-1.99 (m, 2H), 1.86-1.79 (m, 1H), 1.70-1, 27 (m, 10H), 0.93-0.76 (m, 6H) ppm, ¹⁹ F NMR (376

MHz, DMSO _d6 ) δ -73.90, -164.22, -165.02, -186.37 ppm. Anal. HPLC: >99%, dwell time: 7.55 minutes (method A).

Example 84.

This example applies to the compounds in table. 2 and fig. 31.

General Procedure B for preparing compound 6 by substituting phenol for compound 2:

Compound 2 (1 eq.), the corresponding phenol (2.0-2.5 eq.) and potassium carbonate or cesium carbonate (2.0-3.0 eq.) were added to hot acetonitrile or acetone (60-65°C). ). The resulting suspension was refluxed for 2-3 hours under the control of LCMS and TLC. After cooling to room temperature, the volatiles were removed in vacuo and water was added to the residue. The aqueous mixture was extracted with ethyl acetate. The combined organic solution was washed with water and brine, dried over sodium sulfate and concentrated in vacuo. The crude product was used directly for the next step or purified by flash chromatography or preparative HPLC to give pure arylester 6.

Example 85.

This example applies to the compounds in table. 2 and fig. 31. (1S,2S,4R,8S,9S,11S,12R,13S,19S)8-[2-(4-aminophenoxy)acetyl]-12,19-difluoro-11-hydroxy-9,13-dimethyl- 6-propyl-5,7dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (6-I) nh ₂ /-° ⁰⁼ \ isk V ] 11 n ix° n II f I n

F

Following general procedure B, the reaction of 2b (calculated amount 0.17 mmol) with 4-aminophenol (37 mg, 0.34 mmol) synthesized cesium carbonate (0.11 g, 0.34 mmol) in acetone (0.5 ml) , gave the title compound 6-I (6.0 mg, 6.3% yield of 1b) as a white solid after purification by pre-HPLC (Method B). Mass/charge at NMR: 298 (M/2+H) ⁺ , 558 (M+H) ⁺ (10%).

1H NMR (500 MHz, MeOD _d4 ) δ 7.34 (d, J=10.0 Hz, 1H), 6.78-6.71 (m, 4H), 6.37-6.33 (m, 2H ), 5.63-5.49 (m, 1H), 5.10-4.99 (m, 1H), 4.77-4.63 (m, 2H), 4.33 (d, J=9 ,1 Hz, 1H), 2.74-2.57 (m, 1H), 2.39-2.13 (m, 3H), 1.98-1.31 (m, 12H), 1.03- 0.93 (m, 6H) ppm. Anal. HPLC: purity 97.4%, holding time: 7.55 minutes (me

- 179 045579 tod B).

Example 86.

This example applies to the compounds in table. 2 and fig. 31.

(1 S,2S,4R,6S,8S,9S, 11S,12R, 13S,19S)-8-[2-(4-aminophenoxy)acetyl]-12,19-difluoro-11-hydroxy-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 (S-6-I)

Following general procedure B, except replacing 2b with S-2b, provided S-6-I (19 mg, 19% yield in 2 steps from S-2b) as a white solid. Mass/charge at NMR: 558 (M+H)+.

^1H NMR (400 MHz, DMSOd6) δ 7.26 (dd, J=10.2, 1.0 Hz, 1H), 6.65-6.55 (m, 2H), 6.51-6.44 (m, 2H), 6.30 (dd, J=10.2, 1.9 Hz, 1H), 6.11 (s, 1H), 5.74-5.46 (m, 2H), 5, 23 (t, J=4.9 Hz, 1H), 5.14 (d, J=7.2 Hz, 1H), 4.99 (d, J=18.2 Hz, 1H), 4.74- 4.55 (m, 3H), 4.26-4.12 (m, 1H), 2.65-2.53 (m, 1H), 2.29-2.19 (m, 1H), 2. 13-1.94 (m, 2H), 1.86-1.22 (m, 11H), 0.92-0.78 (m, 6H) ppm, ¹⁹ F NMR (376 MHz, DMSOd6) δ -164 .26, -186.38 ppm. Anal. HPLC: > 99%, dwell time: 7.34 minutes (method B).

Example 87.

This example applies to the compounds in table. 2 and fig. 31.

Certain methods were used, as outlined in the source Org. Biomol. Chem., 2014, 12, 7551-7560 the entire contents of which are incorporated herein by reference for all purposes.

Step 1: 4-amino _(2H4 ⁾ phenol:

D

Η ₂ Ν ^/ γΥ

D

A 20 mL microwave tube was charged with 4-hydroxyaniline (0.97 g, 8.9 mmol), deuterium oxide (D2O, 10 mL) and concentrated deuterium chloride (DCl, 125 μL) to obtain a suspension. The tube was filled with a nitrogen atmosphere, sealed and irradiated in a microwave oven (CEM Discover SP) at 180°C for 2.5 hours under control, which is controlled by LCMS. Then the mixture was cooled to room temperature (28-32°C) and kept at this temperature for 18 hours. Volatiles were removed in vacuo to leave a brown residue, which was suspended in deuterium oxide (10 ml) in a 20 ml microwave tube. The tube was filled with nitrogen, sealed and irradiated in a microwave oven at 180°C for 5.5 hours. After cooling to room temperature (28-32°C), the mixture was kept at this temperature for 16 hours. Volatiles were removed in vacuo and the residue was purified by flash chromatography (10-60% ethyl acetate in petroleum ether) to give 4-amino( ^2H4 )phenol (0.50 g, 50% yield) as a brown solid.

1H NMR (500 MHz, DMSO _d6 ) δ 8.31 (s, 1H), 4.36 (s, 2H) ppm.

(1 S,2S,4R,8S,9S, 11S,12R,13S, 19S)-8-{2-[4-amino(2,3,5,6- ^2H4 )phenoxy]acetyl} -12.19 -difluoro-11 hydroxy-9,13-dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (6-I

D)

Step 2: Potassium hydroxide (45 mg, 0.80 _mmol ) was added to a mixture of 4-Amino( ^2H4 ) phenol (0.10 g, 0.88 mmol) in dimethyl sulfoxide (3 ml). After stirring at 28-32°C for 2 minutes, then stirring at 60°C, compound 2b (0.20 g, 0.40 mmol) was added to the mixture in one portion and stirred under nitrogen protection at 60°C for an hour . After cooling to room temperature, the mixture was directly purified by pre-HPLC (Method A) followed by pre-HPLC (Method B) to obtain

- 180 045579

6-II (10 mg, 4.4% yield) as a yellowish solid. Mass/charge at NMR: 562 (M+H)+.

1H NMR (400 MHz, DMSO _d6 ) δ 7.27 (d, J=10.1 Hz, 1H), 6.30 (dd, J=10.1, 1.7 Hz, 1H), 6.12 ( s, 1H), 5.745.45 (m, 2H), 5.03-4.93 (m, 1H), 4.82-4.58 (m, 4H), 4.27-4.14 (m, 1H), 3.33 (s, 1H), 2.70-2.53 (m, 1H), 2.312.20 (m, 1H), 2.14-1.93 (w, 2H), 1.86 -1.70 (m, 1H), 1.67-1.24 (m, 10H), 0.92-0.73 (m, 6H) ppm. ¹⁹ F NMR (376 MHz, DMSO _d6 ) δ -164.24, -165.05, -186.35 ppm. Anal. HPLC: 98.41%, dwell time: 7.34 minutes (method B).

Compound 6-1D is useful, for example, for analytical methods.

Example 88.

This example applies to the compounds in table. 2 and fig. 31.

(1 S,2S,4R,8S,9S, 11S,12R, 13S, 19S)-8-[2-(4-amino-3-methoxyphenoxy)acetyl]-12,19-difluoro-11hydroxy-9,13- dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one trifluoroacetate (6-II) nh ₂ ρ / -o ^{z 0=::} \

BUT. /x I V ] ι 1 n £а° n I I f I n

F

Following general procedure B, 2b (0.50 g, 0.92 mmol) was reacted with 4-amino-3methoxyphenol (0.32 g, 2.3 mmol) to produce carbonate (0.60 g, 1.8 mmol) in acetonitrile (20 ml) gave compound 6-II (0.25 g, 47% yield) as a white solid. Mass/charge at NMR: 588 (M+H)+.

1H NMR (400 MHz, DMSO _d6 ) δ 9.00 (s, 2H), 7.33-7.23 (m, 1H), 7.16-7.08 (m, 1H), 6.77-6 .68 (m, 1H), 6.52-6.41 (m, 1H), 6.35-6.27 (m, 1H), 6.12 (s, 1H), 5.74-5.51 (m, 2H), 5.31-5.11 (m, 2H), 4.98-4.68 (m, 3H), 4.28-4.15 (m, 1H), 3.90-3 .83 (m, 3H), 2.74-2.55 (m, 1H), 2.35-2.21 (m, 1H), 2.17-1.97 (m, 2H), 1.88 -1.75 (m, 1H), 1.67-1.28 (m, 10H), 0.93-0.78 (m, 6H) ppm. Anal. HPLC: >99%, dwell time: 7.68 and 7.72 minutes (method A).

Example 89.

This example applies to the compounds in table. 2 and fig. 31.

Preparation of compound 6-III.

(1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)-8-[2-(4-amino-3-fluorophenoxy)acetyl]-12,19-difluoro-11-hydroxy9,13- dimethyl-6-propyl-5,7-dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-16-one (6-III) °\ /=\ n °< γ ^Ν ” ² iJL ^o— jApTh ⁿ

F

Compound 2e (0.20 g, 0.37 mmol), 4-amino-3-fluorophenol (0.25 g, 2.0 mmol), potassium hydroxide (0.11 g, 2.0 mmol) and dimethyl sulfoxide (3 ml) at room temperature. The resulting mixture was stirred at 60°C for an hour under nitrogen protection until the reaction was complete, under the control of TLC and LCMS. After cooling to room temperature and membrane filtration, the reaction solution was directly purified by pre-HPLC (Method A) to obtain the title compound 6-III (40 mg, 19% yield) as a yellowish solid. Mass/charge at NMR: 576 (M+H)+.

1H NMR (500 MHz, MeOD _d4 ) δ 7.40-7.31 (m, 1H), 7.20 (td, J=9.1, 1.9 Hz, 1H), 6.91-6.84 (m, 1H), 6.806.76 (m, 1H), 6.40-6.30 (m, 2H), 5.57 (ddd, J=48.6, 9.7, 6.8 Hz, 1H ), 5.15 (d, J=18.1 Hz, 1H), 4.90-4.79 (m, 2H), 4.75 (t, J=4.3 Hz, 1H), 4.41 -4.28 (m, 1H), 2.78-2.57 (m, 1H), 2.40-2.12 (m, 3H), 1.98-1.39 (m, 11H), 1 .07-0.92 (m, 6H) ppm. Anal. HPLC: 100%, dwell time: 8.10 minutes (method A).

Example 90.

This example applies to the compounds in table. 2 and fig. 31.

(6S,8S,9S, 10R, 11S,13S,14S,17R)-17-(2-(4-aminophenoxy)acetyl)-11,17-dihydroxy-6,10,13trimethyl-6,7,8,9 ,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3-one (6-VI)

- 181 045579

To a solution of compound 2f (60 mg, 0.13 mmol) in N,N-dimethylformamide (3 ml) was added cesium carbonate (86 mg, 0.26 mmol) and N-Boc-4-aminophenol (28 mg, 0.13 mmol). The reaction mixture was stirred at room temperature for 18 hours, the process was monitored using LCMS. The mixture was diluted with ethyl acetate (10 ml). The organic solution was washed with water (10 ml), dried over sodium sulfate and concentrated. The white residue (50 mg, NMR mass/charge: 566 (M+H)+ was dissolved in dichloromethane (5 ml), trifluoroacetic acid (0.5 ml) was added to the solution. The reaction mixture was stirred at room temperature for 2 hours until complete removal of Boc according to LCMS data.Volatiles were removed in vacuo and the residue was purified by reverse phase flash chromatography (0-25% acetonitrile in water) to give 6-VI (10 mg, 7.5% yield) as white solid. Mass/charge at NMR: 466 (M+H) ⁺ .

^1H NMR (400 MHz, DMSO _d6 ) δ 7.32 (d, J=10.0 Hz, 1H), 6.61-6.58 (m, 2H), 6.51-6.47 (m, 2H), 6.19 (dd, J=10.0, 1.6 Hz, 1H), 5.82 (t, J=1.6 Hz, 1H), 5.39 (s, 1H), 5, 04-5.01 (m, 3H), 4.66 (d, J=3.2 Hz, 1H), 4.58 (d, J=18.0 Hz, 1H), 4.30 (d, J =2.4 Hz, 1H), 2.67-2.50 (m, 2H), 2.13-2.01 (m, 2H), 1.93-1.89 (m, 1H), 1, 67-1.61 (m, 3H), 1.45-1.30 (m, 5H), 1.01 (d, J=3.2 Hz, 3H), 0.95-0.71 (m, 5H) ppm.

Example 91.

This example demonstrates the general methods for synthesizing the payload linker intermediates in Table 1. 4.

This example applies to the compounds in table. 4 and fig. 31 and 33.

Synthesis of payload linkers (LP1-LP16) began with the preparation of L4 carbonates from the reactions of amines (4) or anilines (6) with protected Val-Cit-PAB-PNP (L2a or L2b) followed by N deprotection or the formation of L4 amides between anilines (6) using Boc or Fmoc protected Val-Cit-OH or Fmoc-Val-Ala-OH (L3a-c) followed by N-deprotection. Compounds L4 were directly linked to L9 or L10 to generate the final linker steroids LP1, LP2, LP3, LP13, LP14, LP15, and LP16. Compounds L4 were also coupled to Fmoc-D-Lys-COT L5 followed by de-Fmoc to produce compound L6, which underwent [3+2] cycloaddition with azido-cyclodextrin (7a) or azido sulfonates (7b or 7c) to form L8. Finally, coupling reactions of L8 with PEG4 acid or NHS ester (L9 or L10) were used to produce linker payloads LP5, LP8, LP10, and LP12.

General Procedure C for the synthesis of intermediate L4.

To a solution of payload 4 or 6 (1.0 equiv.) and Boc-vcPAB-PNP (1.1 equiv.) in N,Ndimethylformamide (1 ml per 10 mg payload) was added HOBt (1.0 equiv.) and DIPEA (2.0 equiv.) at room temperature. The resulting mixture was stirred at room temperature (18-30°C) overnight until the payload was consumed under LCMS monitoring. After membrane filtration, the reaction solution was directly purified by pre-HPLC to obtain Boc-L4 (52% yield) as a white solid, which was dissolved in dichloromethane (0.6 ml per 1 mg of Boc-L4 compound). Trifluoroacetic acid (0.2 ml per 1 mg of Boc-L4 compound) was added dropwise to the resulting solution at 0°C. The mixture was stirred at room temperature (18-30°C) for an hour until Boe was removed under LCMS control. Volatiles were removed in vacuo to obtain compound L4, which was then used for the next step without further purification.

General Procedure D for synthesis of intermediate L4:

Fmoc-vcPAB-PNP (1.1 equiv), HOBt (1.5 equiv.) and DIPEA (2.0 equiv.) at room temperature. The mixture was stirred at room temperature (18-30°C) for 3 hours until completely consumed under LCMS control. Piperidine (0.03 ml per 10 mg payload) was added to the reaction mixture and the mixture was stirred at room temperature (18-30°C) for an hour until Fmoc was removed under LCMS control. After membrane filtration, the reaction solution was directly purified by reverse phase flash chromatography or pre-prep-HPLC to obtain compound L4.

General procedure E for the synthesis of intermediate L4.

To a solution of Boc-Val-Ala-OH or Boc-Val-Cit-OH (1.0 equiv.) in dichloromethane (0.2 ml per 10 mg of peptide) was added DIPEA (2.0 equiv.) and HATU (1. 2 equiv.) at 20-25°C. The mixture was stirred at 20-25°C for 30 minutes, after which aniline (1.1 equiv.) was added, the mixture was stirred for 16 until the peptide was completely consumed under LCMS control. Triacetic acid (0.05 ml per 10 mg of peptide) was then added to the reaction mixture. The mixture was stirred at 20-25°C for another hour. Volatiles were removed under reduced pressure and the residue was directly purified by pre-HPLC (Method B) to obtain compound L4. General Procedure F for synthesis of intermediate L4:

DIPEA (3.0 equiv.) and HATU (1.4 equiv.) were added to a solution of Fmoc-Val-Ala-OH (1.2 equiv.) in N,N-dimethylformamide (0.2 ml per 10 mg of peptide). at 20-25°C. The mixture was stirred at 20-25°C for 5 minutes, after which aniline (1.0 equiv.) was added, the resulting mixture was further stirred for 2 hours until the peptide was completely consumed under LCMS control. Piperidine (5.0 equiv) was then added to the reaction mixture. The mixture was stirred at 20-25°C for 2 hours. After membrane filtration, the reaction solution was directly purified by inverse flash chromatography.

- 182 045579 phase (0-100% acetonitrile in aqueous ammonium bicarbonate (10 mmol) or pre-HPLC method (method B) to obtain compound L4.

Example 92.

This example applies to the compounds in table. 4 and fig. 33. Preparation of compound L4a, VA-R

6-VI.

(2S)-2-αmuno-N-[( 1S)-1-[(4-{2-[( 1 S,2S,4R,6R,8S,9S, 11S,12S,13R)-11 -hydroxy- 9,13-dimethyl-16-oxos l η e l l ^2.9 l ^4.8 l'^; ,1 ⁸ 1......... 1 l 1 l o τ _Ιττ ι e

6-propyl-5,7-dioxapentacyclo[10.8.0.0.0.0 ^{13, 8} ]icosa-14,17-dien-8-yl]-2oxoethoxy}phenyl)carbamoyl]ethyl]-3-methylbutanamide

Following general procedure E (65% yield) or F (53% yield) compound L4a was obtained from R-6-YI as a white solid. Mass/charge at NMR: 692 (M+H)+.

^1H NMR (500 MHz, DMSO _d6 ) δ 9.95 (d, J=8.2 Hz, 1H), 8.19-8.09 (m, 1H), 7.54-7.47 (m, 2H), 7.33 (d, J=10.1 Hz, 1H), 6.85 (d, J=9.0 Hz, 2H), 6.22-6.13 (m, 1H), 5, 93 (s, 1H), 5.14-5.04 (m, 1H), 4.86-4.77 (m, 2H), 4.75 (d, J=4.2 Hz, 1H), 4 .70 (t, J=4.3 Hz, 1H), 4.48-4.38 (m, 1H), 4.34 (s, 1H), 3.01 (t, J=5.0 Hz, 1H), 2.58-2.52 (m, 1H), 2.33-2.25 (m, 1H), 2.13-2.06 (m, 1H), 2.03-2.00 ( m, 1H), 1.95-1.89 (m, 1H), 1.88-1.84 (m, 2H), 1.631.53 (m, 5H), 1.45-1.33 (m, 6H), 1.32-1.26 (m, 3H), 1.06-0.93 (m, 2H), 0.92-0.82 (m, 10H), 0.80-0.75 ( m, 3H) ppm.

4b.

Example 93.

This example applies to the compounds in table. 4 and fig. 33. Preparation of compound L4b, vcPAB{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-difluoro-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}carbamate

Following General Procedure D, vcPAB-4b (0.13 g, 73% yield) was obtained from 4b (93 mg, 0.20 mmol) after purification by reverse phase flash chromatography (50-80% acetonitrile in aqueous bicarbonate ammonium (10 mmol)) in the form of a white solid. Mass/charge at NMR: 871 (M+H) ⁺ .

Example 94.

This example applies to the compounds in table. 4 and fig. 33. Preparation of compound L4c, VA-6-I.

(2S)-2-amino-N-[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro -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]-2oxoethoxy}phenyl)carbamoyl]ethyl]-3-methylbutanamide

Following General Procedure E, crude L4c (0.69 g, 72% yield in 2 steps) was obtained from 6-I (0.50 g, 0.90 mmol) with Boc-Val-Ala-OH without purification. as a yellow colored oil which was used directly for the next step. Mass/charge at NMR: 728 (M+H) ⁺ .

Example 95.

This example applies to the compounds in table. 4 and fig. 33. Preparation of compound L4d, VCPAB-6-I.

{4-[(2S)-2-[(2S)-2-amino-3-methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-(4{2-[(1S,2S,4R,8S ,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7- 183 045579 dioxapentacyclo[10.8.0.0 ^2.9 .0 ^4.8 .0 ^13.18 ]icosa-14,17-dien-8-yl]-2-oxoethoxy}phenyl)carbamate

Following general procedure E, compound 6-I (87 mg, 0.15 mmol) gave compound L4d (80 mg, 64% yield) as a white solid after purification by pre-HPLC (Method B). Mass/charge at NMR: 963 (M+H) ⁺ .

^1H NMR (500 MHz, DMSOd6) δ 10.22 (s, 1H), 9.57 (s, 1H), 8.69 (d,J=7.5 Hz, 1H), 8.08 (s, 3H), 7.61 (d, J=6.8 Hz, 2H), 7.36 (d, J=6.8 Hz, 3H), 7.27 (d, J=8.0 Hz, 1H) , 7.22-7.0 (m, 1H), 6.84 (d, J=7.2 Hz, 2H), 6.30 (dd, J=8.0 Hz, J=1.6 Hz, 1H), 6.11 (s, 1H), 6.10-6.0 (m, 1H), 5.72-5.55 (m, 1H), 5.52 (s, 1H), 5.48 (s, 1H), 5.16-5.05 (m, 3H), 4.88-4.80 (m, 1H), 4.80-4.76 (m, 1H), 4.75-4 .70 (m, 1H), 4.55-4.48 (m, 1H), 4.25-4.20 (m, 1H), 3.703.60 (m, 1H), 3.12-2.90 (m, 2H), 2.70-2.55 (m, 1H), 2.40-2.20 (m, 1H), 2.15-2.0 (m, 3H), 1.86-1 .75 (m, 1H), 1.75-1.65 (m, 1H), 1.64-1.54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 4H), 0.97-0.91 (m, 5H), 0.90-0.85 (m, 4H), 0.85-0.80 (m, 3H) ppm.

Example 96.

This example applies to the compounds in table. 4 and fig. 33. Preparation of compound L4e, VA-6II.

(2S)-2-amino-N-[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro -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]-2oxoethoxy}-2-methoxyphenyl)carbamoyl]ethyl]-3-methylbutanamide

Following General Procedure F, compound 6-III (0.10 g, 0.17 mmol) gave crude compound L4e (0.12 g, 82% yield in 2 steps), which was then used for the next step without further purification. Mass/charge at NMR: 758 (M+H) ⁺ .

Example 97.

This example applies to the compounds in table. 4 and fig. 33. Preparation of compound L4f, VA-6III.

(2S)-2-αmino-N-[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro -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]-2oxoethoxy}-2-fluorophenyl)carbamoyl]ethyl]-3-methylbutanamide

Following General Procedure F, compound L4f (95 mg, 0.17 mmol) gave crude compound L4f (0.10 g, 66% yield in 2 steps), which was then used for the next step without further purification. Mass/charge at NMR: 746 (M+H)+.

Example 98.

This example applies to the compounds in table. 4 and fig. 33. Synthesis of intermediate payloads L6.

General procedure: To a solution of compound L5 (1.2 equiv.) in N,N-dimethylformamide (0.2 ml per 10 mg L5) was added HATU (1.4 equiv.) and DIPEA (3 equiv.) at room temperature. The mixture was stirred at room temperature for 5 minutes, after which compound L4 (1.0 equiv.) was added. The reaction mixture was then stirred at room temperature for 2 hours until compound L4 was completely consumed under LCMS monitoring. After filtering through a membrane, the reaction solution is

- 184 045579 direct purified by pre-HPLC to obtain cyclooctyne L6.

Example 99: General Procedure H for the preparation of intermediates 8.

To a solution of L6 in N,N-dimethylformamide (0.5 ml per 10 mg of compound L6) was added an azido compound (L7a(CD-N3), L7b(N3-PEG4-sulfonate) or L7c (N3-2-sulfonate), 1.5 equiv. vs L6) and DIPEA (0.1 ml per 10 mg L6) at room temperature. After stirring at 30°C for 24 hours, most of the starting material was absorbed and the reaction was monitored by LCMS. The reaction mixture was directly purified by pre-HPLC to obtain compound L8 as a white solid.

Example 100.

Preparation of compound L8a, aCDCCK-vcPAB-4b.

{4-[(2S)-2-[(2S)-2-[(2R)-2-amino-b-{2-[(1-{[31,32,33,34,35,3b,37 ,38,39,40,41,42-dodecahydroxy10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29dodecaoxaheptacyclo[ 26.2.2.2 ³ ' ⁶ .2 ⁸ ' ¹¹ .2 ¹³ ' 16 .2 ¹⁸ ' ²¹ .2 ²³ ' ²⁶ ]dotetracotan-5-yl]methyl}-1H,4H, ^5H ,6H,7H,8H,9Hcyclooct[ d] [1,2,3]triazol-4-yl)oxy] acetamido}hexanamido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methyl N-{2-[(1S,2S,4R, 8S ,9S,11S,12R,13S,19S)-12,19-difluoro-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-oxoethyl}carbamate (L8a)

He

Following General Procedure H to prepare L6a (0.12 g, 0.10 mmol) with L7a afforded L8a (0.11 g, 51% yield) as a white solid. Mass/charge at NMR: 1081 (M/2+H)+.

^1H NMR (400 MHz, DMSO _d6 ) δ 10.05 (s, 1H), 8.30-7.80 (m, 3H), 7.80-7.55 (m, 2H), 7.50- 7.40 (m, 1H), 7.40-7.25 (m, 3H), 6.30 (d, J=12.5 Hz, 1H), 6.11 (s, 1H), 6.0 (s, 1H), 5.80-5.35 (m, 16H), 5.25-5.05 (m, 1H), 4.97 (s, 2H), 4.90-4.50 (m , 13H), 4.50-4.00 (m, 5H), 3.95-3.55 (m, 22H), 3.30-3.20 (m, 8H), 3.20-3.00 (m, 4H), 3.00-2.85 (m, 5H), 2.25-2.20 (m, 2H), 2.10-1.95 (m, 4H), 1.80-1 .00 (m, 30H), 1.00-0.90 (m, 4H), 0.90-0.80 (m, 14H) ppm.

Example 101.

Preparation of compound L8d, aCDCCK-VA-2168.

(2R)-2-amino-N-[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S, 19S)-12,19-difluoro-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}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-6-{2-[(1{[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 ' ^p 2 ¹³ ' ¹⁶ .2 ¹⁸ ' ²¹ .2 ²³ ' ²⁶ ]dotetracotan-5ul]methyl}-1H,4H,5H,6H,7H,8H,9H- cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamido}hexanamide (L8d)

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Following General Procedure H, L6b (60 mg, 59 μmol) with L7a gave L8d (40 mg, 34% yield) as a white solid. Mass/charge at NMR: 1009.5 (M/2+H)+.

Example 102.

Preparation of compound L8f, aCDCCK-vcPAB-6-I.

{4-[(2S)-2-[(2S)-2-[(2R)-2-αmino-6-{2-[(1-{[31,32,33,34,35,36,37 ,38,39,40,41,42-dodecahydroxy10,15,20,25,30-pentakis(hydroxymethyl)-2,4,7,9,12,14,17,19,22,24,27,29dodecaoxaheptacyclo[ 26.2.2.2 ^3.6 .2 ^8.11 .2 ^13.16 .2 ^18.21 .2 ^23.26 ]dotetracotan-5-yl]methyl}-1H,4H,5H,6H,7H,8H,9Hcycloocta[ d] [1,2,3]triazol-4-yl)oxy] acetamido}hexanamido]-3-methylbutanamido]-5(carbamoylamino)pentanamido]phenyl}methyl N-(4-{2-[(1S,2S, 4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro11-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-dien8-yl]-2-oxoethoxy}phenyl)carbamate

Following General Procedure H, compound L6c (0.10 g, 80 µmol) with compound L7a gave compound L8f (0.11 g, 58% yield) as a white solid. Mass/charge at NMR: 751 (M/3+H) ⁺ .

Example 103.

Preparation of linkers with payload LP101 - LP116. Preparation of compound LP1: L6a (COTdLys-vcPAB-4b).

{4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]hexanamido]-3methylbutanamido]- 5-(carbamoylamino)pentanamido]phenyl}methyl N-{2[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl -16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ² ' ⁹ -12' ⁸ -12,' ¹⁸ ]icos-14,17-dien-8-yl]-2-oxoethyl}carbamate

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Following General Procedure G for L4b (0.20 g, 0.23 mmol), L6a (0.12 g, 45% yield) was obtained as a white solid by pre-HPLC (Method B). Mass/charge at NMR: 1385 (M+H)+.

1H NMR (400 MHz, MeODd4) δ 7.65-7.55 (m, 2H), 7.40-7.26 (m, 3H), 6.39-6.27 (m, 2H), 5, 65-5.45 (m, 1H), 5.13-5.01 (m, 2H), 4.71-4.50 (m, 2H), 4.40-4.14 (m, 4H), 4.11-3.82 (m, 3H), 3.46-3.39 (m, 1H), 3.29-3.09 (m, 4H), 2.76-2.54 (m, 1H ), 2.41-2.10 (m, 7H), 2.09-1.99 (m, 1H), 1.96-1.80 (m, 5H), 1.78-1.21 (m , 23H), 1,060.82 (m, 12H) ppm.

Example 104.

Preparation of compound LP102: L6b (COT-dLys-VA-6-I).

(2R)-2-amino-6-[2-(cyclooct-2-yne-1 -yloxy)acetamido]-N-[( 1S)-1-{[(1 S)-1 -[(4- { 2[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-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]-2oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]hexanamide

Following General Procedure G for L4c (0.20 g, 0.23 mmol), L6b (0.21 g, 46% yield) was obtained as a white solid by pre-HPLC (Method B). Mass/charge at NMR: 1021.5 (M+H) ⁺ .

¹ H NMR (400 MHz, MeODd4) δ 7.33-7.60 (m, 3H), 6.87-6.91 (m, 2H), 6.32-6.37 (m, 2H), 5 .47-5.65 (m, 1H), 5.07-5.30 (m, 1H), 4.72-4.86 (m, 3H), 4.34-4.51 (m, 3H) , 3.83-4.20 (m, 3H), 3.33-3.49 (m, 1H), 3.14-3.27 (m, 3H), 2.59-2.75 (m, 1H), 1.31-2.39 (m, 33H), 0.93-1.05 (m, 12H) ppm.

Example 105.

Preparation of compound LP103: L6c (COT-dLys-vcPAB-6-I).

{4-[(2S)-2-[(2S)-2-[(2R)-2-amino-6-[2-(cyclooct-2-yn-1-yloxy)acetamido]hexanamido]-3methylbutanamido]- 5-(carbamoylamino)pentanamido]phenyl}methyl N-(4-{2[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-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} phenyl)carbamate

Following General Procedure G for L4d (0.14 g, 0.15 mmol), L6c (0.10 g, 57% yield) was obtained as a white solid by pre-HPLC (Method B). Mass/charge at NMR: 1255.5 (M+H) ⁺ .

1H NMR (400 MHz, MeODd4) δ 7.61 (d, J=8.4 Hz, 1H), 7.32-7.39 (m, 4H), 6.84-6.88 (m, 2H) , 6.31-6.36 (m, 2H), 5.43-5.63 (m, 1H), 5.05-5.16 (m, 3H), 4.71-4.83 (w, 1H), 4.50-4.54 (m, 1H), 4.18-4.33 (m, 3H), 3.002.85 (m, 2H), 3.40-3.51 (m, 1H) , 3.00-3.29 (m, 6H), 1.31-2.35 (m, 34H), 1.29 (t, J=7.2 Hz, 2H), 0.93-1.02 (m, 12H) ppm.

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Example 106.

General Procedure I for payloads LP104 - LP116.

HATU (1.3 equiv.) and DIPEA (5.0 equiv.) were added to a solution of PEG ₄ -acid L9 (1.2-1.3 equiv.) in N,N-dimethylformamide (1 ml per 10 mg L9). at room temperature. The mixture was stirred at room temperature (19°C) for half an hour, after which a solution of compound L4 or L8 (1.0 equiv.) in N,N-dimethylformamide (0.6 mg per 10 mg L4 or L8) was added. The resulting mixture was stirred at room temperature for an additional 2 hours until compound L4 or L8 was consumed under LCMS monitoring. After membrane filtration, the filtrate was directly purified by pre-HPLC to obtain compound L1. (L9a: BCN-PEG ₄ acid, L9b: DIBAC-PEG ₄ acid, L9c: MAL-PEG 4 acid) General Procedure J for LP104 - LP116 payloads

To a solution of compound L4 or L8 (1.0 eq.) in DMF (1 ml per 50 mg) was added compound DIBACPEG4-NHS L10b (1.1-1.2 eq.) and DIPEA (5.0 eq.) at room temperature. The reaction mixture was stirred at room temperature for 3 hours under LCMS control. The reaction mixture was directly purified by preparative HPLC (Method B) to obtain compound L1.

Example 107.

Preparation of compound LP104: L11a (DIBAC-PEG4-aCDCCK-vcPAB-4b.

{4-[(2S)-2-[(2S)-2-[(2R)-2-[ 1 -(4-{2-azatricyclo[ 10.4.0.0 ⁴ ' ⁹ ]hexadeca-1 (12),4 (9),5,7,13,15-hexaen-10in-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-6-{2-[( 1 { [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 ]dotetracotan-5yl]methyl} -1H,4H,5H,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamido}hexanamido]-3methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl }methyl N-{2[(1 S,2S,4R,8S,9S, 11S,12R, 13S,19S)12,19-difluoro-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}carbamate

He

Following general procedure I, compound L8a (0.10 g, 46 μmol) with compound L9b gave compound L1a (26 mg, 22% yield) as a white solid. Mass/charge at NMR: 1349 (M/2+H)+.

^1H NMR (500 MHz, DMSO _d6 ) δ 9.71 (s, 1H), 8.30-8.00 (m, 3H), 8.00-7.74 (m, 2H), 7.70- 7.58 (m, 5H), 7.52-7.20 (m, 12H), 6.35-6.20 (m, 2H), 6.15-5.85 (m, 3H), 5, 80-5.35 (m, 18H), 5.25-4.90 (m, 6H), 4.90-4.50 (m, 14H), 4.40-4.25 (m, 4H), 4.25-4.10 (m, 3H), 4.10-3.95 (m, 2H), 3.95-3.55 (m, 22H), 3.55-3.40 (m, 22H ), 3.203.00 (m, 6H), 3.00-2.85 (m, 3H), 2.65-2.55 (m, 1H), 2.25-2.20 (m, 4H), 2.10-1.95 (m, 6H), 1.80-1.70 (m, 5H), 1.70-1.50 (m, 10H), 1.50-1.45 (m, 9H ), 0.90-0.80 (m, 14H) ppm. Anal. HPLC: >99%, dwell time: 6.23 minutes (method B).

Example 108.

Preparation of compound LP105: L11b (BCN-PEG4-aCDCCK-vcPAB-4b (1R,8S,9S)-bicyclo[6.1.0]non4-in-9-ylmethyl N-(14-{[(1 R)-l- {[(1S)-1-{[(1 S)-4-(carbamoylamino)-1 -[(4- {[({2[(1S,2S,4R,8S,9S,11S,12R,13S, 19S)-12,19-difluoro-11-hydroxy-9,13-dimethyl-16-oxo-6-propyl-5,7dioxapentacyclo[10.8.0.0 ² ' ⁹ .0 ⁴ ' ⁸ .0 ¹³ ' ¹⁸ ]icos- 14,17-dien-8-yl]-2oxoethyl}carbamoyl)oxy]methyl}phenyl)carbamoyl]butyl]carbamoyl}-2-methylpropyl]carbamoyl}-5-{2[(1-{[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 ]dotetrakotan-5yl]methyl}-1H,4H,5H ,6H,7H,8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamido}pentyl]carbamoyl}3,6,9,12-tetraoxatetradecane-1-yl)carbamate

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Following general procedure I, compound L8a (22 mg, 10 μmol) with BCN-PEG ₄ -acid L9a gave compound L1b (10 mg, 38% yield) as a white solid. Mass/charge at NMR: 1293 (M/2+H)+.

¹ H NMR (500 MHz, DMSOd6) δ 9.68 (s, 1H), 8.14-7.08 (m, 11H), 6.30 (d, J=10.0 Hz, 1H), 6, 11 (s, 1H), 5.99 (s, 1H), 5.67-5.31 (m, 15H), 5.21-3.33 (m, 61H), 3.13-2.60 ( m, 22H), 2.30-1.96 (m, 46H), 0.95-0.80 (m, 17H) ppm. Anal. HPLC: dwell time: 7.31 minutes (48%) and 7.41 (52%) (method B).

Example 109.

Preparation of compound LP108: L11e (DIBAC-PEG4-aCDCCK-VA-6-I.

-(4-{2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1 (12),4(9),5,7,13,15-hexaen-10-in-2-yl} -4oxobutanαamido)- N-[(1R)-1-{[(1S)-1-{[(1S)-1-[(4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S, 19S)-12,19-difluoro-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} phenyl)carbamoyl]ethyl]carbamoyl} -2-methylpropyl]carbamoyl} -5- {2-[( 1 {[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 ' 2 ⁶ ]dotetracotan-5ul]methyl}-1H,4H,5H,6H,7H, 8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamido}pentyl]-3,6,9,12tetraoxapentadecane-15-amide

Following General Procedure J, L8d (19 mg, 9.4 μmol) with DIBAC-PEG4-NHS L10b gave L1e (7.0 mg, 29% yield) as a white solid. Mass/charge at NMR: 1276.8 (M/2+H) ⁺ .

^1H NMR (400 MHz, DMSOd6) δ 9.80-9.47 (m, 1H), 8.23-7.91 (m, 3H), 7.83-7.11 (m, 13H), 6 .87-6.66 (m, 2H), 6.32-6.11 (m, 2H), 5.85-5.23 (m, 14H), 5.14-5.01 (m, 3H) , 4.86-3.99 (m, 19H), 3.85-3.40 (m, 38H), 3.272.87 (m, 13H), 2.76-2.55 (m, 3H), 2 .33-2.20 (m, 4H), 2.12-1.91 (m, 6H), 1.83-1.72 (m, 4H), 1.59-0.98 (m, 31H) , 0.89-0.84 (m, 12H) ppm. Anal. HPLC: 100%, dwell time: 7.76 minutes (method B).

Example 110.

Preparation of compound LP110: L11g (DIBAC-PEG4-aCDCCK-vcPAB-6-I.

{4-[(2S)-2-[(2S)-2-[(2R)-2-[ 1 -(4-{2-azatricyclo[ 10.4.0.0 ⁴ , ⁹ ]hexadeca-1 (12),4 (9),5,7,13,15-hexaene-10

- 189 045579 in-2-yl}-4-oxobutanamido)-3,6,9,12-tetraoxapentadecane-15-amido]-6-{2-[(1{[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 .22 ^3.26 ]dotetracotan-5yl]methyl}-1H,4H,5H,6H,7H, 8H,9H-cycloocta[d][1,2,3]triazol-4-yl)oxy]acetamido}hexanamido]-3methylbutanamido]-5-(carbamoylamino)pentanamido]phenyl}methyl N-(4-12[(1S ,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro-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}phenyl)carbamate

Following general procedure I, compound L8d (0.10 g, 44 μmol) with DIBAC-PEG ₄ -acid L9b gave compound L1g (29 mg, 24% yield) as a white solid. Mass/charge at NMR: 1394 (M/2+H)+.

^1H NMR (500 MHz, DMSOd4) δ 9.67 (s, 1H), 9.56 (s, 1H), 8.20-8.05 (m, 2H), 7.85-7.70 (m , 2H), 7.70-7.60 (m, 4H), 7.50-7.25 (m, 12H), 6.90-6.80 (m, 2H), 6.30 (d, J =12.5 Hz, 1H), 6.11 (s, 1H), 6.0 (s, 1H), 5.80-5.35 (m, 16H), 5.25-5.00 (m, 6H), 4.90-4.65 (m, 10H), 4.65-4.45 (m, 4H), 4.40-4.00 (m, 6H), 3.95-3.55 ( m, 22H), 3.503.30 (m, 22H), 3.20-2.85 (m, 12H), 2.65-2.55 (m, 2H), 2.45-2.35 (m, 2H), 2.35-2.20 (m, 3H), 2.15-1.95 (m.5H), 1.90-1.70 (m, 4H), 1.70-1.50 ( m, 10H), 1.50-1.00 (m, 18H), 0.90-0.80 (m, 12H) ppm. Anal. HPLC, dwell time: 7.93 (82%) and 8.02 (18%) minutes (method B).

Example 111.

Obtaining connection LP112: (DIBAC-PEG4_ _a CDCCK-vcPAB-4b.

{4-[(2S)-2-[(2S)-2-[1-(4-{2-azαtricyclo[10.4.0.0 ^4,9 ]hexadeca-1(12),4(9),5,7 ,13,15-hexαene-10-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,12R,13S,19S)-12,19-difluoro-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-oxoethyl}carbamate

Following General Procedure J, from compound L4b (43 mg, 50 μmol) with DIBAC-suc-PEG4-acid (L9b) the title compound L12 (16 mg, 23% yield) was obtained as a white solid after purification by prep. HPLC (method B). Mass/charge at NMR: 1406 (M+H)+.

1H NMR (DMSOd6, 500 MHz) δ 9.99 (s, 1H), 8.11 (d, J=7.5 Hz, 1H), 7.88 (d, J=8.5 Hz, 1H), 7.80-7.75 (m, 1H), 7.70-7.66 (m, 1H), 7.65-7.60 (m, 3H), 7.53-7.33 (m, 6H ), 7.33-7.28 (m, 3H), 6.30 (dd, J=10.0 Hz and 1.5 Hz, 1H), 6.11 (s, 1H), 6.10-6 .00 (m, 1H), 5.72-5.55 (m, 2H), 5.41 (s, 2H), 5.05-5.01 (m, 1H), 4.97 (s, 2H ), 4.80-4.72 (m, 1H), 4.60-4.58 (m, 1H), 4.43-4.33 (m, 1H), 4.25-4.10 (m , 3H), 3.88-3.80 (m, 1H), 3.65-3.55 (m, 3H), 3.503.40 (m, 12H), 3.30-3.25 (m, 2H ), 3.12-2.90 (m, 4H), 2.70-2.55 (m, 2H), 2.48-2.35 (m, 2H), 2.30-2.20 (m , 2H), 2.15-1.95 (m, 4H), 1.86-1.65 (m, 3H), 1.64-1.54 (m, 5H), 1.49 (s, 4H ), 1.46-1.34 (m, 5H), 0.90-0.80 (m, 12H) ppm. Anal. HPLC: 100%, holding time: 7.40 minutes (method B).

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Example 112.

Obtaining connection LP113: MAL-PEG4-VA-R-11-5.

-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-N-[( 1S)-1-{[(1 S)-1-[(4- {2[( 1S,2S,4R,6R,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]-2oxoethoxy}phenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12-tetraoxapentadecan-15-amide

Following General Procedure J, L4a (20 mg, 25 μmol) with MAL-PEG4-NHS L10c gave LP113 (7 mg, 27% yield) as a white solid.

Mass/charge at NMR: 1119 (M+H)+.

1H NMR (400 MHz, DMSOd6) δ 9.89-9.60 (m, 1H), 8.51-6.73 (m, 10H), 6.18 (dd, J=10.1, 1.7 Hz, 1H), 5.93 (s, 1H), 5.17-4.05 (m, 9H), 4.02-3.52 (m, 13H), 2.71-2.54 (m, 1H), 2.46-2.20 (m, 5H), 2.15-1.77 (m, 5H), 1.631.53 (m, 5H), 1.47-1.20 (m, 9H) , 1.10-0.94 (m, 2H), 0.95-0.65 (m, 12H) ppm. Anal. HPLC: 100%, dwell time: 7.46 minutes (method B).

Example 113.

Obtaining connection LP114: L11j (DIBAC-PEG4-VA-6-H.

-(4- {2-azatricyclo[10.4.0.0 ^4,9 ]hexadeca-1 (16),4(9),5,7,12,14-hexaen-10-in-2-yl}-4oxobutanamido)- N-[( 1S)-1-{[(1 S)-1 -[(4- {2-[(1 S,2S,4R,8S,9S, 11S,12R,13S,19S)-12,19 -difluoro-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}-2-methoxyphenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12tetraoxapentadecan-15-amide

F

Following general procedure I, compound L4e (40 mg, 47 μmol) with DIBAC-suc-PEG4 acid L9b gave compound L1j (25 mg, 41% yield) as a white solid. Mass/charge at NMR: 1293 (M+H) ⁺ .

1H NMR (400 MHz, DMSO _d6 ) δ 8.98-8.86 (m, 1H), 8.37-8.30 (m, 1H), 7.94-7.88 (m, 1H), 7 .87-7.72 (m, 2H), 7.70-7.57 (m, 2H), 7.52-7.42 (m, 3H), 7.41-7.22 (m, 4H) , 6.65-6.59 (m, 1H), 6.44-6.34 (m, 1H), 6.33-6.27 (m, 1H), 6.12 (s, 1H), 5 .77-5.49 (m, 2H), 5.18-5.11 (m, 1H), 5.07-4.98 (m, 1H), 4.91-4.70 (m, 3H) , 4.54-4.43 (m, 1H), 4.29-4.16 (m, 2H), 3.79 (s, 3H), 3.65-3.53 (m, 3H), 3 .51-3.38 (m, 12H), 3.30-3.22 (m, 2H), 3.13-3.03 (m, 2H), 2.72-2.54 (m, 2H) , 2.47-2.18 (m, 4H), 2.13-1.91 (m, 4H), 1.85-1.72 (m, 2H), 1.64-1.55 (m, 3H), 1.521.33 (m, 6H), 1.31-1.23 (m, 3H), 0.99-0.77 (m, 13H) ppm. Anal. HPLC: 99%, dwell time: 9.18 and 9.22 minutes (method B).

Example 114.

Preparation of compound LP115: L11k-(DIBAC-PEG4-VA-6-III.

-(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)-1 -[(4- {2-[(1 S,2S,4R,8S,9S, 11S,12R,13S,19S)-12,19 -difluoro-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}-2-fluorophenyl)carbamoyl]ethyl]carbamoyl}-2-methylpropyl]-3,6,9,12-tetraoxapentadecan15-amide

F

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Following general procedure I, compound L4f (82 mg, 0.11 mmol) with DIBAC-suc-PEG4 acid L9b gave compound L1k (50 mg, 35% yield) as a white solid. Mass/charge at NMR: 1280 (M+H)+.

^1H NMR (500 MHz, DMSO _d6 ) δ 9.54 (s, 1H), 8.41-8.15 (m, 1H), 8.01-7.17 (m, 12H), 6.90 ( d, J=10.8 Hz, 1H), 6.73 (s, 1H), 6.31 (d, J=9.9 Hz, 1H), 6.12 (s, 1H), 5.77- 5.46 (m, 2H), 5.28-4.65 (m, 5H), 4.58-4.42 (m, 1H), 4.29-4.11 (m, 2H), 3, 71-3.43 (m, 15H), 3.29 (s, 2H), 3.08 (s, 2H), 2.71-2.54 (m, 2H), 2.47-2.17 ( m, 4H), 2.161.88 (m, 4H), 1.88-1.69 (m, 2H), 1.69-1.19 (m, 13H), 0.95-0.80 (m, 12H) ppm. ¹⁹ F NMR (376 MHz, DMSO) δ 121.11 and -121.92, -165.13 and -165.14, -186.38 and -186.40 ppm. Anal. HPLC: >99%, dwell time: 8.32 minutes (method B).

Example 115.

Preparation of LP116 compound: L11k-(DIBAC-PEG4-VC-PAB-4b).

{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- (4-{2-[(1S,2S,4R,8S,9S,11S,12R,13S,19S)-12,19-difluoro11-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-dien8-yl]-2-oxoethoxy}phenyl)carbamate

Following General Procedure I, from compound L4k (58 mg, 60 μmol) with DIBAC-suc-PEG4 acid L9b, the title compound L1v (20 mg, 22% yield) was obtained as a white solid. Mass/charge at NMR: 1499 (M+H) ⁺ .

1H NMR (400 MHz, DMSOd6) δ 10.02 (s, 1H), 9.59 (s, 1H), 8.14 (d, J=7.6 Hz, 1H), 7.88 (d, J =8.8 Hz, 1H), 7.80-7.75 (m, 1H), 7.70-7.66 (m, 1H), 7.65-7.60 (m, 3H), 7. 53-7.45 (m, 3H), 7.40-7.28 (m, 7H), 6.84 (d, J=9.2 Hz, 2H), 6.30 (dd, J=10, 4 Hz, J=1.6 Hz, 1H), 6.11 (s, 1H), 6.10-6.0 (m, 1H), 5.72-5.55 (m, 1H), 5, 52 (s, 1H), 5.43 (s, 2H), 5.16-5.05 (m, 4H), 4.88-4.70 (m, 3H), 4.43-4.33 ( m, 1H), 4.25-4.20 (m, 2H), 3.65-3.55 (m, 3H), 3.503.40 (m, 12H), 3.30-3.25 (m, 2H), 3.12-2.90 (m, 4H), 2.70-2.55 (m, 2H), 2.48-2.43 (m, 1H), 2.40-2.35 ( m, 1H), 2.30-2.20 (m, 2H), 2.15-1.95 (m, 4H), 1.86-1.75 (m, 2H), 1.64-1, 54 (m, 5H), 1.49 (s, 4H), 1.46-1.34 (m, 4H), 1.23 (s, 2H), 0.90-0.80 (m, 12H) chnm. Anal. HPLC: 100%, dwell time: 7.83 minutes (method B).

Example 116.

Conjugation of ALS conjugates.

Conjugation of steroid-antibody conjugates is indicated in FIG. 33. In one example, site-specific conjugates were prepared using a microbial transglutaminase (MTG EU 2.3.2.13, Zedira, Darmstadt, Germany) (hereinafter MTG-mediated) two-step conjugation of a mutated antibody N297Q or N297D. In the first step, the mutated antibody was functionalized with azido-PEG3-amine through an MTG-mediated enzymatic reaction. See, for example, PCT International Patent Application No. PCT/US17/19537, filed February 24, 2017, incorporated herein by reference in its entirety for all purposes. In the second step, an alkyne-functionalized payload linker was attached to the azido-functionalized antibody via a [2+3] 1,3-dipolar cycloaddition reaction (Figure 33 shows a DIBAC-functionalized payload linker (LP112) conjugated to azido-functionalized antibody prepared by [2+3] cyclization, a process that produced site-specific and stoichiometric conjugates in approximately 50-80% yield.

Preparation of the steroid-antibody conjugate in FIG. 33.

This example generally demonstrates a method for site-specific conjugation of a payload to an antibody or antigen-binding fragment thereof. This example refers to FIG. 33.

The following example demonstrates the method for preparing the azido-functionalized antibody-drug conjugate shown in Table. 16.

An aglycosylated human IgG1 isotype antibody in BupH™ (pH 7.6-7.8) was mixed with >200 molar equivalents of azido-dPEG3-amine (molecular weight 218.26 g/mol). The resulting solution was mixed with transglutaminase (25 units/ml; 5 units of MTG per 1 mg of antibody), which gave a final antibody concentration of 0.5-3 mg/ml, then the solution was incubated at 37°C for 4-24 hours at shaking gently. The reaction was monitored using ESI-MS. After completion of the reaction, excess amine and MTG were removed by size exclusion chromatography to obtain the azido-functionalized antibody. This product was characterized by DNS-PAGE and ESI-MS.

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Azido-dPEG ₃ -amine added to two sites -Q295 and Q297- of the antibody results in an increase of 804 Da for the 4DAR aglycosylated antibody-PEG ₃ -azide conjugate. Conjugation sites were identified and confirmed on EEQ ^Lmker YQ ^{Lm er} STYR for the azido-functionalized 4DAR antibody by mapping the trypsin-digested heavy chain peptide sequence.

The following example demonstrates a method for creating site-specific antibody-drug conjugations using chemical click reactions.

Conjugates of site-specific aglycosylated antibodies with human IgG1 containing the N297Q mutation in Table. 16, described below, was prepared by a [2+3] click reaction between azido-functionalized antibodies with an alkyne-containing payload linker. As shown in table. 16, anti-PRLR-PEG ₃ -N ₃ antibodies were conjugated to payloads LP112, LP104 and LP116;. Anti-Fel D Ab-PEG ₃ -N ₃ antibodies were conjugated to payloads LP112 and LP116.

The detailed conjugation procedure is given below. The site-specific antibody linker-payload (LP) conjugate was prepared by incubating mAb-PEG ₃ -N ₃ (1-3 mg/ml) in an aqueous medium (eg, PBS, PBS with 5% glycerol, GEPES) with > 6 molar equivalents of payload linker dissolved in a suitable organic solvent such as dimethyl sulfoxide, N,N-dimethylformamide or DMA (i.e. reaction mixture contains 5-20% organic solvent, v/v) at 24 to 37°C for more than 6 hours. The progress of the reaction was monitored using ESI-MS; the absence of mAb-PEG ₃ -N ₃ indicated the completion of conjugation. Excess payload linker and organic solvent were removed by size exclusion chromatography eluting with PBS or by protein A column chromatography eluting with acid buffer followed by neutralization with hydroxymethylaminomethane (pH 8.0). Purified conjugates were analyzed using size exclusion chromatography, DS-PAGE and ESI-MS. In table 16 shows a list of steroid-antibody conjugates from the corresponding payloads, their molecular weights and DAR values.

In a specific example, an azido-functionalized antibody (1 mg) in 0.800 ml PBSg (PBS, 5% glycerol, pH 7.4) was treated with six molar equivalents of DIBAC-PEG ₄ -D-Lys (COT-^-CD)VC-PABC- payload (concentration 10 mg/ml in dimethyl sulfoxide) for 6-12 hours at room temperature, excess linker with payload (LP) was removed using size exclusion chromatography (SEC, Superdex 200 HR, GE Healthcare). The final product was concentrated by ultracentrifugation and characterized by UV, size exclusion chromatography, DNS-PAGE and ESI-MS.

Example 117.

Characterization of ALS conjugate by LC-ESI-MS.

Measurement of the intact mass of ADC conjugate samples by LC-ESI-MS was carried out to determine the drug distribution profile and to calculate the average DAR of intact forms of the ALS conjugate. Each test sample (20-50 ng, 5 µl) was loaded onto an Acquity UPLC Protein BEH C4 column (10 kPa, 300 A, 1.7 µm, 75 µm x 100 mm; catalog number 186003810). After 3 minutes of desalting, the protein was eluted and mass spectra were obtained using a Waters Synapt G2-Si mass spectrometer (Waters).

As shown in the following FIG. 34, the deconvoluted mass spectra showed a predominant peak for the alicosylated anti-prolactin receptor antibody with a molecular weight of 144602 Da and a predominant peak for the azido-functionalized anti-prolactin receptor antibody with a molecular mass of 145385 Da, indicating an increase of 783 Da compared to its aglycosylated parent antibody (i.e., matching 4 amino-PEG ₃ -azide conjugations to each aglycosylated antibody). In addition, the predominant peak for the anti-PRLRLP12 conjugate had a molecular weight of 151015 Da, indicating an increase of 5630 Da compared to its azido-functionalized antibody (i.e., matching 4 LP conjugates (molecular mass = 1405.6 Da with each aglycosylated antibody).Similarly, other site-specific anti-PRLR-ADC conjugates had 3,9-4AR.

As shown in the following FIG. 35, the deconvoluted mass spectra show a predominant peak for the alycosylated anti-Fel D1 antibody with a molecular mass of 145,441 Da and a predominant peak for the azido-functionalized anti-Fel d1 antibody with a molecular mass of 146,235 Da, indicating an increase of 794 Da compared to its aglycosylated parent antibody (i.e., matching 4 amino-PEG3-azide conjugations with each aglycosylated antibody). Additionally, the predominant peak for the anti-Fel d1-LP12 conjugate had a molecular weight of 151871.0 Da, indicating an increase of 5635 Da compared to its azido-functionalized antibody (i.e., matching 4 LP conjugates (molecular weight = 1405 .6 Yes for each aglycosylated antibody.) Similarly, other site-specific anti-Fel d1-ADC conjugates had 3.9-4AR.

In table 16 provides a list of noncytotoxic steroidal antibody conjugates (ncADCs) from their corresponding payloads, their molecular weights of unprotected antibodies, azido-functionalized antibodies, payloads and ALS steroid conjugates, and the values

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IER-DAR. In table Ab refers to an antibody, Ab - N3 refers to an azido-functionalized antibody, ncADC refers to a noncytotoxic steroid-antibody conjugate.

Example 118.

In vitro enzyme assay.

Payload cleavage in the Cathepsin B assay.

The payload linker was tested in a Cathepsin B assay. After 4 hours of incubation in Cathepsin B (CaB) with and without a CaB inhibitor (VA074), the payload linker and payload were assessed by LC-MS/MS. The results showed that hydrophilic payload linkers (LP104) could be cleaved by CapB and released more payload (4b) compared to non-hydrophilic payload linkers (LP12).

The following is the procedure for the SarB assay: The linker stock solution (10 mM in DMSO) was added to the incubation buffer (100 mM NaOAc, 10 mM dithiothreitol, pH 5) to obtain a 50 μM substrate solution. 4 µl of 0.47 µg/µl Cathepsin In Human Liver (Athens Research & Technology, Athens, GA) in 50 mmol NaOAc, 1 mmol EDTA, pH 5, was added to 196 µl of a 50 µmol substrate solution. The reaction mixture was incubated at 37°C for 4 hours. Then, 5 μL of acetic acid and 150 μL of acetonitrile (containing osalmide as internal standard) were added to 50 μL aliquots of the reaction mixture. After shaking, the hardened samples were frozen in a freezer at -70°C, followed by thawing and centrifugation at 14,000 rpm. 50 μL aliquots of the resulting supernatants were then diluted with an equal volume of water and analyzed by LC-ESI-MS/MS for the released payload.

Table 17

Results of digestion using cathepsin B

С° ^e Useful With the use of CA074 / Coefficient, dyne- Linker _ ^g pre-load without the use of CA074 / _L /\ ¹ tion (%) Splintered fragment Hydrophilic linker Conjugated fragment LP101 4b vcPAB / Lk-CCK No application Mean 48.20 RSD using Mean <1.00 RSD LP112 4b vcPAB / Lk- DIBAC No application Mean 20.8 RSD using Mean <1.00 RSD LP104 4b vcPAB aCDCCK Lk- DIBAC No application Mean 29.0 RSD using Mean <1.00 RSD LP102 6-1 Val-Ala / Lk-CCK No application Mean 22.72 RSD using Mean <1.00 RSD LP108 6-1 Val-Ala aCDCCK Lk- DIBAC No application Mean RSD using Mean RSD LP116 6-1 vcPAB / Lk- DIBAC No application Mean RSD using Mean <1.00 RSD LP103 6-1 vcPAB / Lk-CCK No application Mean 29.0 RSD using Mean <1.00 RSD

* Mean = mean value; RSD = relative standard deviation.

The experimental analysis using SarB included the following procedure.

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1. Preheat assay buffer: 0.1 mol NaOAc/0.01 mol DTT (pH 5.0).

2. Test Compound Model Mixture Solutions: 25 µM Test Compound Model Mixture Solutions: Add 2 µL of 5 mmol stock solution in 398 µL of 0.1 mol NaOAc/0.01 mol DTT buffer (pH 5.0).

3. Prepare 0.47 µg/µl cathepsin B in 50 mmol NaOAc/1 mmol EDTA (pH 5.0). Place on ice.

4. Without CA074 inhibitor samples: add 4 µl of 0.47 µg/µl cathepsin B to 196 µl of 25 µm model mixture solutions (from step 2), incubate tubes at 37°C.

5. With CA074 inhibitor samples: add 4 µl 0.47 µg/µl cathepsin B with 4 µl 10 mmol inhibitor (CA074) to 196 µl 25 µmol model mixture solutions (from step 2), incubate tubes at 37°C.

6. After 4 hours, 5 μl aliquots were taken (with and without CA074 inhibitor) for enzyme activity testing (step 15-17). Simultaneously, 50 μL aliquots were removed per time point (4 hours), 5 μL acetic acid was added, then 150 μL ACN (IS) was added to stop the reaction.

7. After hardening, shake the plate and centrifuge them at 14000 rpm.

8. Transfer 50 µL of supernatant from each well to a 96-well sample plate containing 50 µL of ultrapure water (Millipore, ZMQS50F01) for LC/MS analysis. The experimental assay using SarB included control compounds and the following procedure.

9. Preheat the assay buffer: 100 mM Na/K phosphate, pH 6.0, 1.33 mM EDTA, and 2 mM DTT.

10. Prepare 0.024 μg/μL Cathepsin B: Add 1 μL of 0.47 μg/μL Cathepsin B stock solution to 19 μL buffer (from step 11).

11. Add 2 µl of 0.024 µg/µl cathepsin B (from step 12) to an opaque 96-well plate.

12. Add 96 µl of buffer to each sample.

13. Add 2 µl of 10 mmol Z-RR-MNA substrate (final concentration 200 µmol). For a negative control (with inhibitor), add 2 µl of 10 mM inhibitor (CA074).

14. Immediately read samples in kinetic mode at 340 nm excitation/425 nm emission (read plate every 30 seconds for 3 minutes). Cathepsin B stability in incubation samples:

15. Take 5 µL of incubation samples in 93 µL of assay buffer (from step 11) and add 2 µL of 10 mM substrate (Z-RR-MNA).

16. Incubate samples at 37°C for 2 minutes.

17. Read samples at 340 nm excitation/425 nm emission.

Example 119.

Cell-free and cellular activity in vitro.

Cell-free glucocorticoid receptor binding using the LanthaScreen TR-FRET GR Competitive Glucocorticoid Receptor (GR) Binding Assay Kit.

To evaluate the ability of novel steroids to bind to the glucocorticoid receptor (GR), a cell-free binding assay was performed using the LanthaScreen TR-FRET GR Competitive Binding Assay Kit (Life Technologies, Cat. No. A15901). The analysis was carried out in accordance with the manufacturer's instructions. Budesonide is a commercial glucocorticoid receptor steroid and was used as a control in the binding assay and other cell-based assays described herein below. Briefly, a threefold serial dilution of budesonide and the derivative compound noted below was prepared in 100% dimethyl sulfoxide, starting at 100 nmol (100X the final value). Serial dilutions were further diluted 50-fold in nuclear receptor F buffer with 5 mM dithiothreitol and 0.1 mM stabilizing peptide and transferred to a 384-well assay plate. Fluormone GS1 Green, GR-LBD (GST), and anti-GST Tb antibody were then added sequentially to a 384-well assay plate. The plate was then incubated at room temperature for 2.5 hours while protected from light. The plate was analyzed on an Envision multilayer reader (PerkinElmer) with excitation set at 340 nm and emission filters at 520 nm and 486 nm. The FRET ratio was calculated as 520 nm/486 nm. IC ₅₀ values were determined using a four-parameter logistic equation on a 12-point response curve (GraphPad Prism).

As shown in table. 18, budesonide competed for GS1 Green fluormone binding in a glucocorticoid receptor assay with an IC ₅₀ value of 10 to 100 nmol. N-analogues of budesonide similarly competed for binding with IC ₅₀ values ranging from less than 10 nmol to more than 100 nmol. The novel steroids tested here performed comparable or better (lower IC ₅₀ values) in this assay and a similar bias for the GR ligand compared to budesonide.

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Table 18

Cell-free binding and cell functional activity

Connection numbers ECso cells HEK293/9xUAS- Luc2P/pBind-GR/PRLR-HA (nmol) IC50 by GR competitive binding assay, (nmol) Budesonide +++ Full activation ++ 4b +++ Full activation +++ 4d ++ Partial activation ++ 4h +++ Full activation ++ 5-1 +++ Full activation NT 6-1 +++ Full activation NT R-6-I +++ Full activation +++ 5-6-1 +++ Full activation +++ 6-1 D +++ Full activation +++ 6-P +++ Full activation +++ 6-SH +++ Full activation NT 6-VI +++ Full activation NT 6-VII ΝΑ Without activation +++

In table 18: +++: < 10 nmol; ++: <100 nmol > 10 nmol; +: > 100 nmol; NT: not tested. NA= no activation. Full activation: > 75% of the degree of activation caused by budesonide. Partial activation: (20%, 75%) of the degree of activation caused by budesonide. No activation: <20% of budesonide-induced activation. A cell-free assay is used to evaluate the direct binding of compounds to the recombinant GR LBD, regardless of their permeability. Cell-based assays are used to measure how compounds activate intracellular glucocorticoid receptor-mediated transcription after crossing the plasma membrane, thus making binding across membrane permeabilization a prerequisite for activity.

Example 120.

Cellular analysis of the glucocorticoid receptor (GR) coactivator luciferase reporter gene.

Glucocorticoid activation assay.

The activity of steroid payloads and anti-PRLR steroid ncADC conjugates was studied using a cell-based luciferase reporter gene assay using either the 293/PRLR/GRE-luc cell line described in Example 62 as an antigen-negative 293 cell line that contains the chimeric receptor , consisting of a glucocorticoid receptor ligand binding domain fused to the yeast CAL4 DNA binding domain (pBind-GR, Promega catalog no. E1581), and a Gal4 activating sequence (9XGal4 UAS-Luc) that drives luciferase expression. The resulting cell line is called 293/GRE-Luc.

The bioassay was performed using these two cell lines, tesgin anti-PRLR-LP112, dexamethasone, budesonide, compound 4b, control payload Ab-LP112, as well as one anti-PRLR Ab, using the assay set up as described in Example 63.

As shown in FIG. 36A and in table. Below, after 72 hours of incubation, the anti-PRLRLP112 payload showed the highest fold in the 293/PRLR/GRE-luc cell line, while the LP112(4b) payload showed a better IC ₅₀ value than budesonide and dexamethasone. The control payload Ab-LP112 and the unconjugated anti-PRLR mAb did not demonstrate activity in this cell line.

As shown in FIG. 36B and in table. Below, after 72 hours of incubation, the anti-PRLRLP112 payload showed no activation in the 293/PRLR/GRE-luc cell line, which does not express the prolactin receptor, indicating that steroid delivery by anti-PRLR-ncADC conjugates is antigen-dependent . The LP112 payload (4b) again showed a better IC ₅₀ value than budesonide and dexamethasone. The control payload Ab-LP112 and the unconjugated anti-PRLR Ab did not demonstrate any activity in this cell line.

The following table refers to FIG. 36A and 36B.

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ECso in 293/PRLR/GRE-Luc (mol) ECso in 293/GRE-Luc (mol) Anti-PRLR Ab-LPl 12 +++ + Ab-LPl12 control + + Anti-PRLR Ab N.A. N.A. 4b (LP112 payload) +++ +++ Budesonide ++ +++ Dexamethasone +++ +++

In table +++<5 nmol, 5 nmol<++<10 nmol, 10 zero<+.

Example 121.

This example describes a mouse model of lipopolysaccharide-induced cytokine release.

The purpose of this study is to test compounds 4b and 6-I to inhibit lipopolysaccharide-induced cytokine release in mice. Test compounds were administered 48 hours, 24 hours and 2 hours before LPS challenge, and levels of cytokines in blood samples, including TNF-α and IL6, were measured at 2 hours and 4 hours after LPS challenge.

Materials and reagents.

Lipopolysaccharide (LPS) derived from E. Coli K12 was purchased from Invivogen (San Diego, CA, USA, cat. no. Tlrl-eklps), dexamethasone was purchased from ADAMAS (Emeryville, CA, USA, cat. no. 50- 02-2). The mouse TNF-α ELISA kit was purchased from eBioscience (ThermoFisher Scientific, cat. no. 88-7324). The mouse ILISA ELISA kit was purchased from eBioscience (ThermoFisher Scientific, cat. no. 88-7064).

Experimental methods.

Keeping animals.

Eighteen naïve C57BL/6J mice were used in this study. The animals were males with a body weight of 18–20 g at the start of the study. The animals were purchased from the Shanghai Laboratory Animal Center, CAS (SLAC) and housed in the ChemPartner animal vivarium in a pathogen-free flora (SPF) environment. Upon arrival, animals were checked for health conditions, including coat, limbs, orifices, and abnormal signs of posture or movement, and acclimatized for more than 7 days.

Animals were kept 3 mice per cage in IVC polycarbonate shoe cages in an environment free of pathogenic flora; Animal house environmental controls were set to maintain a temperature of 20–26°C, humidity of 40–70%, and a lighting cycle of 12 hours light/12 hours dark.

Standard food (SLAC-M01, Shanghai Laboratory Animal Center) and purified water (filtered tap water) were provided ad libitum throughout the study period.

Procedure for conducting the experiment.

Grouping. Animals were randomized into 6 groups (A-F) before the start of the study. Each group consisted of 3 mice. Group A served as naïve controls; Group B received dexamethasone and served as a positive control; Group C was treated with compound 4b and Group D-F was treated with compound 6-I.

Progress of the experiment.

All mice received lipopolysaccharide (LPS) dissolved in PBS at a dose of 0.5 mg/kg by intraperitoneal injection. Mice in group A received PBS, mice in group B received Dex (5 mg/kg body weight), mice in group C received compound 4b (5 mg/kg body weight) by intraperitoneal injection 2 hours before LPS challenge; Mice in groups D, E and F received a dose of 5 mpc intraperitoneally, 2 hours, 24 hours and 48 hours before LPS administration, respectively.

Blood samples were collected 2 hours and 4 hours after LPS administration in heparin-containing tubes. Blood samples were centrifuged, and plasma samples were collected and stored at -80°C until analysis.

Plasma TNFa levels were measured using ELISA kits following standard procedures recommended by the manufacturer.

Pharmacokinetic results are presented in Fig. 37A and in table. 20.

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Table 20

Summary pharmacokinetic parameters of compounds 4B and 6-I

Pharmaco- kinetic parameters Unit Dexamethasone (IP, 1 mg/kg in male C57BL/6 mice) 4b (IP, 1 mg/kg male C57BL/6 mice) 6-1 (IP, 1 mg/kg in male C57BL/6 mice) Mean SD CV(%) Mean SD CV(%) Mean SD CV(%) Tschakh h 0.625 0.433 69.3 0.438 0.375 85.7 0.250 0.00 0.00 Stakh ng/ml 231 7.97 3.45 39.4 1.75 4.43 44.8 4.77 10.6 Terminal. tl/2 h 1.64 0.187 11.4 1.69 0.620 36.7 1.91 0.210 11.0 AUClast h*ng/ml 545 60.6 H,1 84.6 7.71 9.12 107 13.6 12.7 AUCinf h*ng/ml 562 67.2 11.9 89.2 8.20 9.19 113 14.1 12.5

Mean = mean value; SD = standard deviation; CV = coefficient of variation

Pharmacodynamic results.

LPS injection induced TNF-α release in this pharmacodynamic model observed at the 2-h sampling point. The results were consistent with the reported cytokine release kinetics in the mouse LPS challenge model, with TNF-α levels decreasing at the 4-hour time point. Therefore, the effect of the test compounds could not be measured, and therefore no significant difference was observed between groups at the 4 hour time point.

Blood samples were collected at 2 h and 4 h after LPS challenge; plasma TNF-α levels were measured. Data were expressed as mean ± SEM, * p < 0.05, ** p < 0.01 versus group A, according to Oneway.

Shown in FIG. 37A, at time point 2 hours, compound 4b at a dose of 5 MPC significantly inhibited the production of TNF-α; compound 6-I showed time-dependent inhibition, with significant TNF-α production inhibited when dosed 2 hours before LPS administration. Dexamethasone was able to significantly inhibit TNF-α at the 2-h sampling point.

Blood samples were collected at 2 h and 4 h after LPS challenge; plasma TNF-α levels were measured. Data were expressed as mean ± SEM, * p < 0.05, ** p < 0.01 compared with group A, using Oneway ANOVA.

The ANOVA analysis is presented in Fig. 37B and in table. 21.

Table 21 TNF-α Raw Data

TNF-a (pg/ml) A:PBS In: DEX 5 mg/kg (-2h) S:4b 5 mg/kg (-2h) D: 6-1 5 mg/kg (-2h) E) 6-1 5 mg/kg (-24h) F) 6-1 5 mg/kg (-48h) 2h 840.2 644.3 56.5 110.6 23.1 1280.1 1293.6 508.5 10.4 30.8 997.3 1149.4 968.6 487.1 274.8 240.8 55.4 750.0

Example 122.

Mouse dendritic cells.

To determine the effect of compounds 4b on ex vivo LPS-induced inflammatory immune responses, CD11c ⁺ dendritic cells were isolated from the spleen of wild-type C57B1/6 mice (Jackon Labs, Protocol no. 426.0). Spleen dendritic cells 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% fetal calf serum (FCS) in GES-buffered medium RPMI-1640) and incubated at 37°C for 25 minutes. After incubation, spleen tissue was washed with RPMI-1640 medium and filtered through a 70 mm filter, then erythrocyte lysis was performed using ACK lysis buffer (Gibco cat. no. A1049201) for 1 minute. The cell suspension was then washed twice using RPMI-1640 medium. Classical dendritic cells were isolated from mononuclear cell suspensions using CD11c magnetic microbeads (Milteny Biotec cat. no. 130108338). Briefly, the cell suspension was washed twice with autoMACS running buffer (Milteny Biotec cat. no. 130091221), then incubated for 30 min at 4°C with CD11c + microbeads ^.

- 198 045579 according to established Milteny Biotec protocols. CDllc+ cells were isolated by positive selection, washed, suspended in complete RPMI medium [RPMI-1640 (ThermoFisher Scientific, cat. no. 15140122) containing 10% fetal bovine serum (FBS) (ThermoFisher Scientific, cat. no. 10082147) and 1% penicillin -streptomycin (ThermoFisher Scientific), cat. No. 11875093)], and counting was performed before cultivation at 2x105 cells per well. Control complete RPMI medium, compound 4b-treated control complete RPMI medium (at 10 nmol and 100 nM), or dexamethasone-treated (Sigma, cat. no. D4902-25MF) complete RMPI medium (at 10 nmol and 100 nmol) were added to cells at 96- well plate for cultivation. Dendritic/control cells treated with compound 4b 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 compounds 4b on ex vivo LPS-induced inflammatory immune responses in human innate immune system cells, CD14+ monocytes (Lonza cat. no. 2W-4OOC) were isolated and cultured in the presence of 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)], supplemented with human IL-4 (50 ng/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 IL-4 and GM-CSF was changed every three days. Two specific culture conditions were developed: Condition 1: Incubation of CD14+ monocytes with control complete RPMI medium, compound 4b treated complete RMPI medium (at 10 nmol and 100 nmol), or dexamethasone (Sigma) treated complete RMPI medium (at 10 nmol and 100 nmol ) for the entire volume of a 7-day culture; or Condition 2: Incubation of CD14+ monocytes with control complete RPMI medium for 5 days prior to incubation with compound 4b treated complete RMPI medium (at 10 nmol and 100 nmol), or dexamethasone (Sigma cat. no. D4902-25MT) treated complete RMPI medium (at 10 nmol and 100 nmol) until the 7th day. On day 7, different experimental groups were stimulated with 10 ng/ml LPS for 24 hours.

Measurement of cytokines in supernatants 24 h post-infection with LPS ex vivo.

Supernatants were collected into 96-well round-bottom tissue culture plates 24 h after LPS infection and stored at −20°C until further analysis. Cytokine concentrations in the supernatants were measured using the Pro-Inflammatory Panel 1 Multiplex Immunoassay Kit (Mouse) (MesoScale Discovery, Cat. No. K15048D) according to the manufacturer's instructions or the Pro-Inflammatory Panel 1 Multiplex Immunoassay Kit (Human) (MesoScale Discovery, Cat. No. K15048D). No.K15049D). Briefly, 50 μl/well of calibrators and samples (diluted 1:2 in diluent) were added to plates precoated with immobilized antibodies and incubated at room temperature with shaking at 700 rpm for 2 h. The plates were then washed 3 times with 1xPBS containing 0.05% (w/v) Tween-20 surfactant, followed by the addition of 25 μl of detection antibody solution diluted in diluent 45. After a 2-hour incubation at room temperature with shaking, the plates were washed 3 times. times, 150 μl of 2x reading buffer was added to each well. Electrochemiluminescence was immediately read on an MSD Spector® instrument. Data analysis was performed using GraphPad Prism™ software. Statistical significance between groups was determined by one-way ANOVA with post-test multiple comparison test, and the standard error of the mean (SEM±) was calculated.

Review of results and conclusions.

As shown in table. 22, LPS challenge ex vivo induced robust production of IL12p70, IL1e, IL6, KC-GRO and TNF-α and splenic CD11c ⁺ DCs. In contrast, administration of dexamethasone and Compound 4b in vitro at increasing doses over 24 hours significantly reduced LPS-induced cytokine responses in CD11c ⁺ DCs.

Table 22

Compound 4b and dexamethasone (sigma) inhibit LPS-induced cytokine production in CD11C+ dendritic cells.

Cytokine Control Control +LPS Dexamethasone (10 nmol) Dexamethasone (10 nmol) + LPS Dexamethasone (100 nmol) Dexamethasone (100 nmol) + LPS IL-12p70 17.43+2.72 77.02+4.40 8.55+1.08 30.41+3.22 6.05+1.13 9.29+3.28 IL-lb 9.53+0.26 162.48+5.69 6.299+0.33 34.76+4.33 5.25+0.12 10.58+0.88 IL-6 144.49+16.69 1492.12+66.47 98.93+8.98 714.89+77.13 55.07+1.76 303.48+11.37 KC- GRO 111.69+10.96 536.47+49.90 75.75+8.03 399.40+49.75 65.99+2.75 233.49+18.48

- 199 045579

TNF-a 0±0 8644.14±74.73 0±0 2496.27±415.75 0±0 405.49±70.23 4b (10 nmol) 4b (10 nmol) +LPS 4b (100 nmol) 4b (100 nmol) +LPS IL-12p70 5.24±1.12 3.84±2.88 6.43±0.67 8.21±2.79 IL-lb 5.22±0.ll 6.32±0.83 5.65±0.18 4.80±0.99 IL-6 47.90±4.55 184.54±21.59 63.87±4.78 201.10±20.96 KS- GRO 62.85±6.51 186.57±15.71 84.30±6.06 211.96±25.23 TNF-a 0±0 146.46±18.66 0±0 193.29±25.56

As shown in table. 23, LPS challenge ex vivo induced robust expression of IL12p70, IL1β, IL6, and TNF-α by human monocyte-derived dendritic cells. In contrast, monocytes cultured for the entire 7-day conditioning period (condition 1) with compound 4b and dexamethasone (Sigma) caused a significant reduction in the production of proinflammatory cytokines. In addition, conditioning mature monocyte-derived dendritic cells with compound 4b and dexamethasone (Sigma) also significantly reduced the production of IL12p70, IL6, and TNFα compared with LPS stimulation of control dendritic cells.

Table 23

Compound 4b and dexamethasone (SIGMA) inhibit LPS-induced cytokine production in human monocyte-derived dendritic cells

Condition 1 Cytokine Control Control +LPS Dexamethasone (10 nmol) Dexamethasone (10 nmol) + LPS IL-12p70 0.79±0.04 994.23± 19.89 0.65±0.02 4.83±0.05 IL-lb 0.80±0.001 12.97±0.186 0.625±0.006 l.77±0.ll IL-6 8.71±0.23 5669.2±266.12 2.68±0.03 687.45±2.17 IL-8 850.50±0.89 1677.86±20.06 174.59±0.68 8789.98±126.98 TNF-a 44.20±l.08 7476.12±177.0 2.32±0.098 242.34±10.75 4b (10 nmol) 4b (10 nmol) + LPS 4b (100 nmol) 4b (100 nmol)+LPS Condition 1 IL-12p70 0.58±0.002 l.05±0.08 0.66±0.03 0.50±0.19 IL-lb 0.6H0.007 l.18±0.ll 0.64 N0D5 1,ONO,07 IL-6 2.23±0.002 325.46±2.12 3.45±0.13 159.32±7.31 IL-8 166.99±2.01 8670.27±268.97 136.50±0.87 8461.59±522.77 TNF-a l.28±0.087 117.75±6.63 l.46±0.10 123.3H0.ll Condition 2 Cytokine Control Control +LPS Dexamethasone (10 nmol) Dexamethasone (10 nmol) + LPS IL-12p70 5.83±2.55 49.64±2.05 0.64±0.03 6.32±1.69 IL-lb 3.13±0.05 13.03±l.26 0.61 HO.003 37.88±l.90 IL-6 37.74±l.50 6679.97±173.8 14.76±0.267 5747.37±234.08 IL-8 1541.04±82.6 1958.27±5.35 288.23±6.02 3314.0±37.86 TNF-a 61.49±4.82 2690.61±164.5 l.73±0.02 994.92±73.66 IL-12p70 4b (10 nmol) 4b (10 nmol) +LPS 4b (100 nmol) 4b (100 nmol)+LPS IL-lb 0.60±0.01 4.8HO.63 0.6H0.02 2.03±0.16 IL-6 0.59±0.005 30.10±0.93 0.59±0.03 18.20±l.04 IL-8 12.87±0.312 5124.75±114.8 ll.25±0.82 4680.03±104.90 TNF-a 274.75±1.75 3003.52±212.16 317.46± 14.83 2735.36±87.88 2.03±0.14 874.18±30.46 l.37±0.251 696.26±75.10

The embodiments and examples described above are for illustrative purposes only and

- 200 045579 are not restrictive. Those skilled in the art will understand or be able to determine, using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within scope and are included in the appended claims.

Claims (42)

1. Compound of formula (A) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, R ⁴ 0^0 where R ¹ and R ² together form ' where R ⁴ represents -C _1-20 alkyl; R ³ represents -NR ^a R ^b , R ⁵ in each instance is independently -OH, halo, -C _1-20 alkyl or -C _6-20 arylC _1-20 alkyl and each R ⁵ is located on any ring atom; R ^a and R ^b are each independently -H, -C _1-20 alkyl or -C _6-20 aryl, where -C _6-20 aryl is unsubstituted or substituted with an amino group; or R ^a and R ^b cyclize to form heterocycloalkyl with three to six ring atoms, including one heteroatom, which is N, to which they are attached; and n is an integer from 0 to 19. 2. A compound according to claim 1, where the compound with formula (A) has the structure of formula (A ¹ ): where R ¹ -R ³ have the meaning specified in paragraph 1, and R ^5A and R ^5B each independently represent a halo or a hydrogen atom. R ⁴ o^o 3. The compound according to claim 2, wherein R ¹ and R ² together form ' where R ⁴ represents n-propyl. 4. A compound according to any one of claims 1 to 3, wherein R ^a represents H and R ^b represents H or -C _1-20 alkyl. 5. A compound according to claim 1, having the formula (A ⁴ ): N and w O R' (A ⁴ ) where R ⁴ represents -C1-20alkyl and R ³ represents -NR ^a R ^b , where R ^a and R ^b in each case independently represent a hydrogen atom or -C6-20 alkyl, or R ^a and R ^b together form a heterocycloalkyl with 3-6 ring atoms, including one heteroatom, which is N, to which they are attached. 6. The compound according to claim 5, where R ⁴ represents n-propyl. 7. A compound according to claim 5 or 6, where R ³ represents -NH ₂ , -NHCH3 or -N(CH ₃ ) ₂ . 8. A compound according to claim 1, having the formula (A ⁶ ): - 201 045579 where R ⁴ is defined in claim 1 and R ³ represents -NR ^a R ^b , where R ^a and R ^b in each case independently represent a hydrogen atom or -C1_ ₂₀ alkyl. 9. The compound according to claim 1, having the formula 1000: or a pharmaceutically acceptable salt thereof, R ⁴ o^o where R ¹ and R ² together form ' where R ⁴ represents -C1- ₂₀ alkyl; R ³ represents -NR ^a R ^b ; R ⁵ in each case is independently selected from the group consisting of -OH, halo and _-C1-20 alkyl; n is an integer from 0 to 16; and each R ⁵ is located on any atom of the ring; R ^a and R ^b are in each case independently selected from the group consisting of -H and -C1- ₂₀ alkyl; or R ^a and R ^b are cyclized to form a heterocycloalkyl with 3-6 atoms, including one heteroatom, which is N, to which they are attached. 10. A compound according to claim 9, having the formula 1010, 1020, 1030 or 1040: n.2 ABOUT R' F .2 Η O R' 1020 1010 Η.2 O R' 1030 sn ₃ .2 Η O R' 1040 where R1-R ³ are defined in paragraph 9; or a pharmaceutically acceptable salt thereof. 11. The compound according to claim 10, where R ³ represents -NH2, and R ⁴ is defined in claim 9. 12. A compound according to claim 9, having the formula 1110, 1120, 1130 or 1140: where R ³ represents -NR ^a R ^b , R ^a and R ^b each independently represents a hydrogen atom or -C ₆ - ₂₀ alkyl, and R ⁴ as defined in paragraph 9; or a pharmaceutically acceptable salt thereof. 13. The compound according to claim 12, where R ⁴ represents n-propyl. 14. The compound according to claim 13, having the formula 1120. 15. A compound according to claim 12, where R ³ represents -NH ₂ , -N(H)CH ₃ , -N(CH ₃ ) ₂ or 16. The compound according to claim 12, where R ³ represents -NH2. - 202 045579 17. A compound according to any one of claims 15, 16, where R ⁴ represents n-propyl. 18. Connection selected from: or a pharmaceutically acceptable salt thereof. 19. A conjugate compound comprising a binding agent conjugated to a compound according to any one of claims 1 to 18, wherein the binding agent is an antibody or an antigen binding fragment thereof. 20. A conjugated compound comprising a binding agent conjugated to a compound according to any one of claims 1 to 18 and a cyclodextrin (CD), where the binding agent is an antibody or an antigen-binding fragment thereof. 21. A conjugate compound comprising a binding agent conjugated to a compound of formula (A); where the compound of formula (A) is: - 203 045579 or its stereoisomer, where R ¹ and R ² represent, independently, -H, -C _1-20 alkyl, -O-C(O)-C1_ ₂₀ alkyl, -OH or halo; or R1 and ^R2 together form where ^R4 is -C1. ₂₀ alkyl; R ³ represents -NR ^a R ^b , R ⁵ represents in each case independently -OH, halo, -C1. ₂₀ alkyl or -C6. ₂₀ arylS ₁ . ₂₀ alkyl, and each R ⁵ is located on any ring atom; R ^a and R ^b are in each case independently -H, -C1. ₂₀ alkyl or -C ₆ . ₂₀ aryl, where -C ₆ . ₂₀ aryl is unsubstituted or substituted with an amino group; n is an integer from 0 to 19; and wherein the binding agent is an antibody or an antigen binding fragment thereof. 22. The conjugated compound according to claim 20 or 21, where CD is selected from the group consisting of - 204 045579 where ' denotes the bond through which the CD is associated with the conjugated binding agent. 23. The conjugate compound according to any one of claims 20-22, where the binding agent is an antibody or an antigen-binding fragment thereof modified with one or more H ₂ N-LL-N ₃ groups, where LL is divalent polyethylene glycol. 24. The conjugate compound of claim 19, wherein the binding agent is an antibody or an antigen-binding fragment thereof modified with one or more H ₂ N-LL-N ₃ groups, where LL is divalent polyethylene glycol. 25. Conjugate compound having formula 1200: X\| ^VA - ^L gtOO ⁰ \K° he U 1200 or a pharmaceutically acceptable salt thereof, wherein R ¹ and R ² are independently selected from the group consisting of -H, -OH, -C _1-20 alkyl, -O-C(O)-C _1-20 alkyl and halo; or R ⁴ o^o R ¹ and R ² together form ' where R ⁴ represents -C _1-20 alkyl; R ³ represents -NR ^a R ^b ; R ⁵ in each case is independently selected from the group consisting of -OH, halo and -C _1-20 alkyl; n is an integer from 0 to 16; and each R ⁵ is located on any atom of the ring; R ^a and R ^b are in each case independently selected from the group consisting of -H and -C _1-20 alkyl; or R ^a and R ^b are cyclized to form a heterocycloalkyl with three to six atoms, including one heteroatom, which is N, to which they are attached; BA is an antibody or an antigen-binding fragment thereof; each L is a linker; BA or L is covalently linked to R ³ ; and x is an integer from 1 to 30. 26. The conjugate compound according to claim 25, having the formula 1210, 1220, 1230 or 1240: or a pharmaceutically acceptable salt thereof; where R ¹ , R ² , R ³ , L and BA are defined in paragraph 25 and R ³ is covalently linked to L or BA. 27. The conjugate compound according to claim 25, selected from the group consisting of: - 205 045579 or a pharmaceutically acceptable salt thereof; where each L represents a linker; each BA is an antibody or an antigen-binding fragment thereof; and each x represents an integer from 1 to 30. 28. The conjugate compound according to claim 19, selected from the group consisting of: - 206 045579 - 207 045579 - 208 045579 where Ab is an antibody or an antigen-binding fragment thereof; and x is an integer from 1 to 30; or a pharmaceutically acceptable salt thereof. 29. A method of treating a disease, disorder or condition associated with glucocorticoid receptor signaling, comprising administering to a patient suffering from said disease, disorder or condition a therapeutically effective amount of a compound according to any one of claims 118. 30. A method of treating a disease, disorder or condition associated with glucocorticoid receptor signaling, comprising administering to a patient suffering from said disease, disorder or condition a therapeutically effective amount of a conjugated compound according to any one of claims 19 to 28. 31. The method of claim 29 or 30, wherein the disease, disorder or condition is an inflammatory disease, disorder or condition. 32. The method according to claim 29, where a compound according to any one of claims 10-14 is administered. - 209 045579 33. A method of delivering a compound according to claim 1 to a cell, comprising contacting said cell with a conjugate of a compound according to claim 19 or 20, which binds a surface antigen of said cell. 34. A linker compound containing a payload, comprising a compound according to any one of claims 1 to 18, linked to a reactive group via a linker. 35. The linker connection according to claim 34, containing a payload selected from the group consisting of: He - 210 045579 36. A pharmaceutical composition containing a therapeutically effective amount of a compound according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier. 37. A pharmaceutical composition containing a therapeutically effective amount of a conjugate of a compound according to any one of claims 19 to 28 and a pharmaceutically acceptable carrier. 38. The connection according to claim 18, where the connection is 39. The conjugate compound according to claim 27, where the conjugate compound is where L, BA and x have the values specified in paragraph 27. 40. The conjugate compound according to claim 39, wherein BA is an antibody modified with one or more H2N-LL-N3 groups, where LL is divalent polyethylene glycol. 41. A conjugate compound comprising an antibody or an antigen-binding fragment thereof conjugated to a compound of the formula: 42. The conjugated compound according to any one of claims 19 to 28, where the antibody or antigen-binding fragment binds a target selected from AXL, BAFFR, BCMA, BDCA2, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10orf54, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD168, CD177, CD19, 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, - 211 045579 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 класс 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, Kp30, Kp46, PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, Siglec1, Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPA, SLAMF7, TACI, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, TROY, TSLPR, TYRO, VLDLR, VSIG4 and VTCN1.