WO2021161263A1

WO2021161263A1 - Glucocorticoid receptor agonist and immunoconjugates thereof

Info

Publication number: WO2021161263A1
Application number: PCT/IB2021/051204
Authority: WO
Inventors: Adrian D. Hobson; Martin E. Hayes; JR. Axel HERNANDEZ; Claire Louise IHLE; Christopher C. MARVIN; Michael J. Mcpherson; Wendy Waegell
Original assignee: Abbvie Inc.
Priority date: 2020-02-13
Filing date: 2021-02-12
Publication date: 2021-08-19

Abstract

Provided herein are glucocorticoid receptor agonist immunoconjugates, glucocorticoid receptor agonists, pharmaceutical compositiosn including the same, and methods of using the same.

Description

GLUCOCORTICOID RECEPTOR AGONIST AND IMMUNOCONJUGATES THEREOF

SEQUENCE LISTING

[0001] The instant application contains a Sequence Listing which has been submitted herewith in a file in ASCII format, and is hereby incorporated by reference in its entirety. Said file in ASCII format, created on February 5, 2021, is named AbbViel240SL_25.txt and is 11.3 kilobytes in size.

BACKGROUND

[0002] Tumor Necrosis Factor α (TNFα) plays a central role in the pathophysiology of several human disorders, and anti-TNFα agents have clinically validated therapeutic utility in the treatment of autoimmune and inflammatory disorders, such as rheumatoid arthritis, psoriasis and inflammatory bowel disease. Despite their success in the clinic, anti-TNFα biologies are still limited in the maximal efficacy they can achieve in patients, necessitating the identification and develoμment of more potent and effective therapeutics. Patients treated with anti-TNFα biologics may also develop an immunogenic response to the therapeutic thus limiting its effectiveness. Therefore, anti-TNFα therapies with lower immunogenicity and high efficacy would be useful for further controlling disease.

[0003] Synthetic glucocorticoid receptor agonists are a potent class of small molecules used in the treatment of inflammatory disorders, but their utility in the chronic treatment of disease is limited due to severe side effects. There is a need to develop therapeutics with enhanced efficacy and longer duration of action compared to anti-TNF antibodies and with minimal unwanted effects.

SUMMARY

[0004] In one embodiment, disclosed herein is an antibody drug conjugate (ADC) compound having Formula I:

wherein: A is adalimumab; L is a linker; Q is a heterobifunctional group; or Q is absent; and n is 1-10; and SM is a monovalent radical of a glucocorticosteroid as described herein. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7, in some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10. [0005] In some embodiments, SM is selected from the small molecules shown in examples SM-1 through SM-8. In some embodiments, L is a peptide comprising 1, 2, or 3 amino acids each independently selected from: glutamic acid, alanine, glycine, lysine, and serine. In some embodiments

wherein the wavy line indicates the attachment point to A and the asterisk indicates the attachment point to L.

[0006] In one aspect, the present disclosure provides an antibody drug conjugate of formula (II)

wherein:

X is hydrogen or fluoro;

A is an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3; wherein the wavy line indicates the attachment point to A

and the asterisk indicates the attachment point to L;

L is a peptide comprising 1, 2, or 3 amino acids each independently selected from: glutamic acid, alanine, glycine, lysine, and serine;

R^A is fluoro, ehloro or C₁-C₅ alkyl; and n is 2 or 4.

[0007] In certain embodiments, R^A is selected from fluoro, chloro, and CH₃.

[0008] In certain embodiments, the antibody drug conjugate is a compound of Formula II, wherein R^A is fluoro. In some embodiments, the compound is:

wo 2021/161263

6 PCT/IB2021/051204

[0009] In yet another embodiment, the antibody drag conjugate is:

[0010] In yet another embodiment, the antibody drug conjugate is

[0011] In another aspect, the present disclosure provides pharmaceutical compositions that include the antibody drug conjugate of any one of the above embodiments, and a pharmaceutically acceptable carrier.

[0012] In yet another aspect, the present disclosure provides an antibody drug conjugate of formula (II A):

X is hydrogen or fluoro; A is adalimumab;

wherein the wavy line indicates the attachment point to A and the asterisk indicates the attachment point to L;

L is a peptide comprising 1 , 2, or 3 amino acids each independently selected from: glutamic acid, alanine, glycine, lysine, and serine;

R^A is fluoro, chloro or C₁-C₅ alkyl; and n is 2 or 4.

[0013] In certain embodiments, R^A is selected from fluoro, chloro, and CH₃. In certain embodiments, R^A is fluoro.

[0014] In certain embodiments, the antibody drug conjugate is:

[0015] In yet another embodiment, the antibody drug conjugate is:

[0016] In yet another embodiment, the antibody drug conjugate is

[0017] In another aspect, the present disclosure provides pharmaceutical compositions that include any of the above antibody drug conjugates, and a pharmaceutically acceptable carrier.

[0018] In yet another aspect, the present disclosure provides the small molecule compound of formula SM: wherein:

X is hydrogen or fluoro: each R^A is independently selected from fluoro, chloro or C₁-C₅ alkyl; and p is 0 or 1.

[0019] In yet another aspect, the present disclosure provides the small molecule compound of formula

SM-A: wherein:

[0020] In yet another aspect, the present disclosure provides the small molecule compound of formula

SM-B:

wherein:

X is hydrogen or fluoro; each R^A is independently selected from fluoro, chloro or C₁-C₅ alkyl; and p is 0 or 1.

[0021] In one embodiment, the compound is:

[0022] In one embodiment, the compound is:

[0023] In one embodiment, the compound is:

[0024] Referring to Table B below, in yet another embodiment, the compound is SM-4, In yet another embodiment, the compound is SM-5. In yet another embodiment, the compound is SM-6. In yet another embodiment, the compound is SM-7. In yet another embodiment, the compound is SM-8.

[0025] In one embodiment, the present disclosure provides a method of treating a condition selected from rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, and juvenile idiopathic arthritis in a subject, comprising administering an effective amount of the antibody drug conjugate of any preceding embodiment or the pharmaceutical composition of any preceding embodiment to the subject.

[0026] In one embodiment, the present disclosure provides the antibody drug conjugate of any preceding embodiment or the pharmaceutical composition of any preceding embodiment for use in the treatment of a condition selected from rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, and juvenile idiopathic arthritis.

[0027] In one embodiment, the present disclosure provides use of the antibody drug conjugate of any preceding embodiment or the pharmaceutical composition of any preceding embodiment for preparation of a medicament for treating a condition selected from rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis, ulcerative colitis, adult Crohn's disease, pediatric Crohn's disease, uveitis, hidradenitis suppurativa, and juvenile idiopathic arthritis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 depicts exemplary comparative studies of Formula (II- 1) against adalimumab and vehicle control.

[0029] FIG. 2 depicts exemplary comparative studies of Formula (Il-7) against adalimumab and vehicle control.

[0030] FIG. 3 depicts exemplary comparative studies of Formula (II-5) against adalimumab and vehicle control.

[0031] FIG. 4 depicts exemplary comparative studies of Formulas (11-12) and (II-13) against adalimumab and vehicle control.

[0032] FIG. 5 depicts exemplary comparative studies of Formulas (11-19) and (II- 10) against adalimumab and vehicle control.

[0033] FIG. 6 depicts exemplary comparative studies of Formula (II-10) against adalimumab and vehicle control. [0034] FIG. 7 depicts exemplary comparative studies of Formula (II-3) against adalimumab and vehicle control.

DETAILED DESCRIPTION

[0035] Provided herein are glucocorticoid receptor agonist immunoconjugates, glucocorticoid receptor agonists, and methods of making and using the same.

I. Definitions

[0036] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0037] The term "antibody", as used herein, is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as ITCVR or VTI) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR), Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, FR4

[0038] The term "anti-TNFα antibody" or "an antibody that binds to TNFα" refers to an antibody that is capable of binding TNFα, e.g., with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting TNFα. The extent of binding of an anti-TNFα antibody to an unrelated, non-TNFα protein can be less than about 10% of the binding of the antibody to TNFα as measured, e.g., by a radioimmunoassay (RIA). in certain embodiments, an antibody that binds to TNFα has a dissociation constant (Kd) of ≤ 1 μM, ≤ 100 nM, ≤10 liM, ≤ 1 riM, or ≤ 0.1 nM. The anti-TNFα antibody can bind to a target on the surface of a cell and become internalized. The anti-TNFα antibodies bind to soluble TNFα and/or membrane bound TNFαT the anti-TNFα antibody binds to TNFα on the surface of a cell and become internalized

[0039] The ADCs disclosed herein comprise drag molecules linked to antibody moieties in various stoichiometric molar ratios depending on the configuration of the antibody and, at least in part, on the method used to effect conjugation. [0040] The terms “drug load” or “drag loading” refer to the number of drug molecules per antibody in an individual ADC molecule. In embodiments, the drag loading may comprise from 1-8 drag molecules, from 1 -6 drag molecules, from 1-4 drag molecules, from 2-4 drug molecules, from 1 -3 drug molecules, or from 2-3 drug molecules. In embodiments, the drug loading may comprise 1 drug molecule, 2 drag molecules, 3 drug molecules, 4 drag molecules, 5 drag molecules, 6 drag molecules, 7 drag molecules, or 8 drag molecules. In embodiments, the general formula of an ADC molecule may be represented as A(-L-D)_n, wherein A is the antibody, L is the linker, D is the drag, and n is the number of drag molecules linked to the antibody. In embodiments, n is from 1-8, or from 1 -6, or from 1-4, or from 2-4, or from 1-3, or from 2-3. In embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8.

[0041] The terms “drag-to-antibody ratio” or “DAR” refer to the weighted average molar ratio of drug molecules per antibody in a population of at least two ADC molecules.

[0042] Accordingly, the terms “drug loading” and “DAR” are related but distinct concepts. Drug loading primarily has relevance for the construction and design of an individual ADC molecule, whereas DAR primarily has relevance for compositions comprising a population of at least two ADC molecules.

[0043] The term "immunoconjugate," "conjugate," "antibody-drug conjugate," or "ADC" as used herein refers to a compound or a derivative thereof that is conjugated to a protein such as a cell binding agent (e.g., an anti-TNFα antibody). Such immunoconjugates can be defined by a generic formula: (SM-L-Q)_n-A, wherein SM = radical derived from a small-molecule glucocorticoid receptor agonist of the invention, L = linker, Q = heterobifunctional group or is absent, and A = a protein (e.g., an antibody), and n = 1-10. Immunoconjugates can also be defined by the generic formula in reverse order: A-(Q-L-SM)_n.

[0044] In the present disclosure, the term "linker" refers to a chemical moiety capable of linking the anti- TNFα antibody to a glucocorticosteroid. Linkers may be susceptible to cleavage (a "cleavable linker") thereby facilitating release of the glucocorticosteroid. For example, such cleavable linkers may be susceptible to peptidase-induced cleavage, at conditions under which the glucocorticosteroid and/or the antibody remains active. In particular, the cleavable linker component disclosed herein comprises a peptide comprising two to three amino acid residues (a dipeptide or tripeptide) and specifically to dipeptides and tripeptides selected from the group consisting of alanine-alanine (Ala- Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), and glycine- lysine (Gly-Lys). The peptide allows for cleavage of the linker by a protease, thereby facilitating release of the glucocorticosteroid upon exposure to intracellular proteases, such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnoi. 21:778-784). [0045] The term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0046] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0047] An "effective amount" of an immunoconjugate as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined in relation to the stated purpose,

[0048] The term "therapeutically effective amount" refers to an amount of an immunoconjugate effective to "treat" a disease or disorder in a subject or mammal. A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result.

[0049] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow' down, lessen one or more symptoms of, and/or slow or halt progression of a diagnosed pathologic condition or disorder (“therapeutic treatment”). Thus, those in need of therapeutic treatment include those already diagnosed with or suspected of having the disorder. Prophylactic or preventative measures refer to measures that prevent the develoμment of a targeted pathological condition or disorder (“prophylactic treatment”). Thus, those in need of prophylactic treatment include those prone to have the disorder and those in whom the disorder is to be prevented.

[0050] In the present disclosure, the term "C₁-C₅ alkyl" refers to a straight- or branched-chain aliphatic hydrocarbon containing from one to four carbon atoms that is either unsubstituted or substituted with one, two, or three substituents. In some embodiments, the C₁-C₅ alkyl is unsubstituted, and is methyl, ethyl, n-propyl, isopropyl, tert-butyl, isobutyl, or isopentyl. In other embodiments the C₁-C₅ alkyl is substituted with one, two or three substituents. In some embodiments, the alkyl has only one substituent. In some embodiments, the alkyl is a C₁ alkyl and has one substituent. In some embodiments, the alkyl is a C₁ alkyl and has three substituents, which are the same, e.g., CF₃.

II. Proteins for linkage to glucocorticoid receptor agonists

[0051] The present disclosure provides immunoconjugates containing glucocorticoid receptor agonists linked to anti-TNFα antibodies, in some embodiments, the antibody can hind to a target on the surface of a ceil and become internalized. In certain embodiments, the anti-TNFα antibodies bind to soluble TNFα and/or membrane bound TNFα. In some embodiments, the anti-TNFα antibody binds to TNFα on the surface of a ceil and become internalized. For example, US 2014/0294813, discloses anti-TNFα proteins that exhibit cellular internalization upon binding to cell surface human TNFα.

[0052] The full-length amino acid sequence for membrane bound human TNFα is: MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFCLLHFGVIGPQREEFPRDLSLI SPLAQAVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYS QVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEK GDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO:1 ). Soluble human TNFα contains amino acids 77-233 of SEQ ID NO:1.

[0053] In some embodiments, the anti-TNFα antibody binds to human TNFα.

[0054] In certain embodiments, the anti-TNFα antibody has one or more of the following effects: neutralizes human TNFα cytotoxicity in an in vitro L929 assay with an IC50 of 1X10 ' M or less; blocks the interaction of TNFα with p55 and p75 cell surface receptors; and/or lyses surface TNF expressing cells in vitro in the presence of complement.

[0055] In certain embodiments, the anti-TNFα antibody does not bind to TNF-beta.

[0056] Anti-TNFα antibodies include, for example, adalimumab, which is a recombinant human antibody.

[0057] In certain embodiments, an anti-TNFα antibody comprises sequences of adalimumab, e.g,, the complementarity-determining regions (CDRs), the variable heavy domain (VH), and/or the variable light domain (VL). Sequences of adalimumab are provided in Table 1.

Table 1: Adalimumab antibody region sequences

[0058] In certain embodiments, the anti-TNFα antibody comprises a heavy chain comprising the CDR set (CDR1 , CDR2, and CDR3) as set forth in SEQ ID Nos: 6, 7, and 8, and a light chain comprising a CDR set as set forth in SEQ ID Nos: 9, 10, and 11. In certain embodiments, the anti-TNFα antibody comprises the heavy chain of SEQ ID NO:2 and/or the light chain of SEQ ID NO:3. In certain embodiments, the anti-TNFα antibody comprises a heavy chain comprising a variable region as set forth in SEQ ID NO:4 and/or a light chain comprising a variable region as set forth in SEQ ID NO:5.

[0059] The anti-TNFα antibodies of the present disclosure can be recombinant polypeptides or synthetic polypeptides of an antibody. It will be recognized in the art that some amino acid sequences of the disclosure can be varied without significant effect of the structure or function of the protein. Thus, the disclosure further includes variations of the polypeptides which show substantial activity or which include regions of an antibody. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.

[0060] The anti-TNFα antibodies can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties can improve the solubility, the biological half life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for such exemplary moieties can be found in REMINGTON’S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0061] The isolated anti-TNFα antibodies described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g., Zoeller et al., Proc. Natl. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

[0062] In some embodiments a DNA sequence encoding an antibody of interest would be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest.

[0063] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding anti-TNFα antibodies. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coii, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA phages.

[0064] Suitable host ceils for expression of anti-TNFα antibodies include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Additional information regarding methods of protein production, including antibody production, can be found, e.g., in U.S. Patent Publication No. 2008/0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823.

[0065] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins tire generally correctly folded, appropriately modified and completely functional. Examples of suitable mammalian host cell lines include HEK-293 and HEK-293T, the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines including, for example, L cells, Cl 27, 3T3, Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5’ or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

[0066] The proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g,, ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione -S -transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0067] Recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can he employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonieation, mechanical disruption, or use of cell lysing agents.

[0068] Methods for purifying antibodies include, for example, those described in U.S. Patent Publication Nos. 2008/0312425, 2008/0177048, and 2009/0187005. Immuiioconjugates containing glucocorticoid receptor agonists

[0069] imniunoconjugates containing glucocorticoid receptor agonists are also provided. In some embodiments, an immunoconjugate binds to Fc gamma receptor. In some embodiments, an immunoconjugate is active in the GRE transmemhrane TNFα reporter assay (as used herein the "GRE transmembrane TNFα reporter assay” refers to the assay used as in Example 4 below). In some embodiments, an immunoconjugate shows reduced immunogenicity (reduced anti-drug immune response (ADA)) as compared to the protein in the immunoconjugate (e.g., the antibody) alone.

[0070] In one embodiment, disclosed herein is an antibody drug conjugate (ADC) compound having

Formula I:

wherein: A is adalimumab; L is a linker: Q is a heterobifunctional group; or Q is absent; and n is 1-10; and SM is a monovalent radical of a glucocorticosteroid as described herein. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0071] In some embodiments, SM is selected from the small molecules shown in examples SM-1 through SM-8. In some embodiments, L is a peptide comprising 1, 2, or 3 amino acids each independently selected from: glutamic acid, alanine, glycine, lysine, and serine. In some embodiments,

[0072] In some embodiments, ADCs are represented by a compound having Formula II:

wherein:

X is hydrogen or fluoro;

A is an antibody comprising a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 6, 7, and 8, and a light chain comprising a CDR set as set forth in SEQ ID Nos; 9, 10, andll;

R^A is fluoro, chloro or C₁-C₅ alkyl; and n is 2 or 4.

[0073] in certain embodiments, A is an antibody comprising a heavy chain comprising the amino acid sequence set for thin SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, A is adalimumab. In certain embodiments, R^A is selected from fluoro, chloro, and CH₃.

[0074] In certain embodiments, ADCs are represented by a compound having Formula II-A or P-B:

X is hydrogen or fluoro;

A is an antibody comprising a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 6, 7, and 8, and a light chain comprising a CDR set as set forth in SEQ ID Nos: 9, 10, and 11; wherein the wavy line indicates the atachment

point to A and the asterisk indicates the attachment point to L: L is a peptide comprising 1, 2, or 3 amino acids each independently selected from: glutamic acid, alanine, glycine, lysine, and serine;

R^A is fluoro, chloro or C₁-C₅ alkyl; and n is 2 or 4.

[0075] In some embodiments, the ADCs are represented by a compound having Formula II-A. In some embodiments, the ADCs fire represented by a compound having Formula II~B. in certain embodiments, A is an antibody comprising a heavy chain comprising the amino acid sequence set for thin SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3. In certain embodiments, A is adalimumab. in certain embodiments, R^A is selected from fluoro, chloro, and CH₃,

[0076] In some embodiments, the ADC of Formula II is a compound of Formulas II-1 to ll-25:

Table 2 : Antibody Drug Conjugates

Table 2; Antibody Drug Conjugates

Table 2; Antibody Drug Conjugates

Table 2; Antibody Drug Conjugates

Table 2; Antibody Drug Conjugates

[0077] In some embodiments, disclosed herein is a compound having any one of Formulae II-1 to ll-25, wherein A is an antibody comprising a heavy chain comprising the CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 6, 7, and 8, and a light chain comprising a CDR set as set forth in SEQ ID Nos: 9, 10, and 11, and n is 2 or 4. In some embodiments, disclosed herein is a compound having any one of Formulae II-1 to ll-25, wherein A is an antibody comprising a heavy chain comprising the amino acid sequence set for thin SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3, and n is 2 or 4. In some embodiments, disclosed herein is a compound having any one of Formulae II-1 to ll-25, wherein A is adalimumab, and n is 2 or 4.

[0078] In some embodiments, the ADC is the compound of Formulas II-3 or II- 10:

[0079] In some embodiments, the ADC is a compound of Formula II-A:

[0080] In some embodiments of the ADC of Formula II-A, A is adalimumab, R^A is fluoro, X is H or F, L is a peptide comprising 1, 2, or 3 amino acids, and n is 2 or 4.

IV, Methods of use and pharmaceutical compositions

[0081] Provided herein are conjugates having Formula II (e.g., having the formulas shown in Table 2) that can be used in vitro or in vivo. Accordingly, also provided are compositions, e.g., pharmaceutical compositions for in vivo use. These compositions include a conjugate or a glucocorticoid receptor agonist having the desired degree of puri ty in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. [0082] The compositions (e.g., pharmaceutical compositions) to be used for in vivo administration can he sterile, which can be accomplished by filtration through, e.g., sterile filtration membranes. The compositions (e.g., pharmaceutical compositions) to be used for in vivo administration can comprise a preservative.

[0083] The pharmaceutical compositions and antibody drug conjugates can be formulated in dosage forms and administered (e.g., via intravenous administration or infusion) in accordance with knowledge in the art.

[0084] Antibody drug conjugates and/or pharmaceutical compositions comprising antibody drug conjugates conjugates described herein can be useful in lysing a cell expressing surface TNFα (in vitro or in vivo) and/or for the treatment of diseases or disorders characterized by increased TNFα (e.g., increased TNFα in synovial fluid). In some embodiments, the antibody drug conjugates and/or compositions are useful in inhibiting cytokine release (in vitro or in vivo) and/or for the treatment of autoimmune or inflammatory diseases. In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of Crohn's disease (e.g., mild to moderate active Crohn's disease involving the ileum and/or the ascending colon and/or the maintenance of clinical remission of mild to moderate Crohn's disease involving the ileum and/or the ascending colon for up to 3 months). In some embodiments, the antibody drug conjugates and/or compositions Eire used for the treatment of ulcerative colitis (e.g., for the induction of remission in patients with active, mild to moderate ulcerative colitis). In some embodiments, the antibody drag conjugates and/or compositions are used for the treatment of rheumatoid arthritis (RA). In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of juvenile idiopathic arthritis (JA). In some embodiments, the antibody drag conjugates and/or compositions are used for the treatment of psoriatic arthritis (Ps A). In some embodiments, the antibody drag conjugates and/or compositions Eire used for the treatment of a spondyloarthropathy such as ankylosing spondylitis (AS) or axial spondyloarthritis (aixSpA). in some embodiments, the antibody drug conjugates and/or compositions Eire used for the treatment of adult Crohns' disease (CD). In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of pediatric Crohn's disease. In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of ulcerative colitis (UC). In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of plaque psoriasis (Ps). In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of hidradenitis suppurativa (HS). In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of uveitis. In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of Behcets disease. In some embodiments, the antibody drug conjugates and/or compositions are used for the treatment of psoriasis, including plaque psoriasis. Some embodiments comprise use of drag conjugates and/or pharmaceutical compositions for the preparation of a medicament for treating the diseases or disorders described herein.

Examples

[0085] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this disclosure.

[0086] Starting materials are commercially available, may be prepared by the procedures described herein, by literature procedures, or by procedures that would be well known to one skilled in the art of organic chemistry. Reagent/reactant names given are as named on the commercial bottle or as generated by IUPAC conventions, CambridgeSoft^® ChemDraw Ultra 12.0, CamhridgeSoft^®

Chemistry E-Notebook 11, or AutoNom 2000. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this disclosure.

Analytical Methods for Compound Synthesis and Characterization

[0087] Analytical data is included within the procedures below. Unless otherwise stated, all ¹H and ¹³C NMR data were collected on a Varian Mercury Plus 400 MHz or a Bruker AVIII 300 MHz instrument; chemical shifts are quoted in parts per million (pμm). HPLC analytical data are either detailed within the experimental or referenced to the table of LCMS and HPLC conditions, using the method provided in Table A:

Table At LCMS Conditions

[0088] Abbreviations used in the examples that follow are:

Example 1: General Synthetic Methods

Example 1-1. General Cysteine BrAC Conjugation Protocol

[0089] An approximate 5-20 mg/rriL solution of the desired antibody was prepared in PBS buffer, pH 6.0 - 7.4. A reducing agent of choice, such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP), was diluted or dissolved in solvents like H₂O, DMSO, DMA or DMF to give a solution with concentration ranging between 1 to 25 mM. Antibodies (anti-hTNF hlgG1) or anti-mTNF mlgG2a (801; McRae BL et al. J Crohns Colitis 10 (1): 69-76 (2016)) were then partially reduced by adding about 2-3.5 equivalents of reducing agent, briefly mixing, and incubating overnight at 0 - 4 °C. Tris buffer, pH 8- 8.5 (20-50 mM) was then added, followed by the linker-drug in DMSO or DMA (less than 15% total) and the mixture was incubated for 2 - 3 hours at room temperature (rt). Excess linker-drug and organic solvent were then removed by purification. Purified ADC samples were then analyzed by size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC) and reduced mass spectrometry.

Example 1-2. General Cysteine Maleimide Conjugation Protocol

[0090] An approximate 5-20 mg/mL solution of the desired antibody was prepared in PBS buffer, pH 6 - 7.4. A reducing agent of choice, such as TCEP, was diluted or dissolved in solvents like H₂O, DMSO, DMA or DMF to give a solution with concentration ranging between 1 to 25 mM. Antibodies (anti- hTNF higGl) or anti-mTNF mIgG2a (801; McRae BL et al. 1 Crohns Colitis 10 (1): 69-76 (2016)) were then partially reduced by adding about 2-3.5 equivalents of reducing agent, briefly mixing, and incubating overnight at 0 - 4 °C. Linker-drug in DMSO or DMA (less than 15% total) was added and the mixture was incubated for 0.5 - 1 hour at room temperature. Excess linker-drug and organic solvent were then removed by purification. Purified ADC samples were then analyzed by SEC, HIC and reduced mass spectrometry.

Example 1-3. General Maleimide Hydrolysis Protocol

[0091] An approximate 1-15 mg/mL solution of ADC was adjusted to pH 8 - 9 with a hydrolysis buffer of choice, for example, borate buffer. The mixture was incubated for 1 - 3 days at room temperature. Hydrolyzed ADC was then buffer exchanged to a buffer of choice, such as PBS. Final ADC sample was analyzed by SEC, HIC and reduced mass spectrometry.

VI. Methods of making immunoconjugates and synthetic intermediates

[0092] The general synthesis of the immunoconjugates of the disclosure is described in Scheme 1 :

[0093] In Scheme 1, R^A, and A are as described herein; PG refers to a protecting group; R' and R" are each independently selected from a side chain of a naturally occurring amino acid e.g., methyl, isopropyl, or non-natural amino acid, e.g,, -CH₂CH₂CH₂N(H)C(=O)NH₂, and R'" is an unsubstituted or substituted C_{1 -4} alkyl. SMALL MOLECULE SYNTHESIS Synthesis of Intermediates 1-6

[0094] Intermediate 1: Synthesis of (8S,9R,10S,11S, 13S,14S,16R, 17R)-9-fluoro-ll, 17- dihydroxy- 10,13, 16-trimethyl-3-oxo-6, 7, 8, 9, 10,11,12,13,14,15, 16,17~dodeeahydro-

3H-cyclopenta[a]phenanthrene-17-carboxylic add

[0095] Periodic acid (59.2 g, 260 mmol) in water (150 mL) was added to a suspension of (8S,9R, 10S,11S,13S, 14S, 16R,17R)-9-fluoro-l 1 , 17 -dihydroxy- 17-(2-hydroxy acetyl) - 10, 13,16-trimethyl-6,7,8,9, 10,11 , 12, 13,14, 15, 16, 17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one (30 g, 76 mmol) in THF (400 mL). The resulting solution was stirred at 30 °C for 1 h. The THF was removed under reduced pressure to leave an aqueous suspension, which was filtered, washed with water and dried under vacuum to give the title compound (27 g, 71.3 mmol, 93 % yleld) as a white solid. ^!H NMR (400MHz, DMSO-d₆) d 7.28 (d, j=10.1 Hz, 1 H), 6.20 (dd, j=1.8, 10.1 Hz, 1H), 5.99 (s, 1H), 5.22 (d, j=1.8 Hz, 1H), 4.59 (s, 1H), 4.11 (d, j=10.1 Hz, 1H), 2.81 (ddd, j=4.0, 6.9, 10.7 Hz, 1H), 2.70 - 2.53 (m, 1H), 2.41 - 2.20 (m, 2H), 2.06 - 1.91 (m, 2H), 1.81 - 1.71 (m, 1H), 1.61 (q, j=11.5 Hz, 1H), 1.51 (d, j=14.6 Hz, ! H i. 1.48 (s, 3H), 1.33 (dq, j=4.6, 12.7 Hz, 1H), 1.05 (dt, j=4.4, 7.9 Hz, 1H), 0.99 (s, 3H), 0.84 (d, j=7.1 Hz, 3H).

[0096] Intermediate 2; Synthesis of (8S,9R,10S,llS,13S,14S,16R,17R)-9-fluoro~-11,- 17- dihydroxy- 10,13, 16- trimethyl-3-oxo-6, 7, 8, 9, 10, 11,12,13,14,15, 16,17-dodecahydro-

3H-cyclopenta[a]phenanthrene-17-carbothioic S-acid

[0097] 1,1’ -Carbonyldiimidazole (18.85 g, 116 mmol) was added to a solution of

(8S,9R, 10 S , 11S , 13S, 14S,16R, 17R)-9-fluoro-l 1 , 17-dihydroxy - 10, 13,1 6-trimethyl-3- oxo-6, 7, 8, 9, 10,1 l ,12, 13, 14,15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17- carboxylic acid (22 g, 58.1 mmol) in DMF (440 mL) and the mixture was stirred under nitrogen at 30 °C for 4 h. Hydrogen sulfide was bubbled into the reaction mixture for 30 min. After 4 h the reaction mixture was poured into 2 M aqueous HC1 (100 mL) and ice (100 mL), the precipitate was collected and dried to give (13.5 g, 34.2 mmol, 58.9 % yleld) as white solid. LCMS (Table A, method g) Rt = 3.080 min; m/z = 395.1 | VI + H ^' ] . ¹H NMR (400MHz, DMSO-d₆) d 7.27 (d, j=10.1 Hz, 1H), 6.20 (d, j=10.1 Hz, 1 H), 5.98 (s, 1H), 5.27 (br. s., 1H), 4.12 (d, j=10.1 Hz, 1 H), 2.96 - 2.76 (m, 1H), 2.60 (dt, j=5.7, 13.5 Hz, 1H), 2.41 - 2.18 (m, 2H), 2.11 - 1.98 (m, 2H), 1.80 - 1.70 (m, 1H), 1.67 - 1.53 (m, 2H), 1.47 (s, 3H), 1.39 - 1.24 (m, 1H), 1.13 - 1.00 (m, 1H), 0.94 (s, 3H), 0.79 (d, j=7.1 Hz, 3H).

[0098] Intermediate 3: Synthesis of (8S,9R,10S,11S, 13S,14S,16S, 17R)-9-fluoro-ll, 17- dihydroxy- 10, 13, 16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro- 3H-cyclopenta[a]phenanthreiie-17-carboxylic acid

[0099] A solution of periodic acid (40.7 g, 178 mmol) in water (150 mL) was added to a suspension of (8S,9R, 10S,11S,13S, 14S, 16S,17R)-9-fluoro-l 1 , 17 -dihydroxy -17 -(2- hydroxyacetyl)-l 0,13, 16 -trimethyl-6, 7, 8,9, 10, 1 l , 12, 13, 14,15,16, 17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one (20 g, 51.0 mmol) in THF (300 mL). The resulting solution was stirred at 30 °C for 1 h. THF was removed under reduced pressure to leave an aqueous suspension, which was filtered and the solid was washed with w'ater and dried to give the title compound (18 g, 47.6 mmol, 93 % yleld) as a white solid. ¹H NMR (400MHz, DMSO-d₆) d 7.28 (d, j=10.1 Hz, 1H). 6.21 (d, j=10.1 Hz, 1H), 6.00 (br. s., 1H), 5.19 (hr. s., 1H), 4.13 (br. s., 1H), 2,63 (t, j=12.8 Hz, 1H), 2.47 - 2.2,5 (m, 2H), 2.12 - 1.94 (m, 2H), 1.92 - 1.65 (m, 3H), 1.49 (br. s., 4H), 1.36 (d, j=1 1.9 Hz, 1H), 1.13 (d, j=6.6 Hz, 3H), 1.06 (br. s., 3H), 0.99 (d, j=8.4 Hz, 1H).

[0100] Intermediate 4: Synthesis of (8S, 9R,10S,11S,13S, 14S,16S,17R)-9-fluoro-ll, 17- dihydroxy- 10, 13, 16-trimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro- 3H-cyclopenta[a]phenanthrene-17-carbothioic S-acid

[0101] 1,1’ -Carbonyldiimidazole (15.43 g, 95 mmol) was added to a solution of

(8S,9R, 10 S , 118,138, 14S,16S,17R)-9-fluoro-l 1 ,17 -dihydroxy- 10, 13, 16-trimethyl -3- oxo-6, 7, 8, 9, 10,11 ,12,13,14,15,16,17 -dodecahydro-3H-cyclopenta[a]phenanthrene- 17- carboxylic acid (18 g, 47.6 mmol) in N,N-dimethylformamide ( 100 mL). The mixture was stirred under nitrogen at 30 °C for 4 h. After that, hydrogen sulfide was bubbled into the reaction mixture for 30 min and then was stirred for another 4 h. The resulting mixture was poured into a mixture of 2 N aqueous HC1 (50 mL) and ice (50 mL). The precipitate was filtered and dried to give the title compound (8.8 g, 21.41 mmol, 45.0 % yleld) as white solid. LCMS (Table A, method h) Rt = 2.292 min: m/z = 395 [ M+H ⁺ ] . ¹H NMR (400MHz, DMSO-d₆) d 7.28 (d, j=10.1 Hz, 1H), 6.22 (dd, j=1.3,

10.1 Hz, ¹H ). 6.08 - 5.96 (m, ¹H ). 5.23 (hr. s., 1H), 4.14 (d, j=9.3 Hz, 1H), 2.63 (dt, j=5.7, 13.2 Hz, 1 H), 2.49 - 2.27 (m. 2H), 2.24 - 2.03 (m, 2H), 1.99 - 1.75 (m. 3H),

I.56 - 1.44 (m, 4H), 1.43 - 1.30 (m, 1H), 1.05 (d, j=7.5 Hz, 4H), 0.97 (s, 3H).

[0102] Intermediate 5: Synthesis of (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro-

11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carboxylic acid

[0103] A solution of periodic acid (16.66 g, 73.1 mmol) in water (75 mL) was added to a 30 °C solution of (6S,8S,9R, 10S,1 IS, 13S, 14S, 16R,17R)-6,9-difluoro-l 1 , 17-dihydroxy 17-(2-hydroxyacetyl)-10, 13,16-trimethyl-6,7,8,9, 10, 11 , 12, 13,14, 15, 16, 17- dodecahydro-3H-cyclopenta[a]phenanthren-3-one (10 g, 24.36 mmol) in THF (135 mL). Three additional reactions were set up, and all four were stirred at 30 °C for 1 h. The parallel reactions were combined, and THF was removed under reduced pressure. The product was filtered, washed with water (400 mL), then dried under high vacuum to give the title compound (37 g, yleld 91 %) as wb ite solid. ¹H NMR (400 MHz, MeOH-d₄) d 0.92 (d, j=7.21 Hz, 3 H) 1.09 (s, 3 H) 1.23 (ddd, j=12.26, 8.16. 4.16 Hz,

1 H) 1.56 - 1.68 (m, 4 H) 1.68 - 1.86 (m, 2 H) 1.87 - 1.92 (m, 1 H) 2.22 - 2.39 (m. 3 H i

2.45 - 2.66 (m, 1 H) 2.98 - 3.13 (m, 1 H) 3.68 - 3.81 (m, 1 H) 4.22 - 4.33 (m, 1 H) 5.43

- 5.69 (m, 1 H) 6.25 - 6.40 (m, 2 H) 7.36 (dd, j=10.09, 1.41 Hz, 1 H).

[0104] Intermediate 6: Synthesis of (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-difluoro- 11, 17-dihydroxy -10,13,16- trimethyl-3-oxo-6, 7, 8, 9, 10,11,12, 13,14, 15,16,17- dodecahydro-3H-cyclopenta[a]phenanthrene-17-carbothioic S-acid

General Procedure A

[0105] Example Number SM-1: Synthesis of (8S,9R,10S,11S,13S J4S,16R,17R)-9- fluoro- 17- (((fluoromethyl)thio)carbonyl)-11-hydroxy -10,13, 16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]p-he1yna7nthl-reyn l 3 - amino-4-fluorobenzoate

[0107] Di-tert-butyl dicarbonate (20.33 mL, 88 mmol) was added to a solution of 3 -amino- 4-fluorobenzoic acid (4.5 g, 29.0 mmol) and sodium hydroxide (2.321 g, 58.0 mmol) in 1 : 1 1 ,4-dioxane/water (50 mL). Additional di-tert-butyl dicarbonate (19.89 mL, 86 mmol), 1 ,4-dioxane (100 mL) and water (100 mL) were added after 29 h to the heterogeneous suspension, and stirring was continued for another 24 h, whereupon the volatile solvents were removed under reduced pressure. The resulting aqueous mixture was washed with EtOAc (100 mL), cooled in an ice bath, and treated with 1 M aqueous HCl (100 mL) to provide an off-white precipitate. This material was filtered and washed with portions of water (5 x 20 mL) and then dried in the vacuum oven overnight at 60 °C to give the title compound as an off-white powder (4.5185 g, 17.70 mmol, 61.0 % yleld). LCMS (Table A, method b) Rt = 0.61 min; MS m/z = 278 [M+Na⁴] . ^!H NMR (400 MHz, DMSO-t U) d 13.01 (s, 1H), 9.17 (s, 1H), 8.28 (dd, J = 7.8, 2.1 Hz, 1H), 7.68 (ddd, J = 8.5, 4.7, 2.2 Hz, 114), 7.31 (dd, J = 10.6, 8.5 Hz, 1H), 1.47 (s, 9H).

[0108] Step 2; Synthesis of (8S,9R, 10S,11S,13S, 14S, 16R.17R)-9-fluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3 - ((tert--butoxycarbonyl)amino)-4-fiuorobenzoate

[0109] ACA-Diisopropylethy! amine (2.2 mL, 12.60 mmol) was added to a room temperature solution of HATH (4.3514 g, 11.44 mmol^') and 3-((tert-butoxycarbonyl)amino)-4- fluorobenzoic acid (2.97 g, 11.64 mmol) in N,N-dimethylformamide (60 mL). After 15 min, (8S,9R, 10S, 11 S , 13S, 14S, 16R,17R)-9-fluoro-l 1 , 17 -dihydroxy- 10,13, 16-trimethyl - 3-oxo-6,7,8,9,10,l 1 , 12, 13,14, 15,16,17-dodecahydro-3H-cyc1openta[a]phenanthrene-17- carbothioic S-acid (4.76 g, 12.07 mmol) and N,N-diisopropylethylamine (2.2 mL, 12.60 mmol) were added. This mixture was stirred for 150 min, whereupon fiuoroiodomethane ( 1.5 mL, 22.19 mmol) was added in one portion. After 7 min, the reaction mixture was diluted with EtOAc (350 mL), and then washed with 1 M aqueous HCl (2 x 100 mL), brine (10 mL), dried (Na₂SO₄), and solvent was removed under reduced pressure. Purification by chromatography (silica, 330 g) eluting with a gradient of 0-10% MeOH/DCM gave the title compound as a white solid (6.42 g, 78% yleld). LCMS (Table A, method b) Rt = 1.08 min; MS m/z = 608 [M-tBu] , 1327 [2M+H⁺]. ¹H NMR (400 MHz, DMSO-d₆) d 9.22 (s, 1H), 8.32 (d, j= 7.5 Hz, 1H), 7.55 (ddd, J = 8.6, 4.6, 2.2 Hz, 1H), 7.37 (dd, J = 10.5, 8.6 Hz, 1H), 7.25 (d, J = 10.1 Hz, 1H), 6.20 (dd, J = 10.1 , 1.9 Hz, 1H), 5.99 (d, J = 1.9 Hz, 2H), 5.86 (s, 1H), 5.50 (dd, J - 4.5, 1.7 Hz, 1H), 4.23 (dd, j= 8.5, 4.0 Hz, 1H), 3.35 (s, 1H), 2.67-2.55 (m, 1H), 2.47-2.30 (m, 1H), 2.34-2.11 (m, 3H), 1.90 (s, 1H), 1.80 (dt, j= 11.9, 6.5 Hz, 1H),

1.42 (d, J = 37.3 Hz, 13H), 1.24 (ddt, J = 14.3, 10.2, 5.2 Hz, 2H), 1.05 (s, 3H), 0.88 (d, j= 7.1 Hz, 3H).

[0110] Step 3: Synthesis of (8S,9R,10S, 1 lS, 13S,14S, 16R, 17R)-9-fluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16- trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- a m i n o - 4 - 11 u o r o b e n z o a t e

[0111] TFA (IS mL, 64.9 mmol) was added to a room temperature solution of

(8S,9R, 10S,11S,13S, 14S, 16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)-11- hydroxy-10, 13, 16 -trimethyl -3 -ox o-6,7, 8,9,10,1 1 ,12, 13,14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl 3-((½ri-butoxyearbonyl)amino)-4-fluorobenzoate (6.42 g, 9.67 mmol) in DCM (50 mL). After 180 min, solvent was removed under reduced pressure. The residue was redissolved in toluene (250 mL), which was removed under reduced pressure to drive off TFA. This residue was redissolved in EtOAc (300 mL), and then washed sequentially with saturated aqueous NaHCO₃ (150 mL), 10 wt% aqueous NaOH (2 x 100 mL), brine (150 mL), dried (Na₂SO₄), and solvent was removed under reduced pressure to give an off-white foam. Purification by chromatography (silica, 40 g) eluting with a gradient of 20-90% MTBE/heptanes gave the title compound as a white solid that was dried under vacuum for 14 h (3.535 g,

6.27 mmol, 65% yleld). LCMS (Table A, method c) Rt = 0.98 min; MS m/z = 564 [M+H⁴ ]. ¹H NMR (501 MHz, DMSO-d₆) d 7.34-7.27 (m, 2H), 7.17 (dd, J = 11.3, 8.4 Hz, 1 H ). 7.03 (ddd, J = 8.3, 4.4, 2.2 Hz, 1H), 6.26 (dd, J = 10.1 , 1.9 Hz, 1H), 6.05 (d, j= 1.7 Hz, 1H), 5.95 (d, j - 50.2 Hz, 2H), 5.55 (dd, j= 4.5, 1.6 Hz, 1H), 5.50 (s, 2H), 4.31 4.23 (m, 1H), 3.38 (ddd, J 10.6, 6.9, 3.3 Hz, 1H), 2.65 (td, j= 13.6, 6.2 Hz,

1H), 2.44 (ddd. j= 16.8, 11.8, 5.4 Hz. 1H), 2.39 2.32 (m, 1H), 2.26 2.10 (m, 2H), 1.96 1.85 (m, 2H), 1.84 (dd, j= 12.3, 5.7 Hz, 1 H), 1.51 (s, 3H), 1.44 (qd, j= 12.9, 5.2 Hz, 1H), 1.27 (ddd, j= 11.8, 7.8, 3.4 Hz, 1H), 1.06 (s, 3H), 0.90 (d, j= 7.1 Hz, 3H). 11neral Procedure B

[0112] Example Number SM-2: Synthesis of (6S,8S,9R,10S,HS,13S,14S,16R,17R)-6,9- difluoro-17-(((fluoromethyl)thio)carbonyl)-ll-hydroxy-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- amino-4-fluorobenzoate

[0113] Step 1: Synthesis of (6S,8S,9R, 10S, 11 S,13S,14S, 16R, 17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((tert-butoxycarbonyl)amino)-4-fluorobenzoate

[0114] YGY-Diisopropylethylamine (0.2 mL, 1.145 mmol) was added to a room temperature solution of HATH (l -[bis(dimethylamino)methylene]-lH- 1 ,2,3 -triazolo[4,5- bjpyridinium 3-oxid hexafluorophosphate) (0.425 g, 1.118 mmol) and 3 -{{tert- butoxycarbonyl)amino)-4-fluorobenzoic acid (0.285 g, 1.118 mmol) in N,N-DMF (5.5 mL). After 10 min, (6S.8S.9R, 10S.1 IS, 13S, 14S,16R,17R)-6,9-difluoro-l 1 , 17- dihydroxy- 10, 13,16 -trimethyl-3 -oxo-6, 7, 8, 9, 10, 1 l ,12, 13, 14,15, 16, 17-dodecahydro-3H- eyclopenta[a]phenanthrene-17-carbothioic S-acid (0.652 g, 1.623 mmol) was added in two portions, followed by LnU-diisopropylethylamine (0.3 mL, 1.718 mmol). This mixture was stirred for 60 min, whereupon (fluoromethyl)(phenyl)(2,3,4,5 - tetramethylphenyl)sulfonium tetrafluoroborate ( Org . Lett. 2008, 10, 557-560) (0.405 g, 1.1 18 mmol) was added in one portion. After 15 min, the reaction mixture was diluted with EtOAc (50 mL), and then washed with a saturated aqueous, solution of NaHCO₃ (20 niL), brine (20 mL), dried (Na2S(>4), and solvent was removed under reduced pressure. Purification by chromatography (silica, 40 g) eluting with a gradient of 0- 10% MeOH/DCM gave the title compound as a light red foam (0.844 g). LCMS (Table A, method c) Rt = 1.08 min; MS rn/z = 582 [M+H⁺].

[0115] Step 2: Synthesis of (6S,8S,9R, 10S, l lS,13S,14S, 16R, 17R) -6,9--difluoro-17- (((fiuoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16- trimethyl-3 -oxo- 6,7,8,9,10, 11 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- amino-4-fluorobenzoate 2,2,2-trifluoroacetate

[0116] A solution of (6S,8S,9R, 10S,11S, 13S, 14S,16R, 17R)-6,9-difluoro-17- (((fIuoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl~3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-eyclopenta[a]phenanthren-17-yl 3- ((tert-butoxycarbonyl)amino)-4-fluorobenzoate (420 mg, 0.616 mmol) in DCM (0.5 mL) at 0 °C was treated with TFA (1.19 mL, 15.40 mmol) and the mixture was stirred at room temp for 1 h. Solvent was removed under reduced pressure, and the residue was dissolved in MeCN (1.5 mL). Purification by RP HPLC on a Phenomenex Cl 8(2) 10 micron column (250 x 30 mm), eluting with a gradient of MeCN (A) and 0.1 % TFA in water (B) at a flow rate of 60 mL/min (0-5.0 min 15% A, 5.0-15.0 min linear gradient 15-95% A, hold at 95% for 10 min) provided the title compound as a white solid after freeze drylng ( 154 mg, 0.256 mmol, 43 % yleld). LCMS (Table A, method b) Rt = 0.98 min; MS m/z = 582 [M+H⁺]. ¹H NMR (400 MHz, DMSO-d₆ d 7.29 - 7.20 (m, 2H), 7.10 (dd, J = 11.1 , 8.4 Hz, 1H), 6.99 (ddd, j= 8.4, 4,5, 2.2 Hz, 1H), 6.28 (dd, j= 10.2, 1.9 Hz, 1H), 6.10 (d, j= 1.9 Hz, 1 H), 5.88 (d, j= 50.1 Hz, 2H), 5.72 - 5.47 (m, 2 H), 4.22 (d, J = 9.5 Hz, 1H), 3.39 - 3.21 (m, 1H), 2.63 - 2.51 (m, 1H), 2.29 - 2.11 (m, 3H), 1.96 - 1.82 (m, 2H), 1.61 - 1.46 (m, 1H), 1.45 (s, 3H), 1.25 (ddd, j= 11.3, 8.5, 5.1 Hz, 1H), 1.00 (s, 3H), 0.85 (d, j= 7.2 Hz, 3H). General Procedure C

[0117] Example Number SM-3, Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17R)-6,9- difluoro-17-(((fluoromethyl)thio)carbonyl)-ll-hydroxy-10,13,16-trimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- amino-4-methylbenzoate

[0118] Step 1: Synthesis of 4-methyl-3-nitrobenzoyl chloride

[0119] To a solution of 4-methyl-3-nitrobenzoic acid (1.5 g, 8.28 mmol) and thionyl chloride (5 mL, 68.5 mmol) in toluene (30 mL) was added N,N-dimethylformamide (0.1 mL. 0.828 mmol) at room temperature. The resulting mixture was stirred at 50 °C for 12 h and then concentrated under vacuum to give the title compound as a pale yellow oil (1.5 g, 86% yleld).

[0120] Step 2: Synthesis of (6S,8S,9R,10S11 S, 13S, 14S,16R,17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16- trimethyl-3 -oxo- 6,7,8,9,10, 11 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 4- methyl-3-nitrobenz.oate

[0121] To a solution of (6S,8S,9R,10S,1 IS, 13S,14S,16R, 17R)-6,9-difluoro- l 1 ,17- dihydroxy- 10, 13,16 -trimethyl-3-oxo-6,7, 8, 9, 10, 1 l ,12,13,14,15, 16,17-dodecahydro-3H- cyclopenta[a]phenantbrene-17-carbothioic S-acid (intermediate 6, above) (300 mg, 0.727 mmol) and N,N-diisopropylethylamine (0.3 mL, 1.455 mmol) in THF (5 mL) at 0 °C was added 4-methyl-3-nitrobenzylchloride (product of step 1 , above) (145 nig, 0.727 mmol). The resulting mixture was stirred at room temperature for 2 h. A solution of bromof 1 u or ome th an e in 2-butanone (33% w/v solution, 0.5 mL) was added, and the resulting mixture was stirred at for 2 h. The mixture was filtered through Celite^®, and the filtrate was concentrated under reduced pressure to give a crude product that was purified by C18 HPLC using a solvent gradient of 55- 100% MeCN in 0.075% aqueous TFA to give the title compound as a colorless solid (205 mg, 52% yleld).

methylbenzoate

[0122] To a solution of (6S.8S,9R,10S,l lS, 13S,14S,16R.17R) -6,9--difluoro~17- (((fluoromethyl)thio)carbonyl)-11-hydroxy-10, 13, 16-trimethyl -3 -oxo- 6,7,8,9,10, 1 1 , 12,13, 14, 15, 16,17 -dodecah ydro- 3H-cyclopenta [ajphenanthren-17-yl 4- methyl-3-nitrobenzoate (product of example 5, step 2 above) (30 mg, 0.049 mmol) in MeOH (5 mL) and water (1.7 mL) was added iron (41.4 mg, 0.741 mmol) and ammonium chloride (39.6 mg, 0.741 mmol). The resulting mixture was stirred at 80 °C for 2 h. The mixture was filtered through Celite^®, and the filtrate was concentrated under reduced pressure to give a crude product that was purified by C18 HPLC eluting with a solvent gradient of 30- 100% MeCN in 0.075% aq TFA to give the title compound as a colorless solid (25 mg, 85% yleld). LCMS (Table A, method c) Rt =

TABLE B: SMALL MOLECULE EXAMPLES 4-8

[0123] Examples in the following table were prepared according the general methods described above.

DRUG-LINKER SYNTHESIS

METHOD DL-A

[0124] Example DLlSynthesis of (8S,9R,10S,llS,13S,14S,16R,17R)-9-fluoro-17- (((fluoromethyl)thio)carbonyl)-ll-hydroxy-10,13,16-trimethyl-3-oxo-

6,7,8,9,10,11,l2,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-

((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)acetamido)propanamido)propanamido)-4-fluorobenzoate

[0125] Step 1 : Synthesis of Allyl 3-amino-4-fluorobenzoate

[0126] Allyl bromide (5.84 mL, 67.5 mmol) was added to a room temperature suspension of 3-amino-4-fluorobenzoic acid (9.97 g, 64.3 mmol) and potassium carbonate (10.21 g, 73.9 mmol) in N,N-dimethyl formamide (90 mL) and was stirred at room temperature for 72 h. The reaction was diluted with EtOAc (400 mL), washed with a brine solution (3 X 300 mL), dried (NaiSCL), and solvent was removed under reduced pressure. Purification by chromatography (silica, 240 g) eluting with a gradient of 0- 60% EtOAc/heptanes gave the title compound as a light amber oil (11.8 g, 60.5 mmol, 94% yleld). LCMS (Table A, method b) Rt = 0.76 min; m/z = 196 [ M+H ⁺ ]· *H NMR (400 MHz, DMSO-d₆) d 7.45 (dd, j= 8.8, 2.2 Hz, 1H), 7.16 (ddd, J = 8.5, 4.8, 2.2 Hz, 1H), 7.10 (dd, J = 11.1 , 8.4 Hz, 1H), 6.02 (ddt, J = 17.3, 10.6, 5.4 Hz, 1H), 5.45 (s, 2H), 5.38 (dq, J = 17.3, 1.7 Hz, 1H), 5.2,6 (dq, J = 10.5, 1.4 Hz, 1H), 4.75 (dt, J = 5.4, 1 .5 Hz, 2H).

[0127] Step 2: Synthesis of (S)-Allyl 3-(2-((tert-butoxycarbonyl)amino)propanamido)-4- fluorobenzoate

[0128] 1 -Propanephosphonic anhydride (T3P) (50% in EtOAc) (32.7 g, 51.4 mmol) was added drop-wise over 4 min to a -30 °C solution of Boc-Ala-OH (4.59 g, 24.23 mmol), allyl 3-amino-4-fluorobenzoate (4.730 g, 24.23 mmol), and pyridine (6.0 mL, 74.2 mmol) in EtOAc (25 mL). The cold bath was removed once the addition was complete and stirring was continued for 14 h. The reaction mixture was diluted with EtOAc (300 ml.,), washed with 1 M aqueous HC1 (2 x 150 mL), saturated aqueous NaHCO_? (2 x 150 mL), brine solution (50 mL), and dried (Na₂SO₄). Solvent was removed under reduced pressure to give the title compound as an off-white solid (8.829 g, 24.10 mmol, 99% yleld), which was carried forward without further purification. LCMS (Table A, method c) Rt = 0.90 min; m/z = 367 [M+H⁺], *H NMR (400 MHz, DMSO-d₆) d 9.84 is, 1H), 8.64-8.57 (m, 1H), 7.78 (ddd, J = 8.6, 4.8, 2.3 Hz, 1H), 7.42 (dd, J = 10.6, 8.6 Hz, 1H), 7.18 (d, j= 7.2 Hz, 1H), 6.04 (ddt, j= 17.3, 10.7, 5.4 Hz, 1H), 5.40 (dq, j= 17.2, 1.7 Hz, 1H), 5.28 (dq, j= 10.5, 1.4 Hz, 1H), 4.81 (dt, j= 5.4, 1.5 Hz, 2H), 4.30- 4.22 (m, 1H), 1.39 (s, 9H), 1.28 id, J = 7.1 Hz, 3H). [0129] Step 3: Synthesis of allyl 3-((S)-2-((S)-2-((tert- butoxycarbonyl)amino)propanamido)propanamido)-4-fluorobenzoate

[0130] TFA (20 mL, 260 mmol) was added to a room temperature solution of allyl (S)-3-(2-((tert-butoxyearbonyl)amino)propanamido)-4-fluorobenzoate (4.4 g, 12,01 minol) in DCM (60 mL). The reaction was complete within 45 min, whereupon solvent was removed under reduced pressure. The residue was redissolved in toluene (2 x 150 mL), which was removed under reduced pressure to drive off TFA. This crude product was dissolved in N,N-dimethyl formamide (50 mL) and treated sequentially with N,N- diisopropylethylamine (3.0 mL, 17.18 mmol) and Boc-L-alanine Ns-uccinimidyl ester (3.4585 g, 12.08 mmol). The coupling was complete within 30 min. Water (175 mL) was added and the reaction mixture was cooled with an ice bath (2 °C) and stirred for 30 min. The resulting white precipitate was filtered to give the title compound as a white solid, which was dried in a vacuum oven overnight at 60 °C (5.06 g, 1 1 .57 mmol, 96% yleld). LCMS (Table A, method c) Rt - 0.87 min; m/z = 438 [ M+H ⁺ ] . ¹H NMR (400 MHz, DMSO-d₆) d 9.87 (s, 1H), 8.54 (d, J - 7.4 Hz, 1H), 8.03 (d, J = 7.0 Hz,

1H), 7,75 (ddd, j= 8.6, 4.8, 2.3 Hz, 1H), 7.39 (dd, j= 10.5, 8.6 Hz, 1 H), 6.91 (d, j= 7.5 Hz, 1H), 6.00 (ddt, j= 17.2, 10.6, 5.4 Hz, 1H), 5.36 (dq, j= 17.2, 1.7 Hz, 1H), 5.25 (dq, j= 10.5, 1.5 Hz, 1 H), 4.77 (dt, j= 5.5, 1.5 Hz, 2H), 4.51 (t, j= 7.1 Hz, 1H), 3.96 (t, j= 7.3 Hz, 1H), 1.33 (s, 9H), 1.29 (d, J = 7.1 Hz, 3H), 1.15 (d, j= 7.2 Hz,

3H) .

[0131] Step 4: Synthesis of 3-((S)-2-((S)-2-((tert-

Butoxycarbonyl)amino)propanamido)propanamido)-4-fluorobenzoic acid

Morpholine (2.0 mL, 22.86 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.139 g, 0, 120 mmol) were added to a room temperature solution of allyl 3-((S)-2-((S)-2-((tert- butoxycarhony!)amino)propanamido)propanamido)-4-fluorobenzoate (5.06 g, 11.57 mmol) in THF (45 mL). LCMS indicated that the reaction was complete within 3 min. The reaction was diluted with EtOAc (300 mL), washed with 1 M aqueous HC1 (2 x 100 mL), brine solution (100 mL), dried (Na₂SO₄), and solvent was removed under reduced pressure. The title compound was isolated as a white solid (4.763 g, 11.99 mmol, 104% yleld) and was used in the subsequent step without further purification. LCMS (Table A, method c) Kt = 0.73 min; m/z - 397 [M+H⁺], ^!H NMR (400 MHz, DMSO -d₆) d 13.02 (s, 1 H), 9.81 (s, 1H), 8.46 (dd, j= 7.7, 2.1 Hz, 1 H), 8.03 (d„ j= 7.1 Hz, 1H), 7.70 (ddd, j= 8.5, 4.8, 2.2 Hz, 1H), 7.34 (dd, j= 10.6, 8.6 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 4.50 (q, J = 7.2 Hz, 1H), 4.03-3.91 (m, 1H), 1.33 (s, 9H), 1.29 (d, j= 7.0 Hz, 3H), 1.15 (d, j= 7.2 Hz, 3H).

[0132] Step 5: Synthesis of (8S,9R,10S, l lS, 13S,14S, 16R, 17R)-9-Fiuoro-17- (((fIuoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-eyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-((icrt-butoxycarbonyT)amino)propanamido)propanamido)-4- fluorobenzoate

[0133] N,N-Diisopropylethylamine (1.0 mL, 5.73 mmol) was added to a yellow_' solution of HATU (1.817 g, 4.78 mmol) and 3-((S)-2-((S)-2-((tert- butoxycarbonyl)amino)propanamido)propanamido)-4-fluorobenzoic acid (1.996 g, 5.02 mmol) in DMF (25.1 mL). After 7 min, (8S.9R, 10S, 11 S,13S,14S, 16R, 17R)-9-fluoro-

dodecahydro-3H-cyclopenta[a]phenanthrene- 17-carbothioic S-acid (2.1793 g, 5.52 mmol) and N,N- diisopropylethylamine (1.0 mL, 5.73 mmol) were added. The reaction was stirred for 120 min, whereupon (fluoromethyl)(phenyl)(2, 3,4,5- tetramethylphenyl)sulfonium tetrafluoroborate ( Org . Lett. 2008, 10, 557-560) (2.00 g, 5.52 mmol) was added in one portion. The reaction was stirred for an additional 20 min, whereupon it was diluted with EtOAc (300 mL), washed with 1 M aqueous HC1 (2 x 100 mL), brine solution (10 mL), dried (Na₂SO₄), and solvent was removed under reduced pressure. Purification by chromatography (silica, 240 g) eluting with a gradient of 0-10% MeOH/DCM gave the title compound as a white solid (3.076 g, 3.77 mmol, 75% yleld). LCMS (Table A, method c) Rt = 0.99 min; m/z = 806 [M+H⁺], ¹H

[0134] Step 6: Synthesis of (8S,9R, 10S,l lS,13S, 14S, 16R,17R)-9-Fluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl -3 -oxo- 6,7,8,9,10, 1 1 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-(2-(2,5-dioxo-2,5-dihydro-lH-pyrroi-l - yl)acetamido)propanamido)propanamido)-4-fluorobenzoate

[0135] TFA (0.500 mL) was added to a solution of (8S,9R, 10S,l ! S,13S, 14S, 16R,! 7R)-9~ fluoro-17-(((fluoromethyl)thio)carbonyl)-l l-hydroxy-10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-(tert- butoxycarbonyl)amino)propanamido)propanamido)-4- fiuorobenzoate (0.2,875 g, 0.357 mmol) in DCM (2.0 mL). After 40 min, solvent was removed under reduced pressure to give a syrup, which was redissolved in toluene (30 mL) and then concentrated to drive off TFA. The resulting residue was dissolved in N,N- dimethylforinaniide (2.5 mL) and then treated with 2,5-dioxopyrrolidin-1 -yl 2- (2,5-dioxo-2,5-dihydro-lH-pyrrol-1 -yl)acetate (0.168 g, 0.666 mmol) and N,N- diisopropylethylamine (0.4 mL, 2.290 mmol). After 25 min at room temperature, the reaction was cooled to 0 °C and then quenched by drop- wise addition of a 7% w/v aqueous solution of TFA in water (3.0 mL, 2.73 mmol). Purification by preparative RP HPLC on a Phenomenex 08(2) 10 μm column (2,50 mm x 50 mm). A gradient of MeCN (A) and 0.1 % TFA in water (B) was used, at a flow rate of 90 mL/min (0-5.0 min 15% A, 5.0-20.0 min linear gradient 15-95%_' A, hold 10 min). The title compound

METHOD DL-B

[0136] Example DL2 Synthesis of (8S,9R,10S,11S,13S,14S,16R,17R)-9~fIuoro-17-

(((fluoromethyl)thio)carbonyl)-11-hydroxy-10,13,16-trimethyl-3-oxo-

6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3-

((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)-4-fliiorobenzoate

[0137] Step 1 : Synthesis of (8S,9R,10S, l lS, 13S,14S, 16R, 17R)-9-Fluoro-T7- (((fIuoromethyl)thio)carbonyl)-1 1 -hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-eyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-aminopropanamido)propanamido)-4-fluorobenzoate

[0138] A 25 mL flask was charged with (8S,9R,10S, 11S,13S,14S,16R, 17R)-9-fluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3 - ((S)-2-((S)-2-((tert-butoxycarbonyl)amino)propanamido)propanamido)-4- fluorobenzoate (2.79 g, 3.46 mmol) (prepared as example DL1 step 5, above) and a 4 M solution of hydrochloric acid in dioxane (35 mL, 140 mmol). Stirring gave a colorless solution, which was stirred at room temperature for 90 min. Solvent was removed under reduced pressure to give a white solid, which was partitioned between EtOAc (500 mL) and 10 wt% aqueous NaOH (150 mL). The phases were separated and the aqueous was extracted with EtOAc (2 x 50 mL), washed with brine (50 mL), dried (Na2S04), and solvent was removed under reduced pressure to give the free base as a glass, which was sonicated with MTBE/heptanes (1 : 1 , 50 mL) and concentrated to give the title compound as a white solid (2.738 g, 3.49 mmol, 100% yleld). ¹ H NMR indicated that the product was a solvate with EtOAc (0.85 mol eqitiv., 90wt% purity).

[0139] Step 2: Synthesis of (8S,9R, 10S, l lS, 13S, 14S, 16R, 17R)-9-Fluoro- 17- (((fluoromethyl)thio)carbonyl)- 1 1 -hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9, 10,11 , 12, 13,14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)-4-fluorobenzoate

[0140] A solution of bromoacetic acid (0.126 g, 0.907 mmol) and 2-ethoxy-l - ethoxycarbonyl-1 ,2-dihydroquinoiine (EEDQ) (0.2406 g, 0.973 mmol) in N,N- dimethylformamide (1.25 mL) was stirred at room temperature. After 90 min, the solution of activated acid was added to a room temperature solution of (8S,9R, 10S,1 lS,13S, 14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)-11- hydroxy- 10, 13,16-trimethyl-3-oxo-6,7,8,9,10,l 1 ,12, 13,14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl 3-((S)-2-((S)-2-aminopropanamido)propanamido)-4- fluorobenzoate (0.250 g, 0.319 mmol) in N,N-dimethylformamide (1 .0 mL). After 330 min . the reaction mixture was diluted with EtOAc (30 niL), washed with water (2 x 15 mL), dried (Naj_^SCH), and solvent was removed under reduced pressure. Purification by chromatography (silica, 12 g) eluting with a gradient of 0-10% MeOH/DCM gave an off-white solid (158 mg, 60%). Further purification by RP HPLC on a Phenomenex Cl 8(2) 10 mhi column (250 mm x 50 mm), eluting with a gradient of MeCN (A) and 0.1%_' TFA in water (B), at a flow rate of 90 niL/min (0-5.0 min 15% A, 5.0-20.0 min linear gradient 15-95% A, hold for 10 min). The title compound was isolated after freeze drylng as white solid (0.0943 g, 0.114 mmol, 36% yleld). LCMS (Table A, method c) Rt - 0.91 min; MS m/z - 826, 828 [ M+H ⁺ ]. ¹H NMR (500 MHz, DMSO-d₆)

METHOD DL-C

[0141] Example DL3: Synthesis of (6S,8S,9R,10S,llS,13S,14S,16R,17R)-6,9-Dmuoro-

17-(((fluoromethyl)thio)carbonyI)-11-hydroxy-10,13,16-trimethyl-3-oxo-

6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenasithren-17-yl 3-

((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanamido)-4-fluorobenzoate

[0142] Step 1: Synthesis of (6S,8S,9R,10S,-11S-, 13S, 14S,16R,17R)-6,9-Difluoro-17- (((fluoromethyl)thio)carbonyl)-1 1 -hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-aminopropanamido)propanamido)-4-fluorobenzoate 2,2,2 -trifluoroaeetate

[0143] A solution of (6S,8S,9R,10S,11S, 13S, 14S,16R, 17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl -3 -oxo- 6,7,8,9,10, 1 1 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-((r£^,ri-butoxycarbonyl)amino)propanamido)propanamido)-4- fluorobenzoate (2.035 g, 2.470 mmol) in DCM (6 mL) was treated with TFA (2.85 mL, 37.0 mmol). The reaction mixture was stirred at room temperature for 90 min, whereupon solvent was removed under reduced pressure. The residue was redissolved in toluene (2 x 150 mL), which was removed under reduced pressure to drive off TFA. Drylng under high vacuum gave the title compound as a white solid (1.77g, 2.446 mmol, 99 % yleld), which was used in the next step without further purification. LCMS (Table A, method b) Rt - 0.87 min; m/z - 724 [ M+H ⁺ ] ¹H NMR (400 MHz, DMSO-d₆) d 10.06 (s, 1H), 8.64 (d, j= 6.8 Hz, 1H), 8.42 (dd, j= 7.4, 2.3 Hz, 1H), 8.03 (d, j=

5.3 Hz, 3H), 7.62 (ddd, j= 8.6, 4.6, 2.3 Hz, 1H), 7.47 (dd, J = 10.5, 8.6 Hz, 1 H ). 7.24 (dd, j= 10.2, 1.4 Hz, 1H), 6.2,8 (dd, .j= 10.1 , 1.9 Hz, 1H), 6.10 (d, J = 2.1 Hz, 1H), 5.93 (d, j= 50.1 Hz, 2H), 5.73 - 5.51 (m, 2H), 4.55 (p, J = 7.0 Hz, 1H), 4.27 - 4.20 (m, 1H), 3.83 (p, J = 5.9 Hz, 1H), 3.37 (ddd, J = 10.9, 7.3, 3.5 Hz, 1H), 2.67 - 2.48 (m, 1H), 2.21 (ddt, J = 26.3, 10.1 , 4.9 Hz, 4H), 1.90 (t, J = 12.1 Hz, 2H), 1.65 - 1.50 (m, 1H), 1.47 (s, 3H), 1.31 (d, j= 7.0 Hz, 3H), 1.27 (d, j= 7.1 Hz, 3H), 1.04 (s, 3H), 0.89 (d, j= 7.1 Hz, 3H).

[0144] Step 2: Synthesis of (6S,8S,9R, 10S, -11S-,13S,14S, 16R, 17R)-6,9-Difluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-(2-bromoacetamido)propanamido)propanainido)-4-fluorobenzoate

[0145] Bromoacetyl bromide (0.244 niL, 2.81 mmol) was added in one portion to a -78 °C solution of (6S,8S,9R,10S, 11S, 13S,14S,16R, 17R)-6,9-difluoro-17- (((fluoromethyl)thio)carbonyl)-1 1 -hydroxy-10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 1 l , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((S)-2-aminopropanamido)propanamido)-4-fluorobenzoate (1.77 g, 2.446 mmol) and !V,lV-diisopropylethylamine (1.708 mL, 9.78 mmol) in DCM (8 mL) and THF (8 mL). After 30 min, another charge bromoacetyl bromide (0.100 mL, 0.367 mmol) was added. The reaction was stirred for an additional 45 min at -78 °C, whereupon solvents were removed under reduced pressure. The residue was dissolved in DCM (200 mL), washed with 1 M aqueous HC1 (100 mL), which gave an emulsion. Volatile solvents were removed under reduced pressure and the resulting aqueous solution was extracted with EtOAc (200 mL). The organic layer was washed with a saturated aqueous solution of NaHCO₃, dried (Na₂SO₄), and the solvent was removed under reduced pressure. Purification by chromatography (silica, 240g) eluting with 10-40% EtOAc in MTBE gave an off white solid. Further purification by RP HPLC on a Phenomenex 08(2) 10 μm column (250 x 50 mm), eluting with a gradient of MeCN (A) and 0.1 % TEA in water (B), at a flow rate of 90 mL/min (0-5.0 min 15% A, 5.0-20.0 min linear gradient 15-95% A, hold for 5 min). The title compound was isolated after freeze drylng as white solid (620 mg, 0.734 mmol, 30%). LCMS (Table A, method b) Rt = 0.88 min; m/z - 844, 846 [M+H⁺]. ¹H NMR (501 MHz, DMSO-d₆) d 9.89 (s, 1H), 8.45 (dd, j=

7.5, 2.2 Hz, 1H), 8.42 (d, j= 7.5 Hz, 1H), 8.26 (d, j= 6.9 Hz, 1H), 7.62 (ddd, j= 8.7,

4.6, 2.2 Hz, 1H), 7.48 (dd, J = 10.5, 8.6 Hz, 1H), 7.25 (dd, J = 10.2, 1.5 Hz, 1 H ). 6.30 (dd, j= 10.1 , 1.9 Hz, 1H), 6.11 (s, 11-1), 5.94 (d, j= 50.0 Hz, 2H), 5.73 - 5.53 (m, 2H), 4.47 (p, J = 7.1 Hz, 1H), 4.31 (p, j= 7.0 Hz, 1H), 4.25 (d, j= 6.0 Hz, 1H), 3.88 (d, J

= 1.5 Hz, 2H), 2.66 - 2.53 (m, 1H), 2.23 (dt, J - 18.5, 12.7 Hz, 3H), 1 .98 - 1.88 (m, 2H), 1.58 (p, j= 12.1 Hz, 1H), 1.48 (s, 3H), 1.32 (dd, j= 8.8, 4.9 Hz, 1H), 1.25 (d, J = 7.1 Hz, 3H), 1.18 (d, j= 7.0 Hz, 3H), 1.05 (s, 3H), 0.90 (d, J = 7.1 Hz, 3H).

METHOD DL-D

[0146] Example DL4: Synthesis of (S)-4-((S)-2-((S)-6-Amino-2-(2- bromoacetamido)hexanamido)propanamido)-5-((2-fluoro-5- ((((8S,9R,10S,llS,13S,14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)- 11 -by droxy- 10,13, 16-trimethyl -3-oxo-6, 7, 8, 9,10,11,12,13, 14,15,16, 17-dodeeahydro-

acid

[0147] Step 1 : Synthesis of (8S,9R,10S, l lS, 13S,14S, 16R, 17R)-9-Fiuoro-17- (((fiuoromethyl)thio)carbony1)-1 1 -hydroxy - 10, 13, 16-trimethyl~3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5- oxopentanamido) -4-flu orobenzoate

[0148] Phosphoryl chloride (4.56 mL, 49.0 mmol) was added drop-wise to a 0 °C solution of (8S,9R, 10S,1 lS,13S, 14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)-11- hydroxy-10, 13, 16 -trimethyl -3 -oxo-6, 7, 8, 9, 10,1 l ,12, 13,14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl 3-amino-4-fluorobenzoate (9.2 g, 16.32 mmol), (R)-2- ((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (8.33 g, 19.59 mmol) and pyridine (13.20 mL, 163 mmol) in DCM (120 mL) and the solution was stirred at 0 °C for l h. The reaction mixture was diluted with DCM (100 mL), washed with water (2 x 100 mL), 1 M aqueous HC1 (50 mL), brine (1 x 100 mL), dried (Na₂SO₄), and solvent was removed under reduced pressure. Purification by chromatography (silica) eluting with 3:2 PE/EtOAc gave the title compound as a white solid (6.0 g, 6.09 mmol, 37.3 % yleld). LCMS (Table A, method e) Rt = 2.05 min; m/z = 972 [ M+H ⁺ ] . ¹ H NMR (400 MHz, DMSO-d₆) d 10.02 (s, 1H), 8.51 (dd, j= 7.7, 2.2 Hz, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.78 - 7.68 (m, 3H), 7.68 - 7.59 (m, 1H), 7.53 (dd, J - 10.5, 8.6 Hz, 1H), 7.41 (t, j= 7.6 Hz, 2H), 7.37 - 7.26 (m, 3H), 6.25 (dd, j= 10.1, 1.8 Hz, 1H), 6.04 (d, J = 7.8 Hz, 2H), 5.90 (s, 1H), 5.61 - 5.54 (m, 1H), 4.33 - 4.18 (m, 5H), 3.46 - 3.35 (m, 1H), 2.69 - 2.60 (m, 1H), 2.50 - 2.21 (m, 3H), 2.19 (d, J = 11.0 Hz, I H i. 1.99 (s, 4H), 1.89 - 1.79 (m, 1H), 1.57 - 1.40 (m,4H) 1.49 - 1.40 (m, OH), 1.38 (s, 9H), 1.34 - 1.21 (m, 1H), 1.08 (s, 3H), 0.92 (d, .j= 7.0 Hz, 3H).

[0149] Step 2: Synthesis of (8S,9R,10S, 1 lS, 13S,14S, 16R, 17R)-9-Fluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16- trimethyl -3 -oxo-

6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H -cyclopenta[a]phenanthren- 17 -yl 3-

((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)propanamido)-5-(tert- butoxy)-5-oxopentanamido)-4-fluorobenzoate

[0150] Diethylamine (0.4 mL, 3.83 mmol) was added to a room temperature solution of (8S.9R, 10S.11S,13S, 14S,16R,17R)-9-fluoro- 17-(((fluoromethyl)thio)carbonyl)-11- hydroxy- 10, 13, 16 -trimethyl-3 -oxo-6,7, 8,9, 10,1 l ,12, 13,14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl 3-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanamido)-4-fluorobenzoate (0.8369 g, 0.862 mmol) in THF (6.0 mL). The reaction was stirred at room temperature for 14 h, whereupon solvent was removed under reduced pressure. The residue was redissolved in toluene (12 mL), which was removed under reduced pressure to remove the diethylamine. The product was isolated as a crude yellow syrup (0.645 g, 0.862 mmol, 100% yleld), which was used in the subsequent step without further purification. LCMS (Table A, method c) Rt = 0.93 min; m/z = 749 [ M+H ⁺ ] - [0151] 1 -Propanephosphonic anhydride (T3P) (50% in EtOAc) (0.8175 g, 1.285 mmol) was added drop-wise to a room temperature solution of (8S,9R, 10S, 11S,13S,14S,16R, 17R)- 9-fluoro-l 7-(((fiuoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodecahydro-3H-cyclopenta[a]phenanthren-17-yl 3- ( ( S ) - 2 - amino-5 -(tert-butoxy)- 5 -oxopentanamido) -4-fluorobenzoate (0.646 g, 0.862 mmol), lV-(9-fluorenylmethoxycarbonyl)-L-alanine (0.322 g, 1.034 mmol), and pyridine (0.3 mL, 3.71 mmol) in EtOAc (1.0 mL). The reaction was stirred a room temperature for 2.5 h, whereupon it was charged with additional pyridine (0.3 mL, 3.71 mmol) and 1 -propanephosphonic anhydride (T3P) (50% in EtOAc) (1.09 g, 1.713 mmol). Stirring was continued at room temperature for 220 min. The reaction mixture was diluted with EtOAc (100 mL), then washed with 1 M aqueous HC1 (2, x 50 mL), saturated aqueous NaHCO₃ (50 niL), brine solution (50 mL), dried (NaiSCL), and solvent was removed under reduced pressure. Purification by chromatography (silica,

40 g) eluting with a gradient of 0-10% MeOH/DCM gave the title compound as light yellow foam (0.91 11 g, 0.831 mmol, 96% yleld). LCMS (Table A, method c) Rt = 1.16 min; m/z = 1042 [ M+H ⁺ ] , ⁵H NMR (400 MHz, DMSO-d₆) d 9.93 (s, 1H), 8.50 (dd, 3 = 7.6, 2.2 Hz, 1H), 8.15 (d, j= 7.5 Hz, 1H), 7.88 (d, j= 7.5 Hz, 2H), 7.71 (t, 3 = 7.9 Hz, 2H), 7.63 (odd, J = 8.5, 4.6, 2.3 Hz, 1H), 7.57-7.45 (m, 2H), 7.41 (td, j= 7.5, 1.2 Hz, 2H), 7.37-7.27 (m, 3H), 6.26 (dd, j= 10.1 , 1.9 Hz, 1 H), 6.05 (d, j= 1.6 Hz, 1 H), 5.95 (d, 3 = 50.0 Hz, 2H), 5.57 (dd, 3 - 4.6, 1.7 Hz, 1 H), 4.50 (q, 3 = 7.6, 7.2 Hz, 1H), 4.35- 4.01 (m, 5H), 2.65 (dt, 3 = 13.7, 6.6 Hz, 1H), 2.45 (dt, 3 = 11.7, 6.3 Hz, 1H), 2.26 (dddd, j= 41.5, 31.3, 16.0, 10.0 Hz, 4H), 2.03-1.89 (m, 3H), 1.89-1.74 (m, 1H), 1.51 (s, 3H), 1.42 (ddd, j= 21.9, 11.8, 5.3 Hz, 1 H), 1.35 (s, 9H), 1.23 (t, 3 - 6.6 Hz, 4H), 1.08 (s, 3H), 0.91 (d, j= 7.1 Hz, 3H).

[0152] Step 3: Synthesis of (10S, 13S, 16S)-tert-Butyl 10-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-16-((2-fluoro-5-((((8S,9R, 10S, 11 S , 13 S , 14S, 16R, 17R)-9- fluoro-17-(((fluoromethyl)thio)carbonyl)11 -hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12,13, 14, 15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17- yl)oxy)carbonyl)phenyl)carbamoyl) -2, 2, 13 -trimethyl -4,11 , 14-trioxo-3-oxa-5,12, 15- triazanonadecan-19-oate

[0153] Diethylamine (2.6 mL, 24.88 mmol) was added to a room temperature solution of (8S,9R, 10S,11S,13S, 14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)-11- hydroxy-10, 13,16-trimethyl-3-oxo-6,7,8,9,10,-11,-12, 13,14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl 3-((S)-2-((S)-2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)propanamido)-5-(½rt-butoxy)-5-oxopentanamido)-4- fluorobenzoate (0.8966 g, 0.860 mmol) in THF (6.0 mL). The reaction was stirred at room temperature for 90 min, whereupon solvent was removed under reduced pressure. The residue was redissolved in toluene (2 x 12 mL), which was removed under reduced pressure to remove the diethylamine. The product was isolated as a crude yellow' syrup (0.705 g, 0.860 mmol, 100% yleld), which was used in the subsequent step without further purification. LCMS (Table A, method c) Rt = 0.94 min; m/z = 820 [M+H⁺], [0154] 1 -Propanephosphonie anhydride (T3P) (50% in EtOAe) (1.095 g, 1 .720 mmol) was added to a -20 °C suspension of N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert- hutoxycarbonyl)-L-lysine Fmoc-Lys(Boc)-OH (0.484 g, 1.032 mmol),

(8S,9R, 10S,11S,13S, 14S,16R,17R)-9-fluoro- 17-(((fluoromethyl)thio)carbonyl)-11- hydroxy- 10, 13,16-trimethyl-3-oxo-6,7,8,9,10,l 1 ,12, 13,14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-l 7-yl 3-((S)-2-((S)-2-aminopropanamido)-5-(tert-butoxy)-5- oxopentanamido)-4-fluorobenzoate (0.705 g, 0.860 mmol), and pyridine (0.4 rnL, 4.95 mmol) in EtOAe (1.0 mL). The cooling bath was removed after the addition of T3P was complete, and the reaction mixture was stirred at room temperature for 13 h. The reaction mixture was diluted with EtOAe (50 mL), washed with 1 M aqueous HC1 (2 x 20 rnL), saturated aqueous NaHCO₃ (20 mL), brine solution (20 mL), and solvent was removed under reduced pressure. Purification by chromatography (silica, 40 g) eluting with a gradient of 0-10% MeOH/DCM gave the title compound as a white solid (1.093 g, 0.860 mmol, 100% yleld). LCMS (Table A, method c) Rt = 1.18 min; m/z = 1170

Step 4: Synthesis of (10S,13S,16S)-tert-Butyl 10-(2-bromoacetamido)-16-((2-fluoro-5- ((((8S,9R,10S,11S, 13S, 14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16 -trimethyl -3 -oxo-6, 7, 8, 9, 10,11 ,12, 13, 14,15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)phenyl)carbamoyl)-2,2,13-trimethyl-4,l 1 ,14- trioxo-3 -oxa - 5,12,15 -triazanonadecan- 19-oate

[0155] Diethylamine (1.5 mL, 14.36 mmol) was added to a room temperature solution of (10S, 13S, 16S)-/_<?rf-butyl 10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-16-((2- fhioro-5-((((8S,9R, 10S, 11 S,13S,14S, 16R, 17R)-9-fluoro-17- (((fluoromethyl)thio)carbonyl)-11-hydroxy- 10, 13, 16-trimethyl-3-oxo- 6,7,8,9,10, 11 , 12, 13, 14, 15, 16,17 -dodeeahydro-3H-cyclopenta[a]phenanthren- 17- yl)oxy)carbonyl)phenyl)earbamoyl)-2,2, 13 -trimethyl-4,11,14-trioxo-3-oxa-5,12, 15- triazanonadecan- 19-oate (1.093 g, 0.860 mmol). The reaction was stirred at room temperature for 140 min, whereupon solvent was removed under reduced pressure. The residue was redissolved in toluene (2 x 12 mL), which was removed under reduced pressure to drive off diethylamine. The product was isolated as a light yellow foam (0.901 g, 0.860 mmol, 100% yleld), which was used in the subsequent step without further purification. LCMS (Table A, method c) Rt - 0.99 min; m/z -1049 [M+H⁺].

[0156] Bromoacetyl bromide (0.4648 g, 2.303 mmol) was added in one portion to a -78 °C solution of (10S,13S,16S)-tert-butyl 10-amino-16-((2-fluoro-5-

((((8S,9R,10S,11S , 13S, 14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)earhonyl)-11- hydroxy-10, 13,16-trimethyl-3-oxo-6,7,8,9,10,l 1 ,12, 13,14, 15,16,17 -dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)phenyl)carbamoyl)-2,2, 13-trimethyl- 4, 1 l ,14-trioxo-3-oxa-5, 12,15-triazanonadecan-19-oate (0.901 g, 0.860 mmol) and N,N- diisopropylethylamine (0.6 mL, 3.44 mmol) in DCM (9.0 mL). The reaction was stirred for 5 min, whereupon it was diluted with DCM (50 mL), washed with 1 M aqueous HCl (20 mL), dried (Na₂SO₄), and solvent was removed under reduced pressure. The aqueous phase was back extracted with EtOAe (20 mL), which was dried (Na₂SO₄) and combined with the material from the DCM layer. Purification by chromatography (silica, 40 g) eluting with 0-10% MeOH/DCM gave an amber syrup, which was treated with DCM/heptanes. Removal of solvent under reduced pressure gave the title compound as an off-white solid (0.6157 g, 0.527 mmol, 61 % yleld). LCMS (Table A,

[0157] Step 5: Synthesis of (S)-4-((S)-2-((S)-6-Amino-2-(2- bromoacetamido)hexanamido)propanamido)-5-((2-fluoro-5-

[0158] ( 10S , 13 S , 16S)-tert-Butyl 10-(2-bromoaeetamido)-16-((2-fluoro-5-

((((8S,9R,10S,1 lS, 13S, 14S,16R,17R)-9-fluoro-17-(((fluoromethyl)thio)carbonyl)-11- hydroxy- 10, 13, 16 -trimethyl-3 -oxo-6,7, 8,9, 10,1 l ,12, 13, 14, 15, 16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)oxy)carbonyl)phenyT)carbamoyl)-2,2, 13 -trimethyl- 4, 1 l , 14-trioxo-3-oxa-5, 12, 15-triazanonadecan-19-oate (0.6157 g, 0.527 mmol) was dissolved in TFA (3.5 mL) and stirred at room temperature for 15 min. The reaction mixture was diluted with MeCN (8.5 rnL) and purified directly by RP HPLC on a Phenomenex C18(2) 10 mhi column (250 mm x 50 mm). A gradient of MeCN (A) and 0.1 % TFA in water (B) was used, at a flow rate of 90 mL/min (0-5.0 min 15% A, 5.0- 20.0 min linear gradient 15-95% A, hold for 5 min). Freeze drylng gave the title compound as a white solid (0.2543 g, 0.251 mmol, 48 % yleld). LCMS (Table A, method c) Rt = 0.87 min: m/z =1012, 1014 [ M+H ⁺ ] ¹H NMR (400 MHz, DMSO-d₆) d

TABLE C: DRUG-LINKER EXAMPLES DLS - DL24

[0159] The following examples were prepared according to the above procedures.

General Cysteine Conjugation Protocol

[0160] An approximate 5-20 mg/mL solution of the desired antibody was prepared in PBS buffer, pH 6 - 7.4. A reducing agent of choice, such as TCEP, was diluted or dissolved in solvents like H₂O, DMSO, DMA or DMF to give a solution with concentration range between 1 to 25 rnM. Antibodies (anti- hTNF hlgGl (adalimumab) or anti-mTNF mIgG2a (8C11 ; McRae BL et al. J Crohns Colitis 10 (1): 69-76 (2016)) were then partially reduced by adding about 2-3.5 equivalents of reducing agent, briefly mixing, and incubating overnight at 0 - 4 °C. Xris buffer, pH 8-8.5 (20-50 mM) was then added, followed by the linker-drug in DMSO or DMA (less than 15% total) and the mixture was incubated for 2 - 3 hours at rt. Excess linker-drug and organic solvent were then removed by purification. Purified ADC samples were then analyzed by SEC, HIC and reduced mass spectrometry.

Example 2, ADC Analytical Procedures

[0161] ADCs were profiled by either anionic exchange chromatography (AEC) or hydrophobic interaction chromatography (HIC) to determine the degree of conjugation and purity of ADC.

[0162] AEC. Approximately 20 μg of ADC was loaded onto an Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a 4 X 250 mm Propac™ WAX-10 column (Tosoh Bioscience, cat. 054999). Column was equilibrated with 100% buffer A and eluted using a linear gradient from 100% buffer A to 100% buffer B over 18 min at 1.0 mL ,/min . where buffer A is 20 mM MES, pH 6.7 and buffer B is 20 mM MES, 500 sodium chloride, pH 6,7.

[0163] HIC. Approximately 20 μg of the ADC was loaded onto an Ultimate 3000 Dual LC system

(Thermo Scientific) equipped with a 4.6 X 35 mm butyl-NPR column (Tosoh Bioscience, cat. 14947). Column was equilibrated in 100% buffer A and eluted using a linear gradient from 100% buffer A to 100% buffer B over 12 min at 0.8 rnL/min, where buffer A is 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0 and buffer B is 25 mM sodium phosphate, 25% isopropanol, pH 7,0.

[0164] SEC. Size distributions of the ADCs were profiled by size exclusion SEC using an Ultimate 3000 Dual LC system (Thermo Scientific) equipped with a 7.8 X 300 mm TSK-gel 3000SW_XL column (Tosoh Bioscience, cat. 08541). Approximately 20 μg of ADC was loaded onto the column and eluted over 17 min using an isocratic gradient of 100 mM sodium sulfate, 100 mM sodium phosphate, pH 6.8 at a flow rate of 1.0 ml ,/mi n.

[0165] MS. Reduced samples (10 μL) were injected to an Agilent 6550 QTof LC/MS system through a temperature controlled (5°C) CTC autosampler. Sample elution was achieved on a Waters C-4, 3.5 μm, 300 A, 2.1 x 50 trim i.d. HPLC column. The mobile phases were: A: 0.1% formic acid in water, and B: 0.1% formic acid in MeCN; the flow rate was 0.45 mL/min, and the column compartment was maintained at 40°C. The HPLC gradient is as follows:

Exemplary characterization spectra for the ADCs described herein are shown in below for ADCs of Formula (Il-1, Mouse Ab), (ll-2, Human Ah), (IT-8, Mouse Ab), and (IT-13, Human Ah). [0168]

Table 4. ADC Characterization (H = Human Antibody)

Example 3. Generation of human and mouse transmembrane TNF-alpha GRE reporter cell lines [0170] in order to create a parental cell line, K562 cells were seeded onto a 6 well dish (Costar:

3516) with 2 mL of complete growth medium (RPML10%FBS, l%L-glutamine, 1% Na Pyruvate and 1% MEM NEAA) at 500,000 cells per well for 24 hours at 37°, 5% CO2. The next day, 1.5 μg of pGL4.36[Luc2P/MMTV/Hygro] (Promega: E316), 1.5 ug pG14.75 [hRLuc/CMV] (Promega: E639A), and 3 uL of PLUS reagent (Invitrogen: 10964-021) were diluted into 244 μL Opti-MEM (Gibco: 31985-070) and incubated at rt for 15 minutes. The pGL4.36[/nc2P/MMTV/Hygro] vector contains MMTV LTR (Murine Mammary Tumor Virus Long Terminal Repeat) that drives the transcription of the luciferase reporter gene luc2P in response to activation of several nuclear receptors such as glucocorticoid receptor and androgen receptor. The pGLA75[hRluc/CMV] Vector encodes the luciferase reporter gene kRluc ( Renilla reniformis) and is designed for high expression and reduced anomalous transcription.

[0171] After incubation, diluted DNA solution was pre-incubated with 1:1 Lipofectamine LTX solution (Invitogen: 94756) (13.2 uL + 256.8 μL Opti-MEM) and incubated at room temperature for 25 minutes to form DNA-Lipofectamine LTX complexes. After incubation, 500 μL of DNA- Lipofectamine complexes were added directly to the well containing ceils. K562 ceils were transfected for 24 h at 37 °C, 5% CO2. After incubation, ceils were washed with 3 mL of PBS and selected with complete growth medium containing 125 μgmL of hygromycin B (Invitrogen: 10687- 010) for two weeks. "K562 pGL4.36[Luc2P/MMTV7Hygro]pGL4.75[hRLuc/CMVj" cells were produced.

[0172] In order to create a murine transmembrane TNF-alpha GRE reporter cell line, the parental cells, K562 pGL4.36[Luc2P/MMTV/Hygro]pGL4.75[hRLuc/CMV], were seeded onto 6 well dish (Costar: 3516) with 2 mL of complete growth medium (RPMI, 10%FBS, l%L-glutamine, 1% Na Pyruvate and 1% MEM NEAA) at 500,000 ceils per well for 24 h at 37°, 5% CO_?.. The next day, 3 μg of mFLJTNFα DNA (Origene: MC208048), which encodes untagged mouse TNF, and 3 μL of PLUS reagent (Invitogen: 10964-021) were diluted into 244 μL Opti-MEM (Gibco: 31985-070) and incubated at rt for 15 minutes. After incubation, diluted DNA solution was pre-incubated with 1:1 Lipofectamine LTX solution (Invitogen: 94756) (13.2 μL + 256.8 μL Opti-MEM) and incubated at rt for 25 minutes to form DNA-Lipofectamine LTX complexes. After incubation, 500 μL of DNA- Lipofectamine complexes were added directly to the well containing ceils. The parental K562 pGL4.36[Luc2P/MMTV/Hygro]pGL4.75[hRLuc/CMV] cells were transfected for 24 h at 37°C, 5% CO_?. After incubation, ceils were washed with 3 mL of PBS and selected with complete growth medium containing 125 μg /mL of hygromycin B (Invitrogen: 10687-010) and 250 μg /mL G418 (Gibco: 10131-027) for two weeks. "K562 mouse FL-TNFα GRE (pGL4.36[luc2P/MMTV/Hygro])" cells were produced.

[0173] In order to create a human transmembrane TNF-alpha GRE reporter cell line, the parental cells, K562 pGL4.36[Luc2P/MMTV/Hygro]pGL4.75[hRLuc/CMV], were transfected with the plasmid hTNF delta 1-12 C-Myc pcDNA3.1(-) plasmid construct. This plasmid is pcDNA 3.1 (Thermofisher eat# V79020) encoding tace resistant transmembrane TNF (i.e., SEQ ID NO:1 lacking amino acids 77-88). (.See Perez C et al. Cell 63 (2): 251-8 (1990) discussing tace resistant transmembrane TNF.) These cell lines were then used in the TNF-alpha reporter assays described in the subsequent examples.

Example 4. Activity of small molecules aod anti-TNF-alpha immunoconjugates in GRE reporter assays

[0174] K562 parental GRE (pGL4.36[luc2P/MMTV/Hygro]) cells and K562 mFL-TNF-a or hTNF delta 1-12 GRF1 (pGL4.36[ltic2P/MMTV/Hygroj) cells were plated onto 96 well tissue culture treated white plates (Costar: 3917) at 50,000 cells per well in 50 μL of assay medium (RPMI, 1% CSFBS,

1% L-glutamine, 1% Na Pyruvate and 1% MEAA). The cells were treated with 25 μL of 3X serial diluted murine or human anti-TNF-a antibody drug conjugates in assay medium, steroid compound, or media alone and incubated for 48 hours at 37 °C, 5% CO₂. After 48 hours of incubation, ceils were treated with 75 μL of Dual-Gio Luciferase Assay System (Promega-E2920) for 10 min and analyzed for luminescence using the Microbeta (PerkinE!mer). Data were analyzed using a four parameter curve fit to generate ECso values. % maximum activation was normalized to 100 nM dexamethasone. Small molecules are tested using the K562 parental GRE reporter cell line and are shown in Table 4. The results using the murine TNF-alpha cell line are shown in Table 5 below (A refers to 8C11 ), and the results using the human TNF-alpha cell line are shown in Table 6 below (A refers to adalimumab). ADC Drag: Antibody Ratio (DAR) and percent (%) High Molecular Weight Species (HMW) was determined by SEC as previously described (see ADC analytical procedures).

[0175] Glucocorticoid Receptor (GR) binding assay

[0176] Small molecules were tested for GR binding using the Polarscreen^TM Glucocorticoid Receptor Assay Kit, Red (ThermoFisher A 15898) according to the manufacturer's protocol. Briefly, compounds were serially diluted in DMSO then transferred into assay kit buffer at a 1:10 dilution. Compounds were further diluted 1:5 in assay kit buffer and10 μl transferred to a 384 well low volume black walled plate (Corning 4514). 5 μl of 4X Fluormone GS Red stock solution and 5m1 of 4x GR full length stock solution was added to each well containing test compound and plates were incubated protected from light at room temperature for 4h. Fluorescence Polarization (mP) was measured for each plate using an EnVision Multilabel Plate Reader (Perkinelmer # 2104-0010 and data were analyzed using a four parameter curve fit to generate EC50 values.

Table 5: Free Payload ln Vitro Data

Example 5. Activity of ami-mTNF-alpha immunoconjugate in contact hypersensitivity model

[0177] Anti-TNFa steroid ADC was evaluated in an acute contact hypersensitivity model, an elicitation of acute skin inflammation using delayed-type hypersensitivity (DTH) response (T-cell driven) via application of a sensitizing agent (fluorescein isothiocyanate (FITC)). The efficacy of anti-TNFα steroid ADCs was measured by the ability to reduce ear swelling. The steroid biomarkers corticosterone and procollagen type 1 N-terminal propeptide (PINP) were included as readouts to assess the putative impact of anti-TNFa steroid ADC treatment on the Hypothalamus-Pituitary- Adrenal (HPA) axis and bone turnover respectively.

Ear Swelling

[0178] On day 0 mice were placed under general anesthesia and the abdomens were shaved. Using a micropipettor, mice were sensitized by epicutaneous applicaton of 400 mE of FITC solution (1.5% solution in 1:1 acetone:DBP) on the abdomen. Six days later, mice were dosed with vehicle or therapeutic agent 1 hour prior to ear challenge with FITC. For ear challenge, mice were placed under general anesthesia and were challenged with 20 μL FITC applied onto right ear. 24 hours after challenge mice were placed under general anesthesia and their ear thickness is measured by caliper. Difference between challenged and unchallenged ears was calculated. 72 hours after ear challenge, mice were injected with ACTH at 1 mpk IP, and terminally bled at 30 min post- ACTH. Plasma is collected and analyzed PINP, corticosterone, free steroid, and large molecule levels.

Quantification of released free steroid and endogenous corticosterone

[0179] Calibration curve of steroid was prepared in mouse plasma with final concentrations from 0.03 nM to 0.1 mM at 8 different concentration levels. Corticosterone calibration curve ranging from 0.3 nM to 1 mM final corticosterone concentrations was prepared in 70 mg/rnL bovine serum albumin solution in PBS buffer. A solution of 160 μL MeCN with 0.1% formic acid was added to 40 μL study plasma samples or calibration standards. Supernatants were diluted with distilled water and 30 m L final sample solution was injected for LC/MS analysis.

Quantification of released free steroid and corticosterone was conducted on an AB Seiex 5500 triple quadruple mass spectrometer connected to a Shimadzu AC20 HPLC system interfaced with an electrospray ionization source operating in positive mode. A Waters XBridge BEH C18, 2.1x30mm, 3.5 μm column was used for chromatography separation. The mobile phase A was 0.1 % formic acid in Milli Q HPLC water, and mobile phase B was 0.1 % formic acid in MeCN. A linear gradient from 2% of mobile phase B to 98% mobile phase B was applied from 0.6 to 1.2 min. The total tun time was 2.6 min at a flow-' rate of 0.8 mL/min. The mass spectrometer was operated in positive MRM mode at source temperature of 700°C.

Quantification of plasma PINP

[0180] Quantification of plasma PINP was conducted on a LCMS platform based on protein trypsin digestion. Plasma samples were partially precipitated and fully reduced by adding MeCN/O.lM ammonium bicarbonate/DTT mixture. Supernatant was collected and alkylated by adding iodoacetic acid. The alkylated proteins were digested by trypsin and resulting tryptic peptides were analyzed by LCMS. Calibration curve were generated by using synthetic tryptic peptide spiked into horse serum (non-interfering surrogate matrix). Stable isotope labeled flanking peptide (3-6 amino acids extension on both termini of the tryptic peptide) was used as internal standard added in the MeCN/DTT protein precipitation mixture to normalize both digestion efficiency and LCMS injection.

[0181] A Columnex Chromenta BB-C18, 2.1x150 mm, 5 μm column was used for chromatography separation. The mobile phase A was 0.1% formic acid in Milli Q HPLC water and mobile phase B was 0.1% formic acid in MeCN. A linear gradient from 2% of mobile phase B to 65% mobile phase B was applied from 0.6 to 3 min. The total run time was 8 min at a flow rate of 0.45 mL/min. An AB Seiex 4000Q trap mass spectrometer was used in positive MRM mode to quantify PINP peptides, at source temperature of 700 °C. Table 8. Contact Hypersensitivy (CHS) Data

Table 9. CHS Data

Example 6, Activity of anti- mTNF-aipha immunoconjugates in collagen-induced arthritis

[0182] The abili ty of anti-TNFa steroid ADC (ADC1) to impact disease was assessed in the collagen- induced arthritis (CIA) model of arthritis. [0183] In these experiments, male DBA/1J mice were obtained from Jackson Labs (Bar Harbor, ME). Mice were used at 6 to 8 weeks of age. Ail animals were maintained at constant temperature and humidity under a 12-hour light/dark cycle and fed with rodent chow (Lab Diet 5010 PharmaServ, Framingham, MA) and water ad libitum. Abb Vie is AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited, and all procedures were approved by the Institutional Animal Care and Use Committee (LACUC) and monitored by an attending veterinarian. Body weight and condition were monitored, and animals were euthanized if exhibiting >20% weight loss.

[0184] The male DBA/J mice were immunized intradermally (i.d.) at the base of the tail with 100 μL of emulsion containing 100 μg of type II bovine collagen (AID Biosciences) dissolved in 0.1 M acetic acid and 200 μg of heat-inactivated Mycobacterium tuberculosis H37Ra (Complete Freund's Adjuvant, Difco, Laurence, KS). Twenty-one days after immunization with collagen, mice were boosted IP with 1 mg of Zymosan A (Sigma, St. Louis, MO) in PBS. Following the boost, mice were monitored 3 to 5 times per week for arthritis. Rear paws were evaluated for paw swelling using Dyer spring calipers (Dyer 310-115)

[0185] Mice were enrolled between days 24 and 28 at the first clinical signs of disease and distributed into groups of equivalent arthritic severity. Early therapeutic treatment began at the time of enrollment.

[0186] Animals were dosed once intraperitoneally (i.p.) with anti-mTNF mAh (10 mpk) or anti-mTNF steroid ADC (1-30 mg/kg) in 0.9% saline. Blood was collected for antibody exposure by tail nick at 24 and 72 hours after dose. Paws were collected at the terminal time -point for histopathology. Blood was collected at the terminal time -point by cardiac puncture for complete blood counts (Sysmex XT- 2000iV). Statistical significance was determined by ANOVA.

[0187] The results are shown in FIGS. 1-7 and demonstrate that a single dose of anti-TNFα steroid ADC can exhibit an extended duration of action through amelioration of paw' swelling for ~28 days compared to anti-TNFα mAh or small steroid alone. See also Table 10.

Table 10: Percent Inhibition

[0188] it is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections sets forth one or more, but not all, exemplary embodiments of the present disclosure as contemplated by the inventor! s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0189] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can he defined so long as the specified functions and relationships thereof are appropri ately performed.

[0190] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applylng knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on tire teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to he interpreted by the skilled artisan in light of the teachings and guidance. [0191] The breadth and scope of the present disclosure should not be limited by any of the above- described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

INCORPORATION BY REFERENCE

[0192] All publications, including patents and published applications, referred to in the Detailed Description are incorporated by reference herein in their entirety.

Claims

What is claimed is:

1. An antibody drug conjugate of formula (II):

wherein:

X is hydrogen or fluoro;

A is an antibody comprising a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:2 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:3; wherein the wavy line indicates the attachment

point to A and the asterisk indicates the attachment point to L;

R^A is fluoro, chloro, or C₁-C₅ alkyl; and n is 2 or 4.

2. The antibody drug conjugate of claim 1, wherein R^A is fluoro, chloro, or CH₃.

3. The antibody drug conjugate of claim 2, wherein R^A is fluoro.

4. The antibody drug conjugate of claim 1, wherein the compound is:

5. The antibody drug conjugate of claim 1, wherein the antibody drug conjugate is:

II- 10.

6 A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 1-5 and a pharmaceutically acceptable carrier.

7 An antibody drug conjugate of formula (II):

(P) wherein:

X is hydrogen or fluoro; A is adalimumab;

R^A is fluoro, chloro, or C₁-C₅ alkyl; and n is 2 or 4.

8. The antibody drug conjugate of claim 7, wherein R^A is fluoro, chloro, or CH₃.

9. The antibody drug conjugate of claim 8, wherein R^A is fluoro.

10. The antibody drug conjugate of claim 7, wherein the compound is:

11. The antibody drug conjugate of claim 7, wherein the antibody drug conjugate is:

II- 10.

12. A pharmaceutical composition comprising the antibody drug conjugate of any one of claims 7-11 and a pharmaceutically acceptable carrier.