Glutamate carboxypeptidase ii inhibitor conjugates for inflammatory bowel disease

Abstract

Conjugates of corticosteroids and GCPII inhibitors and their use in treating inflammatory bowel disease are disclosed.

Description

GLUTAMATE CARBOXYPEPTIDASE II INHIBITOR CONIUGATES FORINFLAMMATORY BOWEL DISEASECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 700,882 filed September 30, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Corticosteroids are highly effective in inducing remission of inflammatory bowel disease (IBD). They are the mainstay of treatment of IBD flare-ups as they are powerful anti-inflammatory agents that rapidly reduce intestinal inflammation. Corticosteroids, however, can have severe adverse effects and, as such, are unsuitable as a long-term IBD treatment. The side effects are numerous and include increased risk of infection, ulcers or gastrointestinal bleeding, osteoporosis, depression, fatigue, bone loss, and increased risk of fractures. Accordingly, there is a need for improved therapeutic agents for treating IBD.SUMMARY

[0003] The presently disclosed subject matter provides conjugates of corticosteroids and binding moieties derived from GCPII / PSMA inhibitors and their use in treating IBD. In some aspects, the presently disclosed subject matter provides a compound of formula (I):

[0004] C-L-P (I); wherein:

[0005] C is a corticosteroid; L is a linker; and P is a GCPII / PSMA binding moiety; and pharmaceutically acceptable salts thereof.

[0006] In certain aspects, the compound of formula (I) is:

[0008] wherein:

[0009] — - is a bond that can be present or absent;

[0010] R1and R2 arc each independently H or -L-P, provided that one of R1and R2 is -L-P and the other is H;

[0011] R.3 is H or C1-C4alkyl; or Ro and R3 can combine to form:

[0012] wherein n is an integer selected from 0, 1, 2, 3, and 4; and R4 is -C1-C4alkyl;

[0013] R5is -OH or =0;

[0014] Re is H or C1-C4alkyl;

[0015] R7 is H or halogen; and

[0016] stereoisomers and pharmaceutically acceptable salts thereof.

[0017] In certain aspects, the GCPII binding moiety, P, is selected from:

[0020] wherein:

[0021] Z is tetrazole or CO2Q;

[0022] Q is H or a protecting group;

[0023] m is an integer selected from the group consisting of 1, 2, 3, 4, and 5; and

[0024] R8is selected from H or -CH2-R9, wherein R9is selected from substituted aryl, substituted pyridine, and unsubstituted isoquinolinc; and stereoisomers thereof.

[0025] In certain aspects, R9is selected from:

[0027] wherein X is a halogen or a radiohalogen.

[0028] In particular aspects, X is selected from Br, I, At, and radioisotopes thereof.

[0029] In certain aspects, the corticosteroid, C, is selected from:(beclomethasone); and stereoisomers thereof.

[0033] In certain aspects, the linker, L, is:

[0035] wherein:

[0036] y is an integer selected from 1 , 2, 3, 4, 5, 6, 7, and 8;

[0037] x is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0038] Rxcan be the same or different and are each independently an amino acid, wherein the amino acid includes both R- or S- isomers; and

[0039] R26, R27, R28, R29, R30, R31, and R32 are each independently H or C1-C4alkyl, or

[0040] R29 and R30 together can form a 4- to 7-member nitrogen-containing heterocyclic ring with the nitrogen attached to R29; and stereoisomers thereof.

[0041] In certain aspects, the linker, L, is selected from:

[0045] wherein:

[0046] q is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0047] each y is independently an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0048] A is selected from -(CH2)y- -C(=O)-O-(CH2)y- -(CH2)y-O-(CH2)y-, -C(=O)-NRI9-(CH2)y-NR18-C(=O)-O-(CH2)y- -(CH2)y-NR38-C(=O)-O-(CH2)y-

[0049] B is selected from -NR24-(CH2)y-C(=O)-, -NR24-(CH2)y-C(=O)-NR25-(CH2)y-, and-NR33-CH(R34)-C(=O)-NR35-(CH2)y-C(=O)-NR36-(CH2)y-;

[0050] each R10, R13, R14, R15, R16, R17, R21, R22, R23, R24, R25, R34, R37, and R38are independently H or C1-C4alkyl;

[0051] each R11, R12, R18, R19, and R34are independently C1-C4alkyl; and

[0052] R20 is C1-C4alkyl or -(CH2)yN(CH3)2; and stereoisomers thereof.

[0053] In particular aspects, the linker, L, is selected from:

[0054] In particular aspects, the compound of formula (I) is selected from:and stereoisomers thereof.

[0074] In other aspects, the compound of formula (I) is selected from:

[0079] In other aspects, the compound of formula (I) is selected from:stereoisomers thereof.

[0085] In other aspects, the compound of formula (I) is:(C3); and stereoisomers thereof.

[0087] In other aspects, the presently disclosed subject matter provides a formulation comprising a compound of formula (I) and a pharmaceutically acceptable carrier.

[0088] In other aspects, the presently disclosed subject matter provides a method for treating an inflammatory bowel disease, the method comprising administering to a subject in need of treatment thereof, a compound of formula (I) or a formulation thereof.

[0089] In certain aspects, the inflammatory bowel disease is selected from Crohn’s disease (CD), ulcerative colitis (UC), indeterminate colitis inflammatory bowel disease, and combinations thereof.

[0090] In certain aspects, the administering the compound of formula (I) decreases GCPII activity in the subject compared to an untreated control subject or a subject without an IBD.

[0091] In certain aspects, the administering the compound of formula (I) reduces or alleviates one or more symptoms associated with IBD, including abdominal pain, diarrhea, fecal incontinence and / or urgency to have a bowel movement, rectal bleeding, swelling or masses due to inflammation in the intestines, weight loss, fever, anemia, malnutrition, delayed growth, anxiety, depression.

[0092] In certain aspects, the method further comprises administering one or more additional therapeutic agents in combination with the compound of formula (I).

[0093] In certain aspects, the one or more additional therapeutic agents is selected from an immunosuppressant drug, a biologic, an anti-inflammatory agent, an orally available agents for treating IBD, a tumor necrosis factor (TNF)-alpha inhibitor, an antibiotic, an anti-diarrheal medication, a fiber supplement, a pain reliever, a vitamin, and a mineral supplement.

[0094] In certain aspects, the method further comprises one or more of stem cell therapy, surgery, and fecal transplant.

[0095] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in pail by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0096] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0097] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0098] FIG. 1 demonstrates that the presently disclosed conjugates are nM inhibitors of GCPII / PSMA enzymatic activity (ICso= 6-30 nM).

[0099] FIG. 2 demonstrates that the presently disclosed conjugates are active and 500-900 nM binders at the glucocorticoid receptor.

[0100] FIG. 3 demonstrates that representative conjugates (e.g., Cl, C2, C3, C6 and C7) selectively internalize in GCPII expressing cells (PC3-PIP cells) versus non-expressing cells (PC3- Flu cells) and internalization is blocked by the known GCPII inhibitor ZJ43.

[0101] FIG. 4 demonstrates that a representative conjugate (C5) is a substrate of Cathepsin enzymes and cleaved in the presence of Cathepsins B and L.

[0102] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, FIG. 5H, FIG. 51, FIG. 5J, and FIG. 5K show in vivo prednisolone release following administration of prednisolone (FIG. 5A) conjugates Cl (FIG. 5B), C12 (FIG. 5C), C13 (FIG. 5D), C14 (FIG. 5E), C15 (FIG. 5F), C16 (FIG. 5G), C2 (FIG. 5H), C3 (FIG. 51), C8 (FIG. 5 J), or C9 (FIG. 5K) at equimolar dose in micetreated with LPS. FIG. 5A shows the concentration profile of prednisolone and FIG. 5B to 5 K show the concentration profile of prednisolone released from the presently disclosed GCP II- prednisolone conjugates Cl, C12, C13, C14, C15, C16, C2, C3, C8, and C9 in the ileum and plasma following oral administration of 10 mg / kg prednisolone or the GCP II-prednisolone conjugates at a molar equivalent of 10 mg / kg prednisolone.

[0103] FIG. 6 shows the ileum to plasma ratio of prednisolone at 1 hr following administration of prednisolone or GCP II-prednisolone conjugates Cl, C2, C3, C8, C9, C12, C13, C14, C15 and C16.DETAILED DESCRIPTION

[0104] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0105] The folate hydrolase (FOLH1) gene encodes Glutamate Carboxypeptidase II (GCPII), an enzyme that is highly overexpressed in human inflammatory diseases, including inflammatory bowel disease (IBD). It has previously been shown that GCPII enzymatic function is elevated between about 300% to about 3000% in human IBD and that inhibiting GCPII activity using small molecule drugs in IBD mouse models causes significant disease improvement. Rais et al., 2016.

[0106] IBD is an idiopathic, chronic and frequently disabling inflammatory disorder of the intestine, which has two subtypes: Crohn’s disease (CD) and ulcerative colitis (UC), each accounting for approximately 50% of IBD patients (Xavier and Podolsky, 2007 ; Srober et al, 2007 ; Sartor, 2006). Crohn’s disease can affect any part of the gastrointestinal (GI) track from the mouth to the anus, whereas ulcerative colitis can affect any part of the large intestine. A third category of IBD, indeterminate colitis inflammatory bowel disease, refers to IBD that has features of both Crohn’s disease and ulcerative colitis. IBD is a widespread GI disease, with a prevalence of approximately 0.2% in the Western population. In the United States alone, 1.4 million patients are diagnosed IBD patients, resulting in enormous suffering and health-care costs.

[0107] IBD is a complex multifactorial disease with both genetic and environmental contributions, the interaction of which leads to IBD (Xavier and Podolsky; Strobcr ct al, 2007; Sartor, 2006; Kaser et al., 2010). Unfortunately, the etiology of the mucosal dysregulation in UC and CD remain elusive (Kaser et al., 2010). Despite increasing therapeutic options available for the management of IBD, approximately one-third of IBD patients do not respond to any given therapy, and there is no cure for IBD (Hamilton et al, 2012). Anti-tumor necrosis factor (TNF)-based therapies, such as infliximab (IFX), adalimumab and certolizumab pegol, are currently the most effective therapies for severe UC and CD (Hanauer et al, 2002; Kozuch and Hanauer, 2008; Colombel et al, 2007; Schreiber et al, 2007). One-third of patients with CD, however, do not respond to anti-TNF therapies and another third lose responsiveness within six months of initiating therapy (Regueiro et al., 2007; Lawrance, 2014). These non-responders have more aggressive mucosal immune responses and additional treatments are indicated (Schmidt et al, 2007).

[0108] Patients with extensive disease or who are at risk for short gut syndrome due to prior resections are usually poor surgical candidates. Currently, the only approved medication for patients who have failed an anti-TNF agent is natalizumab. Natalizumab, however, has been associated with several cases of progressive and often fatal multifocal leukoencephalopathy (PML) (Van et al, 2005). These shortcomings and complications associated with current treatment regimens emphasize the significance of exploring and identifying new and more effective therapies in patients with IBD.

[0109] In some embodiments, the presently disclosed subject matter provides a mechanism to selectively target corticosteroids not only to the gastrointestinal (GI) system, but specifically to the inflamed cells within the GI tract, thereby minimizing systemic exposures and thus significantly reducing the adverse side effects of corticosteroids.

[0110] This strategy involves conjugating corticosteroids to small molecule inhibitors of the enzyme glutamate carboxypeptidase II (GCPII / PSMA; gene Folhl). We and others have shown that GCPII / PSMA is highly upregulated in inflamed areas of the intestines in patients with IBD. Further, we and others also have shown that when small molecule inhibitors bind to GCPII / PSMA on the surface of cells, it triggers internalization. This internalization property of GCPII inhibitors has been exploited by using GCPII / PSMA as a “trojan horse” for selectively targeting prostate cancer cells where GCPII / PSMA is several-fold upregulated versus normal prostate levels. In fact, over the past few years, several GCPII / PSMA small molecule imaging and therapeutic conjugateshave been developed and FDA-approved, including PYLARIFY® (piflufol stat Fl 8), POSLUMA® (flotufolastat Fl 8), and PLUVICTO® (lutetium177Lu vipivotidc tctraxctan).

[0111] More particularly, in some embodiments, the presently disclosed subject matter includes attaching corticosteroids to GCPII / PSMA inhibitors using various cleavable linkers with the rationale that the conjugate can be internalized using GCPII / PSMA as a targeting modality and then cleaved to deliver the corticosteroid selectively into the inflamed cells in the intestines of patients afflicted with IBD. Without wishing to be bound to any one particular theory, this design is aimed at limiting exposure in tissues where the steroid is not needed, thereby reducing toxicity.

[0112] Accordingly, the presently disclosed subject matter, in some embodiments, describes the synthesis and characterization of several conjugates that link a urea-based GCPII inhibitor to a representative corticosteroid, e.g., prednisolone. The inhibitors are conjugated to the corticosteroids either using self-immolative linkers, such as para-aminobenzoic acid (PAB) or modified PAB, linked to dipeptides, designed to be cleaved by intracellular enzymes, such as cathepsins (B,D,L, and the like). The conjugates also are linked via linkers forming esters or carbamates to be cleaved by intracellular esterases. Importantly, we have shown that the GCPIVPSMA-conjugates are specifically taken up in human cells that express GCPII / PSMA and this uptake can be blocked by known GCPII / PSMA inhibitors. Accordingly, the presently disclosed inhibitor-corticosteroid conjugates represent a previously unexplored strategy to directly and selectively deliver therapeutics to inflamed cells in the intestines.

[0113] Compounds of Formula (I)

[0114] Glutamate Carboxypeptidase II (GCPII), also referred to as N-acetyl-L-aspartyl-L- glutamate peptidase I (NAALADase I), NAAG peptidase, or prostate-specific membrane antigen (PSMA), is a metallopeptidase that catalyzes the hydrolysis of N-acetylated aspartate-glutamate (NAAG) to N-acetyl aspartate (NAA) and glutamate and cleaves terminal glutamate moieties sequentially from folate polyglutamate. As used herein, a GCPII inhibitor is a molecule that decreases or inhibits the activity of GCPII. A GCPII inhibitor also is referred to herein as a PSMA inhibitor.

[0115] The modulation of the activity of GCPII may be detected by use of an assay for the intrinsic N-acetylated alpha-linked acidic dipeptidase (AALADase) activity of GCPII. Inhibition curves may be determined using semi-log plots and IC50 values determined at the concentration at which enzyme activity was inhibited by 50%.

[0116] The GCPII inhibitor may interact with GCPII directly (e.g., via interaction with the binding site of GCPII) or may interact with another molecule that results in a decrease in the activity of GCPII. The binding site of GCPII contains a binuclear zinc ion and two substrate binding pockets, i.e., an SI (nonpharmacophore) pocket and an SI’ (pharmacophore) pocket. The active site also contains a chloride ion in the SI pocket. In the vicinity of the SI pocket resides a funnel-shaped O 0 tunnel with a depth of approximately 20 A and a width of 8-9 A. Similarly, a narrow cavity is present near the S 1 ’ pocket.

[0117] Accordingly, in some embodiments, the presently disclosed subject matter provides a conjugate comprising a corticosteroid and a binding moiety derived from a GCPII / PSMA inhibitor, also referred to herein as a “GCPII / PSMA ligand.” In particular embodiments, the GCPII / PSMA inhibitor is a low-molecular- weight (or small molecule) GCPII / PSMA-binding moieties or ligands having an affinity for GCPII / PSMA, which can be conjugated with corticosteroids for treatingIBD. One of ordinary skill in the art would recognize that the term “low molecular weight” refers to a compound having a molecular weight between about 50 Da to about 1,500 Da.

[0118] Representative classes of GCPII / PSMA low-molecular- weight ligands include, but are not limited to, phosphonates, such as 2-PMPA, phosphinates, phosphoramidates, thiols, such as 2(thioalkyl)pentanedioic acids, hydroximates, urea-based glutamate derivatives, and so-called PSMA-targeted carbamate and reversed carbamate derivatives. See, for example, WO2016065145 and W02017027870, each of which is incorporated herein by reference in its entirety. Exemplary chemical structures of representative low-molecular-weight GCPII / PSMA binding motifs include:; and stereoisomers thereof.

[0120] In some embodiments, the presently disclosed subject matter provides a compound of formula (I):

[0121] C-L-P (I); wherein: C is a corticosteroid; L is a linker; and P is a GCPII / PSMA binding moiety; and pharmaceutically acceptable salts thereof.

[0122] In some embodiments, the GCPII / PSMA-binding moiety is selected from a urea-based glutamate derivatives and a phosphonate. More particularly, in some embodiments, the compound of formula (I) is:

[0124] wherein:

[0125] -— is a bond that can be present or absent;

[0126] R1and R2 are each independently H or -L-P, provided that one of R1and R2 is -L-P and the other is H;

[0127] R3is H or C1-C4alkyl; or R2 and R3can combine to form:; wherein n is an integer selected from 0, 1, 2, 3, and 4; and R4 is -C1-C4alkyl;

[0129] R5is -OH or =0;

[0130] Re is H or C1-C4alkyl;

[0131] R7 is H or halogen; and

[0132] stereoisomers and pharmaceutically acceptable salts thereof.

[0133] In some embodiments, the GCPII / PSMA binding moiety, P, is selected from:

[0136] wherein:

[0137] Z is tetrazole or CO2Q;

[0138] Q is H or a protecting group;

[0139] m is an integer selected from the group consisting of 1, 2, 3, 4, and 5; and

[0140] R8is selected from H or -CH2-R9, wherein R9is selected from substituted aryl, substituted pyridine, and unsubstituted isoquinoline; and stereoisomers thereof.

[0141] In certain embodiments, R9is selected from:

[0142]

[0143] wherein X is a halogen or a radiohalogcn. In particular embodiments, X is selected from bromine (Br), iodine (I), astatine (At), and radioisotopes thereof, including, but not limited to,76Br,77Br,123I,125I,131I, and211At. In such embodiments, the epsilon amino group of lysine of the Lys- Glu-urea moiety is modified with, for example, a p-Br-benzyl group. See, for example, PCT Patent Application Publication No. WO / 2017 / 165473 for Prostate-Specific Membrane Antigen Targeted High-Affinity Agents for Endoradiotherapy of Prostate Cancer, to Ray et al., published Sept. 28, 2017, which is incorporated herein by reference in its entirety.

[0144] In particular embodiments, R9is:

[0146] In certain embodiments, the corticosteroid, C, is selected from:

[0150] In certain embodiments, the linker, L, is:

[0152] wherein:

[0153] y is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0154] x is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0155] Rxcan be the same or different and are each independently an amino acid, wherein the amino acid includes both R- or .S'- isomers; and

[0156] R26, R27, R28, R29, R30, R31, and R32 are each independently H or C1-C4alkyl, or

[0157] R29 and R30 together can form a 4- to 7-member nitrogen-containing heterocyclic ring with the nitrogen attached to R29; and stereoisomers thereof.

[0158] One of ordinary skill in the art would appreciate that the:subunit in the linker is equivalent to, or in some embodiments, can be, 2 to10 peptide units.

[0160] In certain embodiments, the linker, L, is selected from:

[0164] wherein:

[0165] q is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0166] each y is independently an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8;

[0167] A is selected from -(CH2)y- -C(=O)-O-(CH2)y- -(CH2)y-O-(CH2)y-, -C(=O)-NRI9-(CH2)y-NR18-C(=O)-O-(CH2)y- -(CH2)y-NR38-C(=O)-O-(CH2)y-

[0168] B is selected from -NR24-(CH2)y-C(=O)-, -NR24-(CH2)y-C(=O)-NR25-(CH2)y-, and-NR33-CH(R34)-C(=O)-NR35-(CH2)y-C(=O)-NR36-(CH2)y-;

[0169] each R10, R13, R14, R15, R16, R17, R21, R22, R23, R24, R25, R34, R37, and R38are independently H or C1-C4alkyl;

[0170] each R11, R12, R1g, R19, and R34are independently C1-C4alkyl; and

[0171] R20 is C1-C4alkyl or -(CH2)yN(CH3)2; and stereoisomers thereof.

[0172] As provided herein, C1-C4includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0173] In particular embodiments, the linker, L, is selected from:

[0174] As used herein, the term “amino acid” includes moieties having a carboxylic acid group and an amino group. The term amino acid includes both natural amino acids (including proteinogenic amino acids) and non-natural amino acids. The term ‘"natural amino acid” also includes other amino acids that can be incorporated into proteins during translation (including pyrrolysine and selenocysteine). Additionally, the term “natural amino acid” includes other amino acids that are formed during intermediary metabolism, e.g., ornithine generated from arginine in the urea cycle. A non-limiting list of representative amino acids is provided in Table 1.

[0175] In some embodiments, the amino acid is selected from proteinogenic amino acids. Proteinogenic amino acids include glycine, alanine, valine, leucinl e, isoleucine, aspartic acid, glutamic acid, serine, threonine, glutamine, asparagine, arginine, lysine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine and histidine.

[0176] The term amino acid also includes alpha amino acids and beta amino acids, such as, but not limited to, beta alanine and 2-methyl beta alanine. The term amino acid also includes certain lactam analogues of natural amino acids, such as, but not limited to, pyroglutamine. The termamino acid also includes amino acid homologues including, but not limited to, homocitrulline, homoargininc, homoscrinc, homotyrosinc, homoprolinc and homophcnylalaninc.

[0177] Examples of non-proteinogenic amino acids include, but are not limited to citrulline, hydroxyproline, 4-hydroxyproline, p-hydroxyvaline, ornithine, 0-amino alanine, albizziin, 4- amino-phenylalanine, biphenylalanine, 4-nitro-phenylalanine, 4-fluoro-phenylalanine, 2, 3, 4,5,6- pentafluoro-phenylalanine, norleucine, cyclohexylalanine, a- aminoiso butyric acid, a- aminobutyric acid, a-aminoisobutyric acid, 2-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid, selenomethionine, lanthionine, dehydroalanine, y-amino butyric acid, naphthylalanine, aminohexanoic acid, pipecolic acid, 2,3-diaminoproprionic acid, tetrahydroisoquinoline-3- carboxylic acid, tert-leucine, tert-butylalanine, cyclopropylglycine, cyclohexylglycine, 4- aminopiperidine-4-carboxylic acid, diethylglycine, dipropylglycine and derivatives thereof wherein the amine nitrogen has been mono- or di-alkylated.

[0178] The terminal portion of the amino acid residue or peptide may be in the form of the free acid, i.e., terminating in a -COOH group, or may be in a masked (protected) form, such as in the form of a carboxylate ester or carboxamide. In certain embodiments, the amino acid or peptide residue terminates with an amino group. In an embodiment, the residue terminates with a carboxylic acid group -COOH or an amino group -NEb. In another embodiment, the residue terminates with a carboxamide group. In yet another embodiment, the residue terminates with a carboxylate ester.

[0179] As disclosed hereinabove, the term “amino acid” includes compounds having a -COOH group and an -NHi group. A substituted amino acid includes an amino acid which has an amino group which is mono- or di-substituted. In particular embodiments, the amino group may be monosubstituted. (A proteinogenic amino acid may be substituted at another site from its amino group to form an amino acid which is a substituted proteinogenic amino acid). The term substituted amino acid thus includes N-substituted metabolites of the natural amino acids including, but not limited to, N-acetyl cysteine, N-acetyl serine, and N-acetyl threonine.

[0180] For example, the term “N-substituted amino acid” includes N-alkyl amino acids (e.g., Ci- 6 N-alkyl amino acids, such as sarcosine, N-methyl-alanine, N- methyl-glutamic acid and N-tert- butylglycine), which can include C1-6N-substituted alkyl amino acids (e.g., N-(carboxy alkyl) amino acids (e.g., N-(carboxymethyl)amino acids) and N-methylcycloalkyl amino acids (e.g., N- methylcyclopropyl amino acids)); N,N-di-alkyl amino acids (e.g., N,N-di-C1-6alkyl amino acids(e.g., N,N-dimethyl amino acid)); N,N,N-tri-alkyl amino acids (e.g., N,N,N-tri- C1-6alkyl amino acids (e.g., N,N,N-trimcthyl amino acid)): N-acyl amino acids (e.g., C1-6N-acyl amino acid): N- aryl amino acids (e.g., N -phenyl amino acids, such as N-phenylglycine); N-amidinyl amino acids (e.g., an N-amidine amino acid, i.e., an amino acid in which an amine group is replaced by a guanidino group),

[0181] The term “amino acid” also includes amino acid alkyl esters (e.g., amino acid C1-6alkyl esters); and amino acid aryl esters (e.g., amino acid phenyl esters). For amino acids having a hydroxy group present on the side chain, the term “amino acid” also includes O-alkyl amino acids (e.g., C1-6O-alkyl amino acid ethers); O-aryl amino acids (e.g., O-phenyl amino acid ethers); O- acyl amino acid esters; and O-carbamoyl amino acids. For amino acids having a thiol group present on the side chain, the term “amino acid” also includes S-alkyl amino acids (e.g., C1-6S- alkyl amino acids, such as S-methyl methionine, which can include C1-6S-substituted alkyl amino acids and S-methylcycloalkyl amino acids (e.g., S -methylcyclopropyl amino acids)); S-acyl amino acids (e.g., a C1-6S-acyl amino acid); S-aryl amino acid (e.g., a S-phenyl amino acid); a sulfoxide analogue of a sulfur-containing amino acid (e.g., methionine sulfoxide) or a sulfoxide analogue of an S-alkyl amino acid (e.g., S-methyl cysteine sulfoxide) or an S-aryl amino acid.

[0182] The presently disclosed subject matter also envisages derivatives of natural amino acids, such as those mentioned above which have been functionalized by simple synthetic transformations known in the art (e.g., as described in “Protective Groups in Organic Synthesis” by T W Greene and P G M Wuts, John Wiley & Sons Inc. (1999)), and references therein.

[0183] In one embodiment, an amino acid side chain is bound to another amino acid. In a further embodiment, the side chain is bound to the amino acid via the amino acid's N-terminus, C- terminus, or side chain. Examples of natural amino acid sidechains include hydrogen (glycine), methyl (alanine), isopropyl (valine), sec -butyl (isoleucine), -CH2CH(CH3)2(leucine), benzyl (phenylalanine), p-hydroxybenzyl (tyrosine), -CH2OH (serine), -CH(OH)CH3, (threonine), -CH2- 3-indoyl (tryptophan), -CH2COOH (aspartic acid), -CH2CH2COOH (glutamic acid), - CH2C(O)NH2(asparagine), -CH2CH2C(O)NH2(glutamine), -CH2SH, (cysteine), CH2CH2SCH3(methionine), -(CH2)4NH2(lysine), -(CH2)3NHC(=NH)NH2(arginine) and -CH2- 3-imidazoyl (histidine).

[0184] In some embodiments, the amino acid can be substituted with a monocyclic ring. Exemplary monocyclic rings and bicyclic rings include, without limitation, benzene, pyrimidines.and purines, and more generally aryl and heteroaryl rings. Exemplary heteroaryls include, but are not limited to, pyridyl, pyrimidinyl, pyrazinyl, furanyl, thienyl, pyrazolyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, pyrrolyl, imidazolyl, indolyl, indolinolyl, and imidazopyridazinyl. Aryls include phenyl (C&), benzyl, naphthyl (Cio), and biphenyl (C12). Exemplary pyrimidines include, without limitation, cytosine, thymine, and uracil. Exemplary purines include, without limitation, purine, adenine, N-substituted adenine, guanine, hypoxanthine, xanthine, theobromine, caffeine, uric acid, and isoguanine. Exemplary purine nucleosides include, without limitation, adenine and guanine.

[0185] The term “peptide” refers to an amino acid chain consisting of 2 to 9 amino acids, unless otherwise specified. In preferred embodiments, the peptide used in the present invention is 2 or 3 amino acids in length. In one embodiment, a peptide can be a branched peptide. In this embodiment, at least one amino acid side chain in the peptide is bound to another amino acid (either through one of the termini or the side chain). The term “N-substituted peptide” refers to an amino acid chain consisting of 2 to 9 amino acids in which one or more NH groups are substituted, e.g., by a substituent described elsewhere herein in relation to substituted amino groups. Optionally, the N-substituted peptide has its N-terminal amino group substituted and, in one embodiment, the amide linkages are unsubstituted.

[0186] In particular embodiments, the compound of formula (I) is selected from:and stereoisomers thereof

[0206] In other embodiments, the compound of formula (I) is selected from:

[0211] In other embodiments, the compound of formula (I) is selected from:stereoisomers thereof

[0217] In other embodiments, the compound of formula (I) is:and stereoisomers thereof.

[0219] Other Representative GCPII inhibitors

[0220] One of ordinary skill in the art would appreciate that derivatives of other GCPII inhibitors could be included as the GCPII / PSMA binding moiety of the compounds of formula (I). Known GCPII inhibitors representative of the general classes of GCPII inhibitors include, but are not limited to, 2-(phosphonomethyl) pentanedioic acid (2-PMPA) (phosphonates), 2-(3- mercaptopropyl)pentanedioic acid (2-MPPA) (thiols), 2-(2-(hydroxyamino)-2- oxoethyl)pentanedioic acid (JHU 241) (hydroxamates), see also Rais et al., 2017, for other hydroxamate-based glutamate GCPII inhibitors, including, 4-carboxy-alpha-[3-(hydroxyamino)- 3-oxopropyl]-benzenepropanoic acid, and A-[N-[(S)]-l,3-dicarboxypropyl] carbamoyl] -L-leucine(ZJ-43) (ureas):and stereoisomers thereof.

[0223] Other phosphonate-based GCPII inhibitors include GPI-5232 and VA-033:stereoisomers thereof.

[0225] Representative examples of the general classes of GCPII inhibitors are presented herein below.

[0226] Hydroxamate-based GCPII Inhibitors

[0227] Hydroxamate-based GCPII inhibitors include 2-(2-(hydroxyamino)-2- oxocthyl)pcntancdioic acid (JHU 241) and 4-carboxy-alpha-[3-(hydroxyamino)-3-oxopropyl]- benzenepropanoic acid. See Stoermer et al., 2003; Novakova et al., 2016; and Rais et al., 2017, for other hydroxamate-based glutamate GCPII inhibitors.

[0228] In some embodiments, the GCPII inhibitor is a hydroxamate-based GCPII inhibitor of formula (Ha):COOHHO'N"^M^COOH

[0229] H n(Ila);

[0230] wherein n is an integer selected from 0, 1, 2, and 3. In particular embodiments, the hydroxamate-based GCPII inhibitor comprises:

[0232] Phosphonate-based GCPII Inhibitors

[0233] As provided hereinabove, in some embodiments, the GCPII inhibitor is 2-PMPA.

[0234] Other phosphonate-based GCPII inhibitors include GPI-5232, Jackson and Slusher, 2001, and VA-033, Ding et al., 2004:

[0236] L-DOPA, D-DOPA, and Caffeic acid

[0237] In some embodiments, the presently disclosed subject matter provides L-DOPA, D-DOPA, and caffeic acid as GCPII inhibitors. Representative prodrugs of L-DOPA, D-DOPA, and caffeic acid are disclosed in International PCT Patent Application Publication No. WO2023064783 for DOPA and Caffeic Acid Analogs As Novel GCPII Inhibitors, to Rais et al., published April 20, 2023, which is incorporated herein by reference in its entirety.

[0238] More particularly, the presently disclosed subject matter provides prodrugs of L-DOPA, D-DOPA, and caffeic acid as compounds of formula (IV):

[0240] wherein:

[0241] - : = indicates that the bond can be a single or a double bond;

[0242] R1is:

[0243] -OR5, wherein R5is selected from the group consisting of H, Ci-Cs alkyl, and -O-(CH2)n- Re, wherein n is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8 and Re is substituted or unsubstituted aryl or heteroaryl; or

[0244] -NR7R8, wherein R7 and Rg are each independently selected from the group consisting of H, C1-C4alkyl, C3-C6 cycloalkyl, Ci-Cs alkoxyl, unsubstituted or substituted aryl or heteroaryl, - (CH2)m-R9, wherein R9is -OR10 or CHX2, wherein R10is H or C1-C4alkyl, and each X is halogen, and -(CH2)m-CH(NH2)(COOH), wherein each m is independently an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0245] R2is H or -NR11R12, wherein R11and R12 are each independently selected from the group consisting of H, C1-C4alkyl, and -C(=O)-R13, wherein R13is C1-C4alkyl or

[0246] -C(NH2)-(CH2)P-R14, wherein R14 is C1-C4alkyl or -NR15R16, wherein R15 and R16are each H or C1-C4alkyl, and p is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8;

[0247] R3and R4are each independently H or -C(=O)- R17, wherein R17is C1-C8alkyl or

[0248] -(CH2)t-O-C(=O)-O-R18, wherein R1s is Ci-Cs alkyl, and t is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8; and

[0249] stereoisomers and pharmaceutically acceptable salts thereof.

[0250] In certain embodiments, R1is -O R5, and R5is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, and n-octyl.

[0251] In certain embodiments, R1is -OR5, and R5is H or -O-(CH2)n-R6, wherein R6is substituted or unsubstituted phenyl.

[0252] In certain embodiments, R1is -NR7R8, and R7 is H or C1-C4alkyl and R8is selected from the group consisting of H, C1-C4alkyl, C3-C6cycloalkyl, unsubstituted or substituted phenyl, - (CH2)m-R9, wherein R9is -OR10 or CHX2, wherein R10is H or C1-C4alkyl, and each X is halogen, and -(CH2)m-CH(NH2)(COOH), wherein each m is independently an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8.

[0253] In certain embodiments, R2 is -NR11R12, wherein Ru is H and R12is H or -C(=O)-R13, wherein R13is C1-C4alkyl or -C(NH2)-(CH2)P-R14, wherein R14 is C1-C4alkyl or

[0254] -NR15R16, wherein R15and R16are each H or C1-C4alkyl, and p is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, and 8.

[0255] In certain embodiments, R3and R4 are each H.

[0256] In certain embodiments, if R1is -OR5, then R5cannot be H.

[0257] In certain embodiments, if R1is -OR5, then R3, R4, and R5cannot all be H.

[0258] In certain embodiments, R3and R4 arc each independently selected from the group consisting of -C(=O)-CH3, -C(=O)-C(CH3)3, and -CH2-O-C(=O)-O-CH(CH3)2.

[0259] In particular embodiments, the compound of formula (IV) is selected from the group consisting of:

[0265] Phosphinate-based GCP1I Inhibitors

[0266] Representative phosphinate-based GCP11 inhibitors include, but are not limited to:

[0267] 2-[[methylhydroxyphosphinyl]methyl]pentanedioic acid;

[0268] 2-[[ethylhydroxyphosphinyl]methyl]pentanedioic acid;

[0269] 2-[[propylhydroxyphosphinyl]methyl]pentanedioic acid;

[0270] 2-[[butylhydroxyphosphinyl]methyl]pentanedioic acid;

[0271] 2-[[cyclohexylhydroxyphosphinyl]methyl]pentanedioic acid;

[0272] 2- [[phenylhydroxyphosphinyl] methyl] pentanedioic acid;

[0273] 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]pentanedioic acid;

[0274] 2-[[((2-phenylethyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;

[0275] 2-[[((3-phenylpropyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;

[0276] 2-[[((3-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;

[0277] 2-[[((2-phenylbutyl)methyl)hydroxyphosphinyl]methyl]pentanedioic acid;

[0278] 2- [ [(4-pheny Ibuty l)hy droxypho sphiny 1] methyl] pentanedioic acid;

[0279] 2-[[(aminomethyl)hydroxyphosphinyl]methyl]pentanedioic acid;

[0280] 7-(L-2-amino-2-carboxyethylthio)-2-(2,2-dimethylcyclopropanecarboxamide)-2- hcptcnoic acid; 2-(phosphonomcthyl)pcntancdioic acid;

[0281] N-[methylhydroxyphosphinyl]glutamic acid;

[0282] N-[ethylhydroxyphosphinyl]glutamic acid;

[0283] N-[propylhydroxyphosphinyl]glutamic acid;

[0284] N-[butylhydroxyphosphinyl]glutamic acid;

[0285] N-[phenylhydroxyphosphinyl]glutamic acid; and

[0286] N-[(phenylmethyl)hydroxyphosphinyl]glutamic acid.

[0287] See U.S. Patent No. 11,167,049, for Organ protection in PSMA-targeted radionuclide therapy of prostate cancer, to Babich et al., issued Nov. 9, 2021, which is incorporated herein by reference in its entirety.

[0288] Phosphoramidate-based GCPII Inhibitors

[0289] Phosphoramidate-based GCPII inhibitors include compounds of formula (V):

[0290] (V).

[0291] Representative phosphoramidate-based GCPII inhibitors are disclosed in Ferraris et al., 2012, and include compounds of formula (V’):

[0293] wherein R is H or C1-C4alkyl, and R’ is benzyl; or a compound of formula (V”):

[0295] wherein R is selected from H, 4-fluorobenzoyl, and 6-(fluorescein-5- carboxamido)hexanoyl.

[0296] Thiol-based GCPII Inhibitors

[0297] Representative thiol-based GCPII inhibitors include 3-(2-mercaptoethyl)biphenyl-2,3- dicarboxylic acid (E2072) and GPI-5693:

[0299] See Wozniak et al., 2012b; Slusher et al., 2001. Other thiol-based are provided in Bafinka et al., 2012, and International Patent Application No. W02002057222 for Thiol-Based NAALADASE Inhibitors, to Tsukamoto et al., published July 25, 2002, which is incorporated herein by reference in its entirety.

[0300] Urea-based GCP1I Inhibitors

[0301] Urea-based GCPII inhibitors include MIP-1555, MIP-1519, MIP-1545, MIP-1427, MIP-1428, MIP-1379, MIP-1072, MIP-1095, MIP-1558, MIP-1405, and MIP-1404. See U.S. PatentNo. 11,167,049, for Organ protection in PSMA-targeted radionuclide therapy of prostate cancer, to Babich et al., issued Nov. 9, 2021, which is incorporated herein by reference in its entirety.

[0302] Other urea-based GCPII inhibitors include PSMA I&T, Weineisen et al., 2015, PSMA- 617, Benesova et al., 2015, PSMA-11, Eder et al., 2012, DCIBzL, Chen et al., 2008,18F-DCFPyl,Chen et al., 2011, ZJ 38, GCPII-IN-1, and JB-352, Knedlrk et al., 2017:

[0305] Other GCPI1 Inhibitors

[0306] Other representative GCPII inhibitors include quisqualate and P-citryl-L-glutamate:

[0308] See Knedlik et al., 2017.

[0309] In some embodiments, the GCPII inhibitor includes:

[0310] (S)-2-((N-((S)-l,2-dicarboxyethyl)sulfamoyl)amino)pentanedioic acid:

[0312] (S)-2-((((S)-5-(4-bromo-2-fluorobenzamido)-l- carboxypentyl)carbamoyl)oxy)pentanedioic acid:

[0314] (S)-2-((5)- 1 -carboxy-3-methylbutylcarbamoyloxy)pentanedioic acid:

[0317] In some embodiments, the presently disclosed subject matter provides a method for treating an inflammatory bowel disease (IBD), the method comprising administering to a subject in need of treatment thereof a compound of formula (I) as provided herein.

[0318] In general, using the presently disclosed methods to treat the IBD in a subject results in a decrease in the severity of the IBD. As used herein, the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disease or condition, such as IBD, and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease or condition, such as IBD, does not require that the disorder, condition or symptoms associated therewith be completely eliminated. The term “decrease” is meant to inhibit, suppress, attenuate, diminish, arrest, or stabilize at least one symptom of IBD (e.g., rectal prolapse, gut inflammation, colonic hypertrophy, stool inconsistency, and the like).

[0319] As provided hereinabove, GCPII activity is markedly elevated in the diseased intestinal mucosa of subjects with IBD. As used herein, the term “elevated GCPII activity” means an increase of GCPII activity in a subject with IBD as compared to the GCPII activity in a subjectwithout IBD, such as an increase of approximately 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more.

[0320] In some embodiments, the presently disclosed subject matter provides methods for inhibiting GCPII activity. In some embodiments, the presently disclosed subject matter provides methods for inhibiting GCPII activity in a subject afflicted with IBD. As used herein, the term “inhibit” means to decrease or diminish GCPII activity in a subject in need thereof. The term “inhibit” also may mean to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or condition, such as IBD. Inhibition may occur, for e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or even 100% compared to an untreated control subject or a subject without the disease or disorder.

[0321] IBD has been classified into the broad categories of Crohn’s disease and ulcerative colitis. Accordingly, as used herein, “a subject having inflammatory bowel disease” is synonymous with the term “a subject diagnosed with having an inflammatory bowel disease,” and means a patient having Crohn’s disease or ulcerative colitis. Crohn’s disease (regional enteritis) is a disease of chronic inflammation that can involve any part of the gastrointestinal tract. Commonly, the distal portion of the small intestine (ileum) and cecum are affected. In other cases, the disease is confined to the small intestine, colon or anorectal region. Crohn’s disease occasionally involves the duodenum and stomach, and more rarely the esophagus and oral cavity.

[0322] The variable clinical manifestations of Crohn’s disease are, in part, a result of the varying anatomic localization of the disease. The most frequent symptoms of CD are abdominal pain, diarrhea and recurrent fever. CD is commonly associated with intestinal obstruction or fistula, which is an abnormal passage between diseased loops of bowel, for example. Crohn’s disease also includes complications such as inflammation of the eye, joints and skin; liver disease; kidney stones or amyloidosis. In addition, CD is associated with an increased risk of intestinal cancer.

[0323] Several features are characteristic of the pathology of Crohn’s disease. The inflammation associated with CD, known as transmural inflammation, involves all layers of the bowel wall. Thickening and edema, for example, typically also appear throughout the bowel wall, with fibrosis also present in long-standing disease. The inflammation characteristic of CD also is discontinuous in that segments of inflamed tissue, known as “skip lesions,” are separated by apparently normal intestine.

[0324] Furthermore, linear ulcerations, edema, and inflammation of the intervening tissue lead to a “cobblestone” appearance of the intestinal mucosa, which is distinctive of CD. A hallmark of Crohn’s disease is the presence of discrete aggregations of inflammatory cells, known as granulomas, which are generally found in the submucosa. Some Crohn’s disease cases display the typical discrete granulomas, while others show nonspecific transmural inflammation. As a result, the presence of discrete granulomas is indicative of CD, although the absence of granulomas also is consistent with the disease. Thus, transmural or discontinuous inflammation, rather than the presence of granulomas, is a preferred diagnostic indicator of Crohn’s disease (Rubin and Farber, 1994).

[0325] Ulcerative colitis (UC) is a disease of the large intestine characterized by chronic diarrhea with cramping abdominal pain, rectal bleeding, and loose discharges of blood, pus and mucus. The manifestations of ulcerative colitis vary widely. A pattern of exacerbations and remissions typifies the clinical course of most UC patients (70%), although continuous symptoms without remission are present in some patients with UC. Local and systemic complications of UC include arthritis, eye inflammation such as uveitis, skin ulcers and liver disease. In addition, ulcerative colitis and especially long-standing, extensive disease is associated with an increased risk of colon carcinoma.

[0326] Several pathologic features characterize UC in distinction to other inflammatory bowel diseases. Ulcerative colitis is a diffuse disease that usually extends from the most distal part of the rectum for a variable distance proximally. The term left-sided colitis describes an inflammation that involves the distal portion of the colon, extending as far as the splenic flexure. Sparing of the rectum or involvement of the right side (proximal portion) of the colon alone is unusual in ulcerative colitis. The inflammatory process of ulcerative colitis is limited to the colon and does not involve, for example, the small intestine, stomach or esophagus. In addition, ulcerative colitis is distinguished by a superficial inflammation of the mucosa that generally spares the deeper layers of the bowel wall. Crypt abscesses, in which degenerated intestinal crypts are filled with neutrophils, also are typical of ulcerative colitis (Rubin and Farber, 1994).

[0327] In comparison with Crohn’s disease, which is a patchy disease with frequent sparing of the rectum, ulcerative colitis is characterized by a continuous inflammation of the colon that usually is more severe distally than proximally. The inflammation in ulcerative colitis is superficial in that it is usually limited to the mucosal layer and is characterized by an acute inflammatory infiltrate with neutrophils and crypt abscesses. In contrast, Crohn’s disease affects the entire thickness ofthe bowel wall with granulomas often, although not always, present. Disease that terminates at the ileocecal valve, or in the colon distal to it, is indicative of ulcerative colitis, while involvement of the terminal ileum, a cobblestone-like appearance, discrete ulcers or fistulas suggest Crohn’s disease.

[0328] Common symptoms of IBD include, but are not limited to, abdominal pain, diarrhea, fecal incontinence and / or urgency to have a bowel movement, rectal bleeding, weight loss, fever, anemia, malnutrition, delayed growth, anxiety, and depression. The condition also can cause swelling or masses, due to inflammation in the intestines.

[0329] If inflammation due to IBD is not controlled, IBD can further damage the intestines causing abscesses, strictures, and fistulas. Patients afflicted with IBD also are at a higher risk of developing colon cancer. IBD also can affect other organs, including, but not limited to, episcleritis or uveitis of the eye; stomatitis, sores, and ulcers of the mouth; steatosis of the liver; gallstones and sclerosing cholangitis of the bile duct system; kidney stones, hydronephrosis, fistulas, and urinary tract infections; erythema nodosum and pyoderma gangrenosum of the skin; spondylolysis, sacroiliitis, and arthritis in the vertebrae, limbs, and joints; and phlebitis (inflammation of blood vessels).

[0330] There are no curative treatments for IBD. As such, current treatment regimens seek to reduce inflammation, reduce the frequency and severity of flare ups, and to treat symptoms. Current treatment regimens include, but are not limited to, immunosuppressants, antiinflammatories, and steroids. Immunosuppressant drugs include, but are not limited to, azathioprine (AZASAN, IMURAN), mercaptopurine (PURINETHOL, PURIXAN) and methotrexate (TREXALL). Anti-inflammatories include, but are not limited to, aminosalicylates, such as mesalamine (DELZICOL, ROWASA, and others), balsalazide (COLAZAL) and olsalazine (DIPENTUM). Orally available agents for treating IBD also include tofacitinib (XELJANZ), upadacitinib (RINVOQ), and ozanimod (ZEPOSIA). Other therapies for IBD include stem cell therapy and fecal transplant.

[0331] The intestines can become damaged if medications do not reduce inflammation. In such cases, surgery can be required to remove an area of the intestine, repair blockages, strictures, abscesses, or fistulas.

[0332] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0333] In general, the “effective amount” of an active agent or drug delivery device refers to the amount necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an agent or device may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the makeup of the pharmaceutical composition, the target tissue, and the like.

[0334] In some embodiments, the presently disclosed subject matter is a pharmaceutical composition, comprising at least one compound of general formula (I) alone or in combination with one or more additional therapeutic agents in admixture with a pharmaceutically acceptable excipient. The one or more additional therapeutic agents can include other agents useful for treating inflammatory bowel disease.

[0335] In some embodiments, the other therapeutic agent comprises an immunosuppressant drug including, but not limited to azathioprine (AZASAN, IMURAN), mercaptopurine (PURINETHOL, PURIXAN), cyclosporine (GENGRAF, NEORAL, SANDIMMUNE), and methotrexate (TREXALL).

[0336] In some embodiments, the other therapeutic agent comprises another biologic, including, but not limited to, natalizumab (TYSABRI), vedolizumab (ENTYVIO) and ustekinumab (STELARA).

[0337] In some embodiments, the other therapeutic agent comprises an anti-inflammatory agent. Representative anti-inflammatory agents include aminosalicylates, such as mcsalaminc (ASACOL HD, DELZICOL, ROWASA, and others), balsalazide (COLAZAL) and olsalazine (DIPENTUM).

[0338] In some embodiments, the other therapeutic agent comprises an orally available agents for treating IBD including, but not limited to, tofacitinib (XELJANZ), upadacitinib (RINVOQ), and ozanimod (ZEPOSIA).

[0339] In some embodiments, the other therapeutic agent comprises a tumor necrosis factor (TNF)-alpha inhibitor including, but not limited to, infliximab (REMICADE), adalimumab (HUMIRA) and golimumab (SIMPONI).

[0340] In some embodiments, the other therapeutic agent comprises an antibiotic including, not limited to, ciprofloxacin (CIPRO) and metronidazole (FLAGYL).

[0341] In some embodiments, the other therapeutic agent comprises an anti-diarrheal medication, such as loperamide (IMODIUM A-D).

[0342] In some embodiments, the other therapeutic agent comprises a fiber supplement including, but not limited to, psyllium powder (METAMUCIL) and methylcellulose (CITRUCEL).

[0343] In some embodiments, the other therapeutic agent comprises a pain reliever, e.g., acetaminophen.

[0344] In some embodiments, the other therapeutic agent comprises a vitamin or a mineral supplement including, but not limited to, an iron supplement, a calcium supplement, and a vitamin D supplement.

[0345] In other embodiments, the presently disclosed methods can be used in combination with other therapeutic treatments for IBD including, but not limited to, stem cell therapy, surgery, and fecal transplant.

[0346] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a compound of formula (I) and at least one additional therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agentsare combined and administered in a single dosage form. In another embodiment, the active agents arc administered in separate dosage forms (c.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0347] Further, the compounds of formula (I) described herein can be administered alone or in combination with adjuvants that enhance stability of the compounds of formula (I), alone or in combination with one or more antibacterial agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0348] The timing of administration of a compound of formula (I) and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound of formula (I) and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a compound of formula (I) and at least one additional therapeutic agent can receive compound of formula (I) and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0349] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the compound of formula (I) and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a compound of formula (I) or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0350] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agentwhen administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0351] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound of formula (I) and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0352] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0353] Qa / QA + QB / QB = Synergy Index (SI)

[0354] wherein:

[0355] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0356] Qais the concentration of component A, in a mixture, which produced an end point;

[0357] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0358] Qb is the concentration of component B, in a mixture, which produced an end point.

[0359] Generally, when the sum of QH / QA and Qb / Qs is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0360] Formulations

[0361] Depending on the specific conditions being treated, the “agent(s)” may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may bedelivered, for example, in a timed- or sustained-slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra- sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

[0362] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0363] Use of pharmaceutically acceptable inert carriers to formulate the compounds herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0364] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.

[0365] In particular' embodiments, the compound disclosed herein is administered intranasally in a form selected from the group consisting of a nasal spray, a nasal drop, a powder, a granule, a cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures.

[0366] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients arc contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the compounds according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the compound is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the compound(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the compound(s), and the preference and experience of the attending physician.

[0367] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0368] Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0369] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0370] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, scaled capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.

[0371] Further, one of ordinary skill in the art will recognize that the presently disclosed compounds, and pharmaceutical compositions thereof, include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and include salts of active compounds that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. The parent form of the compound can differ from the various salt forms in certain physical properties, such as solubility, and the like.

[0372] When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like.

[0373] When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Compounds containing both basic and acidic functionalities allow such compounds to be converted into either base or acid addition salts.

[0374] Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate,carbonate, monohydrogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, galactonatc, gluceptate, gluconate, glutamate, glycollylarsanilatc, hcxylrcsorcinatc, hydrabaminc, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate, monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate, succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (+)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p- toluenesulfonate and trifluoroacetate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0375] Chemical Definitions

[0376] Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0377] The term “hydrocarbon” as used herein, refers to any chemical group comprising hydrogen and carbon. A hydrocarbon group may be substituted or unsubstituted. As would be known to one of ordinary skill in the art, all valencies must be satisfied in making any substitutions. The hydrocarbon may be unsaturated, saturated, branched, unbranched, cyclic, polycyclic, or heterocyclic.

[0378] The term “aliphatic” or “aliphatic group”, as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocyclyl”, “cycloaliphatic”, or “cycloalkyl”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments,“cycloaliphatic” (or “carbocyclyl” or “cycloalkyl”) refers to a monocyclic C3-C7 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl, or (cycloalkyl)alkenyl.

[0379] The term “alkane” refers to acyclic branched or unbranched hydrocarbons having the general formula CnH2n+2, and therefore consisting entirely of hydrogen atoms and saturated carbon atoms.

[0380] The term “alkyl” refers to a univalent group derived from an alkane by removal of a hydrogen atom from any carbon atom and having the chemical formula of -CnEhn+i. The groups derived by removal of a hydrogen atom from a terminal carbon atom of unbranched alkanes form a subclass of normal alkyl (n-alkyl) groups H(CH2)n. The groups RCH2, R2CH (R H), and R3C (R H) are primary, secondary and tertiary alkyl groups, respectively.

[0381] An alkyl can be a straightchain (i.e., unbranched) or branched acyclic hydrocarbon having the number of carbon atoms designated (i.e., C1-10 means one to ten carbons, including 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 carbons). In particular embodiments, the term “alkyl” refers to C1-20 inclusive, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. In other embodiments, the alkyl can be a C1-C4alkyl, including 1, 2, 3, and 4 carbons. In yet other embodiments, the alkyl can be a Ci-Ce alkyl, including 1, 2, 3, 4, 5, and 6 carbons. In even yet other embodiments, the alkyl can be a Ci-Cs alkyl, including 1, 2, 3, 4, 5, 6, 7, and 8 carbons.

[0382] “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms (i.e., a Cns alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, “alkyl” refers to straight-chain alkyls. In other embodiments, “alkyl” refers to branched alkyls. In certain other embodiments, “alkyl” refers to straight-chain and / or branched alkyls. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain.

[0383] Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, rz-pentyl, sec-pentyl, isopentyl, neopentyl, n- hexyl, sec-hexyl, n-heptyl, 71-octyl, w-decyl, n-undecyl, and dodecyl.

[0384] Alkyl groups can optionally be substituted (a “substituted alkyl”) with one or more substituents, which can be the same or different. Such substituent groups include, but arc not limited to, alkyl, substituted alkyl, cycloalkyl, halogen, acyl, carboxyl, oxo, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, cyano, and mercapto.

[0385] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain having from 1 to 20 carbon atoms or heteroatoms consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen, phosphorus, and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) O, N, P and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, - CH2-S-CH2-CH3, -CH2-CH2-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, - CH2-CH=N-OCH3, -CH=CH-N(CH3)- CH3, O-CH3, -O-CH2-CH3, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3and -CH2-O-Si(CH3)3.

[0386] The term “cycloalkane” refers to saturated monocyclic hydrocarbons (with or without side chains), e.g., cyclobutane. Unsaturated monocyclic hydrocarbons having one endocyclic double or one triple bond are called cycloalkenes and cycloalkynes, respectively. Those having more than one such multiple bond are cycloalkadienes, cycloalkatrienes, and the like. The inclusive terms for any cyclic hydrocarbons having any number of such multiple bonds are cyclic olefins or cyclic acetylenes.

[0387] The term “cycloalkyl” refer to a univalent group derived from a cycloalkane by removal of a hydrogen atom from a ring carbon atom. Cycloalkyls can be a mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group also can be optionally substituted with a substituent group provided hereinabove for alkyl groups. Representative monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl, and fused ring systems, such as dihydro- and tetrahydronaphthalene, and the like.

[0388] The term “cycloalkylalkyl” as used herein, refers to a cycloalkyl group, which is attached to the parent molecular moiety through an alkylene moiety, also as defined above, e.g., a Ci-20alkylene moiety. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cy clopcnty lethy 1.

[0389] The terms “cycloheteroalkyl” and “heterocycloalkyl” (or more generally “heterocyclic”) are used interchangeably and refer to an unsaturated ring system, such as a 3- to 10-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), and silicon (Si), in which the nitrogen, sulfur, and phosphorus heteroatoms may be oxidized and the nitrogen heteroatom may be quatemized. The cyclohetero alkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and / or non-aromatic hydrocarbon rings. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidinyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like.

[0390] The terms “cycloalkylene” and “heterocycloalkylene” refer to the divalent derivatives of cycloalkyl and heterocycloalkyl, respectively.

[0391] As used herein the terms “bicycloalkyl” and “bicycloheteroalkyl” refer to two cycloalkyl or cycloheteroalkyl groups that are bound to one another. Non-limiting examples include bicyclohexane and bipiperidine.

[0392] An “unsaturated hydrocarbon” has one or more double bonds or triple bonds. As used herein, the term “alkene” refers to an acyclic branched or unbranched hydrocarbons having one carbon-carbon double bond and the general formula CnHin. Acyclic branched or unbranched hydrocarbons having more than one double bond are alkadienes, alkatrienes, and the like.

[0393] More particularly, the term “alkenyl” as used herein refers to a monovalent group derived from a C2-20 inclusive straight or branched hydrocarbon moiety having at least one carbon-carbon double bond by the removal of a single hydrogen molecule. Alkenyl groups include, but are not limited to, ethenyl (i.e., vinyl), 2-propenyl, butenyl, l-methyl-2-buten-l-yl, pentenyl, 2- isopentenyl, hexenyl, octenyl, allenyl, butadienyl, crotyl (but-2-en-l-yl), 2-(butadienyl), 2,4- pentadienyl, 3-(l,4-pentadienyl), and the like, including higher homologs and isomers.

[0394] The term “cycloalkenyl” as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl, 1 ,3-cyclohexadiene, cycloheptenyl, cyclohcptatricnyl, and cyclooctcnyl.

[0395] The term “alkyne” as used herein refers to an acyclic branched or unbranched hydrocarbons having a carbon-carbon triple bond and the general formula CnH2n-2, RC=CR. Acyclic branched or unbranched hydrocarbons having more than one triple bond are known as alkadiynes, alkatriynes, and the like.

[0396] The term “alkynyl” as used herein refers to a monovalent group derived from a straight or branched C2-20 hydrocarbon of a designed number of carbon atoms containing at least one carboncarbon triple bond. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, pentynyl, hexynyl, and heptynyl groups, and the like.

[0397] As used herein, the term “alkylene” refers to an alkanediyl group having the free valencies on adjacent carbon atoms, e.g. -CH(CH.3)CH2- propylene (systematically called propane-1, 2- diyl). More particularly, the term “alkylene” by itself or a part of another substituent refers to a straight or branched bivalent aliphatic hydrocarbon group derived from an alkyl group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and / or substituted with one or more “alkyl group substituents.” There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as “alkylaminoalkyl”), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH2- ); ethylene (-CH2-CH2-); propylene (-(CFhj -); cyclohexylene (-CeHio-); -CH=CH-CH=CH-; -CH=CH-CH2-; -CH2CH2CH2CH2-, -CH2CHCHCH2-, -CH2CSCCH2-, CH2CH2CH(CH2CH2CH3)CH2-, -(CH2)q-N(R)-(CH2)r- , wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-O-CH2-O-); and ethylenedioxyl (-O-(CH2)2-O-). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being some embodiments of the present disclosure. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0398] The term “heteroalkylene” by itself or as part of another substituent means a divalent group derived from hctcroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and -CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, heteroatoms also can occupy either or both of the chain termini (e.g., alkyleneoxo, alkylenedioxo, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)OR’- represents both -C(O)OR’- and -R’OC(O)-.

[0399] The term “arene” refers to a monocyclic and polycyclic aromatic hydrocarbon.

[0400] The term “aryl” refers to a group derived from arenes by removal of a hydrogen atom from a ring carbon atom. Groups similarly derived from heteroarenes are sometimes subsumed in this definition. An aryl group can include, for example, a single ring or multiple rings (such as from 2 to 3 rings), which are fused together or linked covalently.

[0401] The term “heteroaryl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)i(OH)m(1 40), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group.

[0402] The term “heteroaryl” refers to the class of heterocyclyl groups derived from heteroarenes by removal of a hydrogen atom from any ring atom. A “heteroaryl” group can include from one to four heteroatoms (in each separate ring in the case of multiple rings) selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 -naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. The terms “arylene” and “hetero arylene” refer to the divalent forms of aryl and heteroaryl, respectively.

[0403] For brevity, the term “aryl” when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and hctcroaryl rings as defined above. Thus, the terms “arylalkyl” and “heteroarylalkyl” are meant to include those groups in which an aryl or heteroaryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, furylmethyl, and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like). However, the term “haloaryl,” as used herein is meant to cover only aryls substituted with one or more halogens.

[0404] Where a heteroalkyl, heterocycloalkyl, or heteroaryl includes a specific number of members (e.g., “3 to 7 membered”), the term “member” refers to a carbon or heteroatom.

[0405] Each of above terms defined hereinabove (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl, and “heterocycloalkyl”, “alkenyl”, “alkynyl,” “aryl,” “heteroaryl,” as well as their divalent derivatives) are meant to include both substituted and unsubstituted forms of the indicated group. Optional substituents for each type of group are provided below.

[0406] As used herein, the term “acyl” refers to a group formed by removing one or more hydroxy groups from oxoacids that have the general structure RkE(=O)i(OH)m(1 0), and replacement analogues of such acyl groups. In organic chemistry an unspecified acyl group is commonly a carboxylic acyl group. For example, in some embodiments, the term acyl includes an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent and has the general formula RC(=O)-, wherein R is an alkyl, alkenyl, alkynyl, aryl, carbocylic, heterocyclic, or aromatic heterocyclic group as defined herein). As such, the term “acyl” specifically includes arylacyl groups, such as a 2-(furan-2-yl)acetyl)- and a 2-phenylacetyl group. Specific examples of acyl groups include acetyl and benzoyl. Acyl groups also are intended to include amides, -RC(=O)NR’, esters, -RC(=O)OR’, ketones, -RC(=O)R’, and aldehydes, -RC(=O)H.

[0407] The terms “alkoxyl” or “alkoxy” are used interchangeably herein and refer to a saturated (i.e., alkyl-O-) or unsaturated (i.e., alkenyl-O- and alkynyl-O-) group attached to the parent molecular moiety through an oxygen atom, wherein the terms “alkyl,” “alkenyl,” and “alkynyl” are as previously described and can include C1-20 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl,isopropoxyl, n-butoxyl, sec-butoxyl, tert-butoxyl, and n-pentoxyl, neopentoxyl, n-hexoxyl, and the like.

[0408] The term ‘‘alkoxy alkyl” as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

[0409] “Aryloxyl” refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term “aryloxyl” as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl.

[0410] “Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl.

[0411] “Aralkyloxyl” refers to an aralkyl-O- group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl, i.e., C6H5-CH2-O-. An aralkyloxyl group can optionally be substituted.

[0412] “Alkoxycarbonyl” refers to an alkyl-O-C(=O)- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and tert-butyloxycarbonyl.

[0413] “Aryloxycarbonyl” refers to an aryl-O-C(=O)- group. Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

[0414] “Aralkoxy carbonyl” refers to an aralkyl-O-C(=O)- group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

[0415] The term “acyloxyl” refers to an oxygen-centered radicals consisting of an acyl radical bonded to an oxygen atom, e.g., an acyl-O- group wherein acyl is as previously described.

[0416] The term “amine” refers to a compound formally derived from ammonia by replacing one, two or three hydrogen atoms by hydrocarbyl groups, and having the general structures RNH2(primary amines), R2NH (secondary amines), R3N (tertiary amines). In some embodiments, the term amino refers to the -NH2group. More generally, the amino group is -NR'R”, wherein R' and R” are typically selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0417] The terms “acylamino” and “alkylamino” refer to specific N-substituted organic radicals with acyl and alkyl substituent groups, respectively.

[0418] An “aminoalkyl” as used herein refers to an amino group covalently bound to an alkylene linker. More particularly, the terms alkylamino, dialkylamino, and trialkylamino as used herein refer to one, two, or three, respectively, alkyl groups, as previously defined, attached to the parent molecular moiety through a nitrogen atom. The term alkylamino refers to a group having the structure -NHR’ wherein R’ is an alkyl group, as previously defined; whereas the term dialkylamino refers to a group having the structure -NR’R”, wherein R’ and R” are each independently selected from the group consisting of alkyl groups. The term trialkylamino refers to a group having the structure -NR’R”R”’, wherein R’, R”, and R’” are each independently selected from the group consisting of alkyl groups. Additionally, R’, R”, and / or R’” taken together may optionally be -(CH2)k- where k is an integer from 2 to 6. Examples include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl, methylethylamino, isopropylamino, piperidino, trimethylamino, and propylamino.

[0419] The terms alkylthioether and thioalkoxyl refer to a saturated (i.e., alkyl-S-) or unsaturated (i.e., alkenyl-S- and alkynyl-S-) group attached to the parent molecular moiety through a sulfur atom. Examples of thioalkoxyl moieties include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0420] “Acylamino” refers to an acyl-NH- group wherein acyl is as previously described. “Aroylamino” refers to an aroyl-NH- group wherein aroyl is as previously described.

[0421] The term “carbonyl” refers to a compound containing the carbonyl group, -C(=O)-. The term is commonly used in the restricted sense of aldehydes (R-C(=O)H) and ketones, although it actually includes carboxylic acids and derivatives.

[0422] The term “carboxylic acid” refers to an oxoacids having the structure RC(=O)OH. The term is used as a suffix in systematic name formation to denote the -C(=O)OH group including its carbon atom. In some embodiments, the term “carboxyl” refers to the -COOH group. Such groups also are referred to herein as a “carboxylic acid” moiety.

[0423] “Carbamoyl” refers to an amide group of the formula -COOjNIE.

[0424] “Alky Icarbamoyl” refers to a R’RN-C(=O)- group wherein one of R and R’ is hydrogen and the other of R and R’ is alkyl and / or substituted alkyl as previously described.

[0425] “Dialkylcarbamoyl” refers to a R’RN-C(=O)- group wherein each of R and R’ is independently alkyl and / or substituted alkyl as previously described.

[0426] The term carbonyldioxyl, as used herein, refers to a carbonate group of the formula -O- C(=O)-OR.

[0427] The term “cyano” refers to the -C=N group.

[0428] The terms “halo,” “halide,” or “halogen” as used herein refer to fluoro, chloro, bromo, and iodo groups. Additionally, terms such as “haloalkyl,” are meant to include monohaloalkyl and polyhaloalkyl. For example, the term “halo(Ci-4)alkyl” is mean to include, but not be limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0429] The term “hydroxyl” refers to the -OH group.

[0430] The term “hydroxy alkyl” refers to an alkyl group substituted with an -OH group.

[0431] The term “mercapto” refers to the -SH group.

[0432] The term “oxo compound” refers to a compounds containing an oxygen atom, =0, doubly bonded to carbon or another element. The term thus embraces aldehydes, carboxylic acids, ketones, sulfonic acids, amides and esters. Oxo used as an adjective (and thus separated by a space) modifying another class of compound, as in oxo carboxylic acids, indicates the presence of an oxo substituent at any position. To indicate a double-bonded oxygen that is part of a ketonic structure, the term keto is sometimes used as a prefix, but such use has been abandoned by IUPAC for naming specific compounds. A traditional use of keto is for indicating oxidation of CHOH to C=O in a parent compound that contains OH groups, such as carbohydrates, e.g., 3-ketoglucose. In some embodiments, the term “oxo” as used herein means an oxygen atom that is double bonded to a carbon atom or to another element.

[0433] The term “nitro” refers to the -NO2 group.

[0434] The term “thio” refers to replacement of an oxygen by a sulfur, e.g., PhC(=S)NH2, thiobenzamide.

[0435] The term “thiol” refers to a compounds having the structure RSH (R H), e.g., MeCH2SH ethanethiol. A thiol also is known by the term “mercaptan.”

[0436] The term “thiohydroxyl” or “thiol,” as used herein, refers to a group of the formula -SH.

[0437] The term “sulfate” refers to the -SO4 group.

[0438] The term “sulfide” refers to a compound having the structure RSR (R H) and also are referred to as “thioethers.”

[0439] The term “sulfone” refers to a compound having the structure, RS(=O)2R (R t H), e.g., C2H5S(=O)2CH3ethyl methyl sulfone.

[0440] The term “sulfoxide” refers to a compound having the structure R2S=O (R H), e.g., Ph2S=O diphenyl sulfoxide.

[0441] The term “ureido” refers to a urea group of the formula -NH — CO — NH2.

[0442] One of ordinary skill in the art would recognize that a structure represented generally by, for example, the formula;

[0444] as used herein refers to a ring structure, for example, but not limited to a 3-carbon, a 4- carbon, a 5-carbon, a 6-carbon, a 7-carbon, and the like, aliphatic and / or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the variable “n,” which is an integer generally having a value ranging from 0 to the number of carbon atoms on the ring available for substitution. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:

[0445]

[0446] and the like.

[0447] A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.

[0448] The symbol (lWVWWW) denotes the point of attachment of a moiety to the remainder of the molecule.

[0449] When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being “absent,” the named atom is replaced by a direct bond.

[0450] Throughout the specification and claims, a given chemical formula or name shall encompass all tautomers, congeners, and optical- and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

[0451] Certain compounds of the present disclosure may possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as D- or L- for amino acids, and individual isomers are encompassed within the scope of the present disclosure. The compounds of the present disclosure do not include those which are known in art to be too unstable to synthesize and / or isolate. The present disclosure is meant to include compounds in racemic, scalemic, and optically pure forms. Optically active (R)- and (S)-, or D- and L-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefenic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.

[0452] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the disclosure.

[0453] As used herein, the term “congener” refers to one of two or more substances related to each other by origin, structure, or function.

[0454] The term “enantiomer” refers to one of a pair of molecular entities which are mirror images of each other and non-superposable.

[0455] The term “stereoisomer” refers to an isomer that possess identical constitution, but which differ in the arrangement of their atoms in space.

[0456] The term “racemate” refers to an equimolar mixture of a pair of enantiomers. It does not exhibit optical activity. The chemical name or formula of a racemate is distinguished from those of the enantiomers by the prefix (+)- or rac- (or racem-) or by the symbols RS and SR.

[0457] The term “diastereoisomerism” refers to stereoisomerism other than enantiomerism. Diastereoisomers (or diastereomers) are stereoisomers not related as mirror images.Diastereoisomers are characterized by differences in physical properties, and by some differences in chemical behavior towards achiral as well as chiral reagents.

[0458] It will be apparent to one skilled in the art that certain compounds of this disclosure may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the disclosure. The term “tautomer,” as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0459] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures with the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by13C- or14C-enriched carbon are within the scope of this disclosure.

[0460] The compounds of the present disclosure may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine- 125 (125I) or carbon- 14 (14C). All isotopic variations of the compounds of the present disclosure, whether radioactive or not, are encompassed within the scope of the present disclosure.

[0461] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / - 15%, + / - 10%, + / -5%, + / -4%, + / -3%, + / - 2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0462] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0463] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that donot preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.c., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0464] The following Examples have been included to provide guidance to one of ordinary skill in the ait for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be constmed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 1

[0465] Methods

[0466] Uptake Studies lb PCS -PIP Versus PC3-Flu Cells

[0467] Cell uptake assays were performed with GCPII-expressing (PIP) or non-expressing (Flu) PC-3 cells as described previously with modifications. Choy et al., 2017. Briefly, cells were maintained in RPMI containing 10% FBS and 1% antibio tic / antimycotic. Cells were plated in 6- well plates at a density of 250,000 cells / well in a final volume of 1 mL of growth media and incubated overnight. Twenty-four hours later, cells were incubated for 30 minutes at 37 °C in 0.5 mL / well internalization buffer (phosphate and glutamine-free RPMI + 1% FBS) with or without 10 M ZJ-43 to block GCPII binding. After this incubation, the internalization buffer was replaced with fresh buffer containing 100 or 300 nM test conjugates, with or without 10 pM ZJ 43, and incubated for 2 hours at 37 °C. Cells were then rinsed with 1 mL HBSS, followed by the addition and immediate removal of 1 mL 2.5% Trypsin-EDTA, to release extracellularly bound conjugates. Cells were suspended in 1 mL ice-cold rinse buffer (NaCl [154 mM], KC1 [5.64 mM], CaCh [2.16mM], MgCh [1 M], dextrose [11.1 mM], NaHCCh [2.38 M] + 10% FBS), transferred to centrifuge tubes, and centrifuged at 1000 x g for 5 minutes at 4°C. Supernatants were discarded. The rinse / centrifugation steps were repeated total three times to remove unbound / residual conjugates. Cell pellets were lysed with 200 pL methanol and dried under vacuum at 45 °C for one hour. Lysates were resuspended in 30 pL methanol containing 0.5 pM losartan (as an internal standard) by water bath sonication (10 minutes on ice) and vigorous vortexing. Lysates were then centrifuged for 5 minutes at 16,000 x g at 4 °C. Supernatants (20 pL) were transferred to a 96- well plate, diluted with 40 pL water, and conjugates were quantified by LC-MS.

[0468] Two microliters were injected onto a QExactive Focus orbitrap mass spectrometer coupled to an UltiMate 3000 LC system using an Agilent Eclipse Plus PhenyLHexyl 2.1 x 100 mm column. A gradient of mobile phases (20 / 80 to 5 / 95, water + 0.1% formic acid / acetonitrile + 0.1% formic acid) was run at 0.4 mL / min over 4 minutes. Data acquisition and analysis were performed using Xcalibur software.

[0469] GCPII Enzymatic Activity

[0470] Inhibition potencies against GCPII (IC50 values) were determined using published methods. Hollinger et al., 2022; Su, Y., et al., 2024. Briefly, reactions were carried out in the presence of NAA-[3H]-G and human recombinant GCPII enzyme in Tris-HCl and CoCh at 37 °C for 20 min. Reactions were stopped with ice-cold sodium phosphate buffer containing 1 mM EDTA. Aliquots were then transferred to 96-well spin columns containing AG 1X8 ion-exchange resin and centrifuged. NAA-[3H]-G was bound to the resin and [3H]-G eluted in the flow-through. Columns were washed with formate to ensure complete elution of [3H]-G. The flow-through and the washes were collected, and aliquots were transferred and dried to completion in a solid scintillator-coated 96-well plate. The radioactivity corresponding to [3H]-G was determined with a scintillation counter. Subsequently, IC50 curves were generated from CPM results.

[0471] Glucocorticoid Receptor ( GR) Assay

[0472] The PolarScreen™ Glucocorticoid Receptor Competitor Assay Kit was performed as per the manufacturers’ protocol. In brief, conjugates are added recombinant human GR in a dilution half-log series (10’11to 10'5M final concentration) in 384 well plates. A fluorescent glucocorticoid ligand (Fluormone GS Red) is then added to start the experiment. When the conjugate prevents the formation of the GS Red / GR complex a decrease is measured in the polarization value onClariostar Plus with excitation / emission wavelengths 535 / 590 nm. The shift in polarization value in the presence of test compounds is used to determine relative affinity of test compounds for GR.

[0473] Stability in recombinant cathepsin enzymes

[0474] Compounds (20 pM) were incubated at 37 °C in triplicate in 50 pL of 250 mM MES, 5 mM DTT pH 5, 10 pg / mL Cathepsin B (R&D systems 953-CY-010) or 500 mM MES, 5 mM DTT pH 6 with 10 pg / mL Cathepsin L (R&D systems 952-CY-010). At time 0, 15 minutes, 30 minutes, 1 hour and 3 hours 5 pL was removed from each incubation and proteins were precipitated with 25 pL methanol containing 0.5 pM losartan (internal standard) by centrifugation at 16000 x g for 5 minutes. Supernatants (20 pL) were diluted with water (80 pL) and injected (10 pL) and analyzed by LCMS.

[0475] In Vivo Pharmacokinetic study

[0476] The pharmacokinetic study in mice was conducted according to protocols reviewed and approved by the Johns Hopkins Institutional Animal Care and Use Committee in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals (PHS Policy). Briefly, naive male and female C57BL / 6 mice (weighing between 25 and 30 g) 6-8 weeks of age were used. The animals were maintained on a 12 h light-dark cycle with ad libitum access to food and water. 2.5 mg / kg of LPS was pre-treated before Ihr treatment, Prednisolone and Cl were dissolved in DMSO / Tween 80 / PBS (5:5:90, v / v / v) and were administered to mice as a single oral dose of 10 mg / kg and 10 mg / kg equivalent to prednisolone. The mice were euthanized with carbon dioxide at the time-points indicated for pk analysis. Blood samples (approximately 0.8 mL) were collected in heparinized microtubes by cardiac puncture, and ileum and colon were removed and flash-frozen on dry ice. Blood samples were centrifuged at a temperature of 4 °C at 3000g for 10 min. All samples were kept chilled throughout processing. Plasma samples (approximately 300 pL) were collected in polypropylene tubes and stored at -80 °C until bioanalysis. Flash-frozen ileum and colon samples were also stored at -80 °C until bioanalysis.

[0477] To quantify prednisolone in the pharmacokinetic samples, plasma samples (40 pL) and tissue homogenates (50 pL) were processed with liquid-liquid extraction using 1 mL of methyl tert-butyl ether (MTBE). For plasma and ileum, the mixture was vortex-mixed and centrifuged at 16,000g for 5 min at 4 °C. Supernatant (1 mL) was transferred to a new tube and dried undervacuum at 40 °C for 1 h. The samples were reconstituted with 100 pL of methanol: water mixture (1:1, v / v). Samples arc analyzed using LC-MS / MS system following previously published reports.EXAMPLE 2

[0478] Experimental Data and Synthesis Schemes

[0479] Representative compound of formula (I) are provided in Table 2.

[0480] *denotes prophetic examples

[0481] Scheme 1: Synthesis of conjugate Cl|00482| (75.1 LSj-7,1 l-Bis( / er / -butoxycarbonyl)-2.2-dimethyl-4.9.17- trioxo-3, 20,23,26,29,32- hexaoxa-8,10,16-triazapentatriacontan-35-oic acid (4).(2) (2.88 g, 5.91 mmol) in anhydrous DCM (60 mL) were added EDCI (1.13 g, 5.91 mmol, 1 equiv.) and DMAP (144 mg, 1.18 mmol, 0.2 equiv.) and the resulting mixture was stirred at 20 °C for 12 hours. After the reaction completion, the volatiles were removed under reduced pressure to give a crude product, that was purified by preparative-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 μm); mobile phase: [water (FA)-MeCN]; gradient:35%-65% B over 25 min) togive compound 4 (3.20 g) as a colorless oil in 34% yield.1H NMR (400 MHz, DMSO-d6) d 12.35 - 11.90 (m, 1H), 7.80 (t, J = 5.2 Hz, 1H), 6.28 (dd, J = 8.4, 14.8 Hz, 2H), 4.06 - 4.00 (m, 1H),3.98 - 3.90 (m, 1H), 3.62 - 3.55 (m, 4H), 3.54 - 3.40 (m, 16H), 3.05 - 2.96 (m, 2H), 2.43 (s, 2H), 2.31 - 2.24 (m, 2H), 2.24 - 2.16 (m, 2H), 1.91 - 1.80 (m, 1H), 1.71 - 1.61 (m, 1H), 1.60 - 1.53 (m, 1H), 1.53 - 1.47 (m, 1H), 1.44 - 1.36 (m, 27H), 1.35 - 1.32 (m, 1H), 1.30 - 1.21 (m, 3H). ESI MS: 808.7 ([M+H]+).

[0484] (24S,28S)-24,28,30-Tri-terf-butyl l-(2-((8S,9S,10R, 11S,13S,14S,17R)-11,17- dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,l 1.12.13.14.15 J6.17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethyl) 18,26-dioxo-3,6,9,12,15-pentaoxa-19,25,27- triazatriacontane-l,24,28,30-tetracarboxylate (5).

[0485] To a solution of compound 4 (1.20 g, 1.49 mmol, 1 equiv.) and prednisolone (642 mg, 1.78 mmol, 1.2 equiv.) in anhydrous DCM (20 mL) were added EDCI (427 mg, 2.23mmol, 1.5 equiv.) and DMAP (18.1 mg, 0.149 mmol, 0.1 equiv.) and the mixture was stirred at 20 °C for 12 hours. Volatiles were removed under reduced pressure and the residue was purified by preparative-HPLC (column: Welch Ultimate XB-SiOH 250*50*10 μm; mobile phase: [Hexane- EtOH (0.1% IP A)]; gradient:8%-36% B over 19 min) to compound 5 (1.40 g) as a colorless solid in 82% yield.1H NMR (400 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.32 (d, J= 10.0 Hz, 1H), 6.27 (dd, J= 8.4, 14.4 Hz, 2H), 6.16 (dd, 7 = 1.6, 10.0 Hz, 1H), 5.91 (s, 1H), 5.40 (s, 1H), 5.08 (d, J= 17.6Hz, 1H), 4.76 (d, J = 17.6 Hz, 1H), 4.72 (d, 7= 3.6 Hz, 1H), 4.34 (t, 7 = 5.2 Hz, 3H), 4.31 - 4.27 (m, 1H), 3.98 - 3.91 (m, 1H), 3.66 (t, 7 = 6.4 Hz, 2H), 3.58 (t, 7 = 6.4 Hz, 2H), 3.52 - 3.52 (m, 1H), 3.52 - 3.46 (m, 15H), 3.05 - 2.97 (m, 2H), 2.63 (s, 2H), 2.33 - 2.31 (m, 1H), 2.24 - 2.16 (m, 3H), 2.06 - 1.99 (m, 2H), 1.92 - 1.82 (m, 3H), 1.70 - 1.60 (m, 5H), 1.43 - 1.35 (m, 32H), 1.29 - 1.22 (m, 4H), 0.89 (dd, 7 = 2.0, 10.4 Hz, 1H), 0.78 (s, 3H). ESI MS: 1150.8 ([M+H]+).

[0486] (28S,32S)-l-((8S,9S,10R, 11S,13S, 14S,17R)-ll,17-Dihydroxy -10,13-dimethyl-3-oxo- 6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H -cyclopenta[a]phenanthren-17-yl)-l,4,22,30- tetraoxo-3,7,10, 13,16.19-hexaoxa-23.29.31-triazatetratriacontane-28.32.34-tricarboxylic acid (Cl).

[0487] To a solution of compound 5 (1 .00 g, 0.869 mmol) in anhydrous DCM (20 mL) was added TFA (10 mL) (15.4 g, 135 mmol, 155 equiv.) and the mixture was stirred at 20 °C for1 hour. DCM and TFA were evaporated and the residue was purified by preparative-HPLC(column: Phenomenex luna C18 (250*70 mm, 10 μm); mobile phase: [water (FA)-MeCN]; gradient:23%-53% B over 64 min) to give compound Cl (550 mg, 98.7% purity) as a colorless solid in 64% yield.1H NMR (400 MHz, DMSO-d6) δ 12.78 - 12.09 (m, 3H), 7.80 (s, 1H), 7.32 (d, J= 10.0 Hz, 1H), 6.30 (dd, J = 8.4, 12.8 Hz, 2H), 6.16 (dd, J= 1.6, 10.0 Hz, 1H), 5.91 (s, 1H), 5.41 (s, 1H), 5.08 (d, J= 17.6 Hz, 1H), 4.83 - 4.65 (m, 2H), 4.29 (s, 1H), 4.16 - 3.98 (m, 2H), 3.66 (t, J = 6.4 Hz, 2H), 3.61 - 3.55 (m, 2H), 3.54 - 3.44 (m, 16H), 3.00 (d, J= 6.0 Hz, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.33 - 2.16 (m, 5H), 2.09 - 1.98 (m, 2H), 1.96 - 1.85 (m, 2H), 1.77 - 1.57 (m, 5H),1.57 - 1.42 (m, 2H), 1.41 - 1.34 (m, 5H), 1.33 - 1.22 (m, 3H), 1.08 - 0.95 (m, 1H), 0.93 - 0.86(m, 1H), 0.84 - 0.74 (m, 3H). ESI MS: 982.5 ([M+H]+).

[0488] Scheme 2: Synthesis of conjugate C2.

[0489] 1 -tert- Butyl 4-(2-((8S,9S,10tf ,11S,13S,14S,17R)-11, 17-dihydroxy-10,13-dimethyl-3- oxo-6, 7, 8, 9, 10,11,12,13,14,15,16, 17-dodecahydro-3H-cycIopenta[a]phenanthren- 17-yl)-2- oxoethyl) piperazine- 1,4-dicarboxylate (7).

[0490] To a solution of prednisolone (1) (3.00 g, 8.32 mmol, 1 equiv.) and 4-nitrophenyl carbonochloridate (2.52 g, 12.5 mmol, 1.5 equiv.) in anhydrous DCM (90 mL) was added pyridine (987 mg, 1 01mL, 12-5 mmol, 1.5 equiv.) at 0 °C and the mixture wasstirred at 20 °C for 1 h. Triethylamine (2.48 g, 3.42 mL, 24.5 mmol, 2.95 equiv.) and tert-butyl piperazine- 1 -carboxylate (3.05 g, 16.4 mmol, 2 equiv.) were added inone portion and the resulting mixture was stirred at 20 °C for another 1 h. The reaction mixture was poured into saturated NaHCO3(50 mL) and extracted with DCM (3x100 mL), the organic phase was washed with brine (100 mL), dried over anhydrous Na2SO4and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (petroleum ether / EtOAc, 1:1 to 1:2) to give product 7 (4.00 g) as a yellow solid in 86% yield.1H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J = 10.0 Hz, 1H), 6.16 (dd, J = 1.6, 10.0 Hz, 1H), 5.91 (s, 1H), 5.36(s, 1H), 5.05 (d, J = 17.8 Hz, 1H), 4.84 - 4.64 (m, 2H), 4.28 (s, 1H), 3.42 - 3.32 (m, 8H), 2.29 (dd, J = 3.2, 12.8 Hz, 1H), 2.07 - 1.99 (m, 2H), 1.92 - 1.82 (m, 1H), 1.72 - 1.57 (m, 3H), 1.43 - 1.39 (m, 9H), 1.39 - 1.37 (m, 3H), 1.34 - 1.23 (m, 2H), 1.04 - 0.98 (m, 1H), 0.93 - 0.86 (m, 1H), 0.82 - 0.77 (m, 3H). ESI MS: 573.3 ([M+H]+).

[0491] 2-((8S,9S,10R,11S,13S,14S,17R)-ll,17-Dihydroxy-10,13- dimethyl-3-oxo-6,7,8,9,10,11 , 12,13,14,15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yI)-2- oxoethyl piperazine-l-carboxylate (8).

[0492] To a solution of compound 7 (4.00 g, 6.98 mmol, 1 equiv.) in anhydrous DCM (40 mL) was added TFA (15.3 g, 10 mL, 134 mmol, 10 mL) and the resulting mixture was stirred at 20 °C for 1 h. Volatiles were removed under reduced pressure and the residue wasneutralized with sat. NaHCO3(50 mL) and extracted with DCM (3x50 mL). The combined organic phase was washed with brine (3x50 mL), dried over anhydrous Na SCU and concentrated under reduced pressure to give compound 8 (3.00 g) as a yellow solid in 91% yield.1H NMR (400 MHz, DMSO-d6) <5 7.32 (d, J = 10.0 Hz, 1H), 6.16 (dd, J = 2.0, 10.0 Hz, 1H), 5.91 (s, 1H), 5.35 (s, 1H), 5.02 (d, J = 17.8 Hz, 1H), 4.78 - 4.67 (m, 2H), 4.28 (s, 1H), 4.14 - 4.04 (m, 1H), 3.38 - 3.29 (m, 4H), 2.72 - 2.60 (m, 4H), 2.29 (dd, J = 2.8, 13.3 Hz, 1H), 2.03 (d, J = 9.2 Hz, 2H), 1.86 (d, J = 3.2 Hz, 1H), 1.70 - 1.60 (m, 3H), 1.47 - 1.41 (m, 1H), 1.39 (s, 3H), 1.32 - 1.23 (m, 1H), 1.06 - 0.98 (m, 1H), 0.93 - 0.86 (m, 1H), 0.79 (s, 3H). ESI MS: 473.3 ([M+H]+).

[0493] (3.S.7.S)-Tri-tert-bu tyl 31-(4-((2-((8S,9S,10R,11S,13S,14S, 17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)piperazin-l-yl)-5,13,31-trioxo- 16,19,22,25,28-pentaoxa-4,6,12-triazahentriacontane-l,3,7-tricarboxylate (9).DMAP (22.6 mg, 185.6 μmol, 0.1 equiv.) and the mixture was stirred at 20 °C for 1 h. Volatiles were removed under reduced pressure and the residue was purified by preparative-HPLC (column: Welch Ultimate XB-SiOH 250*50*10 μm; mobile phase: [Hexane-EtOH (0.1% IPA)]; gradient: 12%-42% B over 20 min) to give product 9 (1.50 g) as a colorless oil in 64% yield.1H NMR (400 MHz, DMSO-76) 37.80 (s, 1H), 7.70 (s, 2H), 7.32 (d, J= 10.0 Hz, 1H), 6.28 (dd, J = 8.4, 14.2 Hz, 2H), 6.16 (dd, J= 1.6, 10.0 Hz, 1H), 5.91 (s, 1H),5.38 (s, 1H), 5.06 (d, J= 17.6 Hz, 1H), 4.79 - 4.73 (m, 1H), 4.72 - 4.69 (m, 1H), 4.03 (dt, 7= 5.2,8.4 Hz, 1H), 3.98 - 3.91 (m, 1H), 3.63 (t, J = 6.4 Hz, 2H), 3.58 (t, J = 6.4 Hz, 2H), 3.50 - 3.49 (m, 8H), 3.46 - 3.41 (m, 16H), 3.04 - 2.97 (m, 2H), 2.60 (s, 2H), 2.32 - 2.25 (m, 4H), 2.24 - 2.17 (m,2H), 2.07 - 1.97 (m, 2H), 1.91 - 1.81 (m, 2H), 1.71 - 1.58 (m, 5H), 1.56 - 1.44 (m, 2H), 1.42 - 1.35 (m, 30H), 1.31 - 1.19 (m, 6H), 0.79 (s, 3H). ESI MS: 1262.9 ([M+H]+).

[0495] (3S,7S)-31-(4-((2-((8S,9S,10R ,115,13S,14S,17 / ?)- ll,17-Dihydroxy-10,13-dimethyl-3- oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3 / / -cyclopenta[a]phenanthren-17-yl)-2- oxoethoxy)carbonyl)piperazin-l-yl)-5, 13, 31-trioxo-16, 19, 22,25, 28-pentaoxa-4, 6,12- triazahentriacontane-l,3,7-tricarboxylic acid (C2).the resulting mixture was stirred at 20 °C for 1 h.Solvents were removed under reduced pressure and the residue was purified by preparative-HPLC (column: Phenomenex luna C18 (250*70 mm, 10 μm); mobile phase: [water (FA)-MeCN]; gradient: 20%-50% B over 20 min) to give product C2 (640 mg) as a colorless solid in 52% yield. ‘H NMR (400 MHz, DMSO-76) 3 12.95 - 11.77 (m, 3H), 7.80 (t, J = 5.2 Hz, 1H), 7.32 (d, 7 = 10.0 Hz, 1H), 6.30 (dd, 7 = 8.4, 12.4 Hz, 2H), 6.16 (dd, 7 = 1.6, 10.1 Hz, 1H), 5.91 (s, 1H), 5.37 (s, 1H), 5.06 (d, 7 = 17.6 Hz, 1H), 4.80 - 4.73 (m, 1H), 4.73 - 4.70 (m, 1H), 4.28 (s, 1H), 4.15 - 3.98 (m, 2H), 3.63 (s, 2H), 3.58 (s, 2H), 3.53 - 3.43 (m, 21H), 3.43 - 3.35 (m, 3H), 3.00 (d, 7 = 6.0 Hz, 2H), 2.60 (s, 2H), 2.28 (t, 7 = 6.4 Hz, 5H), 2.03 (d, J = 8.6 Hz, 2H), 1.96 - 1.84 (m, 2H),1 .77 - 1 .57 (m, 5H), 1 .56 - 1 .42 (m, 2H), 1.42 - 1 .34 (m, 5H), 1 .33 - 1 .22 (m, 3H), 1 .07 - 0.94 (m, 1H), 0.93 - 0.85 (m, 1H), 0.79 (s, 3H). ESI MS: 1094.6 ([M+H]+).

[0497] Scheme 3: Synthesis of prednisolone precursor 13.

[0498] Methyl 4-imino-4-methoxybutanoate (10).

[0499] To a solution of hydrogen chloride in 2-isopropoxypropanc (10 M, 20 mL) was added drop wise methyl 3-cyanopropanoate (5.00 g, 44.2 mmol, 1equiv.) and MeOH (2.83 g, 3.58 mL, 88.4 mmol, 2 equiv.) at 0 °C and the resulting mixture was stirred at same temperature for 2 h and at 20 °C for 10 h. The mixture was diluted with 2- isopropoxypropane (100 mL) to form precipitate and the product 10 was collected by filtration to obtain colorless solid (7.00 g) as a hydrochloric acid. *H NMR (400 MHz, DMSO-cfa) d 12.40 - 11.28 (m, 2H), 4.07 (s, 3H), 3.62 (s, 3H), 2.95 - 2.86 (m, 2H), 2.79 - 2.73 (m, 2H).

[0500] Methyl 4,4,4-trimethoxybutanoate (11).

[0501] Compound 10 (6.00 g, 41.3 mmol, 1 equiv.) was dissolved in anhydrous MeOH (20 mL) and stirred at 20 °C for 24 h. The reaction was quenched with sat.NaHCO3(100 mL) and the resulting mixture was extracted with EtOAc (3x100 mL), the combined organic phases were dried over anhydrous Na2SO4 and concentrated under reduced pressure to give compound 11 (7.00 g) as a colorless oil in 88% yield.1H NMR (400 MHz, CDC13) 63.66 - 3.62 (m, 3H), 3.19 (s, 9H), 2.38 - 2.25 (m, 2H), 2.11 - 1.97 (m, 2H).

[0502] Methyl 3-((2'S,4'S,8S,9S,10R,l 1S,13S,14S)-1 l-hydroxy-2'-methoxy-10,13-dimethyl- 3,5'-dioxo-3,6,7,8,9,10,H,12,13,14,15,16-dodecahydrospiro [cyclopenta[a]phenanthrene- 17,4'-[l,3]dioxan]-2'-yl)propanoate (12).

[0503] To a solution of prednisolone (3.00 g, 8.32 mmol, 1 equiv.) in anhydrous toluene (35 mL) and anhydrous DMF (5 mL) were mcthyl compound 11 (4.80 g, 24.9 mmol, 3 equiv.) and 4-methylbenzenesulfonic acid (143.3 mg, 832 μmol, 0.1 equiv.) at 0°C, the mixture was heated to room temperature and stirred at 20 °C for 12 h. The reaction mixture was treated with tricthylaminc (5 mL), poured into sat. NaHCCh (100 mL) and extracted with EtOAc (3x100 mL). The combined organic phases were washed with brine (50 mL) and dried over anhydrous MgSO i. the organic phase was concentrated under reduced pressure, and the residue was purified by column chromatography on silica (PE / EtOAc; 1:1) to give compound 12 (4.50 g) as a colorless oil.1H NMR (400 MHz, CDCI3) <56.30 (d, J = 10.0 Hz, 1H), 6.04 (s, 1H), 3.66 (s, 3H), 3.23 (s, 3H), 2.72 (d, 7 - 12.4 Hz, 1H), 2.58 (dd, 7 = 4.4, 5.4 Hz, 1H), 2.47 - 2.41 (m, 2H), 2.35 (dd, 7 = 3.6, 12.8 Hz, 1H), 2.25 - 2.18 (m, 1H), 2.17 - 2.08 (m, 4H), 2.05 (s, 6H), 1.72 (d, 7 = 14.6 Hz, 2H), 1.46 (s, 3H), 1.41 - 1.34 (m, 1H), 1.20 - 1.09 (m, 3H), 0.92 (s, 3H). ESI MS: 489.3 ([M+H]+).

[0504] 3-((2'S,8S,9S,10R,llS,13S,14S,17S)-ll-Hydroxy-2'-methoxy-10,13-dimethyl-3,5'- dioxo-3,6,7,8,9,10,ll,12,13,14,15,16-dodecahydrospiro[cyclopenta[a]phenanthrene-17,4'- [l,3]dioxan]-2'-yl)propanoic acid (13).12 h. Volatiles were removed under reduced pressure and the residue was purified by preparative-HPLC (column: CD06-Waters Xbridge C18 150*40*10 μm; mobile phase: [water (NH4HCO3)-MeCN]; gradient: 12%-42% B over 10 min) to compound 13 (1.50 g) as a colorless solid in 39% yield.XH NMR (400 MHz, CDCh) 3 7.33 (d, J = 10.0 Hz, 1H), 6.35- 6.25 (m, 2H), 6.03 (s, 2H), 4.54 - 4.45 (m, 2H), 4.32 - 4.25 (m, 1H), 4.17 - 4.09 (m, 1H), 4.02- 3.93 (m, 1H), 3.23 (s, 3H), 2.51 - 2.43 (m, 2H), 2.03 - 1.99 (m, 2H), 1.64 - 1.55 (m, 3H), 1.46 (d, 7= 3.2 Hz, 6H), 1.21 - 1.06 (m, 4H), 0.92 (s, 3H).

[0506] Scheme 4: Synthesis of conjugates C3 and C4.

[0507] (24S,28S)-Tri-tert-butyl l-((3-((2'S, 4'S,8S,9S,1OR,11S,13S,14S)- ll-hydroxy-2'- methoxy-10,13-dimethyl-3,5'-dioxo-3,6,7,8,9,10,ll,12,13,14,15,16- dodecahydrospiro[cyclopenta[a]phenanthrene-17,4'-[l,3]dioxan]-2'-yl)propanoyl)oxy)-18,26-dioxo-3,6,9,12,15-pentaoxa-19,25,27-triazatriacontane-24,28,30-tricarboxylate (14).

[0508] To a solution of compound 4 (1.20 g, 1.54 mmol, 1 equiv.) and compound 12 (803 mg, 1.69 mmol, 1.1 equiv.) in anhydrous DCM (30 mL) were added EDCI (589 mg, 3.08 mmol, 2 equiv.) and DMAP (18.8 mg, 153 μmol, 0.1 equiv.) and the resulting mixture was stirred at20 °C for 12 h. Volatiles were removed under reduced pressure and the residue was purified by preparative-HPLC (column: Welch Ultimate XB-Diol 250*50*10 μm; mobile phase: [Hexane-EtOH (0.1% IP Am)]; gradient: 6%-36% B over 15 min) to give compound 13 (1.30 g) as a colorless oil in 68% yield.1H NMR (400 MHz, DMSO-d6) δ 7.78 (t, 7= 5.4 Hz, 1H), 7.34 (d, J = 10.0 Hz, 1H), 6.26 (dd, J = 8.4, 14.6 Hz, 2H), 6.17 (dd, J = 1.6, 10.1 Hz, 1H), 5.92 (s, 1H), 4.67 (d, 7 = 3.6 Hz, 1H), 4.31 (s, 1H), 4.11 - 4.04 (m, 10H), 3.95 (d, 7 = 5.2 Hz, 1H), 3.60 - 3.46 (m, 18H), 3.05 - 2.97 (m, 2H), 2.41 - 2.16 (m, 8H), 2.04 (t, 7 = 7.2 Hz, 4H), 1.93 (dd, 7= 2.8, 13.7 Hz, 2H), 1.66 (d, 7= 7.2 Hz, 10H), 1.41 - 1.38 (m, 27H), 1.33 - 1.19 (m, 5H), 1.13 - 0.89 (m, 3H), 0.80 (s, 3H). ESI MS: 1205.2 ([M+H]+).

[0509] Synthesis of conjugates C3 and C4.

[0510] To a solution of compound 14 (1.10 g, 889 μmol, 1 cquiv.) in anhydrous DCM (10 mL) was added TFA (7.68 g, 5 mL, 67.3 mmol, 61 equiv.) and the resulting mixture was stirred at 20 °C for 12 h. Volatiles were removed under reduced pressure and the residue was purified by preparative-HPLC (column: CD02-Waters Xbidge BEH C18 150*25*10 μm; mobile phase: [water (NH4HCO3)-MeCN]; gradient: 56%-86% B over 10 min) to give compounds C3 (140 mg,) in 15% yield as a colorless solid and compound C4 (100 mg) in 10% yield as a colorless solid.

[0511] (35,75)-35-(((8S,95,10R,115,135,145)-ll-Hydroxy-17- (2-hydroxyacetyl)-10,13- dimethyl-3-oxo-6,7,8,9,10,l 1.12.13.14.15.16.17-dodecahydro-3 / 7-cyclopenta|a]phenanthren- 17-yl)oxy)-5,13,32,35-tetraoxo-16,19,22,25,28,31-hexaoxa-4,6,12-triazapentatriacontane- 1,3,7-tricarboxylic acid (C3).(q, J = 6.6 Hz, 2H), 2.74 (s, 1H), 2.61 - 2.52 (m, 5H), 2.34 - 2.19 (m, 5H), 2.04 (s, 2H), 1.96 - 1.85 (m, 2H), 1.66 (t, J = 13.2 Hz, 5H), 1.50 (dd, 7 = 7.2, 13.2 Hz, 2H), 1.43 - 1.33 (m, 6H), 1.31 - 1.22 (m, 2H), 1.00 (s, 2H), 0.81 (s, 3H). ESI MS: 1054.7 ([M+H]+).

[0513] (325, 365)-l-((8S, 95, 10R, 115,135, 145)-ll,17-Dihydroxy-10, 13- dimethyl-3-oxo-6,7,8,9,10,11, 12, 13, 14, 15, 16,17-dodecahydro-3H-cyclopen ta| alphenant hren- 17-yl )- l,4,7,26,34-pentaoxo-3,8,ll,14,17,20,23-heptaoxa-27,33,35-triazaoctatriacontane-32,36,38- tricarboxylic acid (C4).3.54 - 3.45 (m, 16H), 3.00 (d, 7= 6.0 Hz, 2H), 2.70 - 2.53 (m,8H), 2.33 - 2.20 (m, 5H), 2.11 - 1.98 (m, 2H), 1.95 - 1.84 (m, 2H), 1.75 - 1.59 (m, 5H), 1.56 -1.40 (m, 3H), 1.39 (s, 3H), 1.33 - 1.21 (m, 3H), 0.89 (dd, J= 2.8, 10.9 Hz, 2H), 0.78 (s, 3H). ESI MS: 1054.7 ([M+H]+).

[0516] (2,5-Dioxopyrrolidin-l-yl) 8-[[(lS)-l-[[(lS)-l-[[4-(hydroxymethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-8- oxo-octanoate (17).butanoyl] amino] -A-[4-(hydroxymethyl)phenyl]-5-ureido-pentanamide (16) (5.00 g, 13.2 mmol, 1 equiv.) in anhydrous DMF (50 mL) during 15 minutes and the resulting mixture was stirred at 25°C for further 2 h. The reaction mixture was diluted with PE / EtOAc; 3:1 (2.1 L), and stirred for 10 min. The precipitate was filtered, suspended in DCM (500 mL) at 25 °C for 1 h, solvent was removed by filtration and product was dried under vacuo to give compound 17 (8.0 g, as a colorless solid in96% yield.1H NMR (400 MHz, DMSO-76) 6 9.89 (s, 1H), 8.06 (d, 7 = 7.6 Hz, 1H), 7.81 (d, 7 = 8.8 Hz, 1H), 7.54 (d, 7 = 8.4 Hz, 2H), 7.23 (d, 7 = 8.4 Hz, 2H), 6.02 - 5.91 (m, 1H), 5.40 (s, 2H), 5.09 (t, 7= 5.6 Hz, 1H), 4.50 - 4.31 (m, 3H), 4.24 - 4.13 (m, 1H), 3.10 - 2.90 (m, 2H), 2.81 (s, 4H), 2.67 - 2.60 (m, 2H), 2.25 - 2.08 (m, 2H), 2.03 - 1.92 (m, 1H), 1.72 - 1.21 (m, 12H), 0.93 - 0.76 (m, 6H). ESI MS: 633.3 ([M+H]+).

[0518] I)i- / ( / 7-butyl (25)-2-[[(15)-l-tert-butoxycarbonyl-5-[[8-[[(lS) -l-[[(lS)-l-[[4-(hydroxymethyl)phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-8- oxo-octanoyl]amino]pentyl]carbamoylamino]pentanedioate (18).

[0519] To a solution of compound 17 (2.00 g, 3.16 mmol, 1 equiv.) and DIPEA (817 mg, 6.32 mmol, 2 equiv.) in anhydrous DMF (40 mL) was added compound 2 (1.54 g, 3.16 mmol, 1 equiv.) and thereaction mixture was stirred at 25 °C for 12 h. Volatiles were removed under reduced pressure and the residue was purified by reverse phase flash chromatography (0.1% FA) to give compound 18 (1.50 g) as a colorless solid in 47% yield.1H NMR (400 MHz, DMSO-76) >' 9.89 (s, 1H), 8.05 (d, 7= 7.6 Hz, 1H), 7.80 (d, 7= 8.4 Hz, 1H), 7.71 (t, 7 = 5.6 Hz, 1H), 7.54 (d, 7 = 8.4 Hz, 2H), 7.23 (d, 7 = 8.4 Hz, 2H), 6.33 - 6.21 (m, 2H), 5.97 (t, 7= 6.0 Hz, 1H), 5.40 (s, 2H), 5.09 (t, 7= 5.6 Hz, 1H), 4.42 (d, 7= 5.6 Hz, 2H), 4.40 - 4.34 (m, 1H), 4.22 - 4.15 (m, 1H), 4.07 - 3.99 (m, 1H), 3.99 - 3.90 (m, 1H), 3.07 - 2.90 (m, 4H), 2.27 - 2.09 (m, 4H), 2.05 - 1.93 (m, 3H), 1.92 - 1.80 (m, 1H), 1.73 - 1.43 (m, 10H), 1.42 - 1.38 (m, 27H), 1.37 - 1.32 (m, 3H), 1.29 - 1.17 (m, 6H), 0.91 - 0.77 (m, 6H). ESI MS: 1005.7([M+H]+).

[0520] Di- / (77-butyl (2S)-2-[[(lS)-l-ter / -butoxycarbonyl-5-[[8-[[(lS)-2-methyl-l-[[(lS)-l-[[4- [(4-nitrophenoxy)carbonyloxymethyl]phenyl]carbamoyl]-4-ureido- butyl]carbamoyl]propyl]amino]-8-oxo- octanoyl]amino]pentyl]carbamoylamino]pentanedioate (19).

[0521] To a solution of compound 18 (400 mg, 0.398 mmol, 1 equiv.) and DMAP (48.6 mg, 0.398 mmol, 1 equiv.) in anhydrous DMF (5 mL) was added bis(4-nitrophenyl) carbonate (363 mg, 1.19 mmol, 3 equiv.) and the reaction mixture was stirred at 25 °C for 2 h. DMF was evaporated and the residue was purified by reverse phase flash chromatography (0.1% FA) to give compound 19 (340 mg) as anoff-white solid in 73% yield.1H NMR (400 MHz, DMSO-d6) d 10.06 (s, 1H), 8.34 - 8.28 (m, 2H), 8.10 (d, 7= 7.6 Hz, 1H), 7.80 (d, 7= 8.8 Hz, 1H), 7.72 (t, 7 = 5.6 Hz, 1H), 7.65 (d, 7= 8.4 Hz, 2H), 7.59 - 7.54 (m, 2H), 7.41 (d, 7= 8.4 Hz, 2H), 6.34 - 6.21 (m, 2H), 6.02 - 5.94 (m, 1H), 5.42 (s, 2H), 5.24 (s, 2H), 4.45 - 4.34 (m, 1H), 4.23 - 4.16 (m, 1H), 4.08 - 3.99 (m, 1H), 3.98 - 3.90 (m, 1H),3.07 - 2.92 (m, 4H), 2.28 - 2.08 (m, 4H), 2.04 - 1.80 (m, 4H), 1.73 - 1.43 (m, 10H), 1.41 - 1.36 (m, 29H), 1.30 - 1.16 (m, 7H), 0.91 - 0.79 (m, 6H). ESI MS: 1170.7 ([M+H]+).

[0522] (2S)-2-[[(1S)-l-terf-Butoxycarbonyl-5-[[8-[[(1S)-l-[[(1S)-l-[[4-[[2- [(8S,9S,10R,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo- ethoxy]carbonyloxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl- propyl]amino]-8-oxo-octanoyl]amino]pentyl]carbamoylamino]pentanedioate (20)

[0523] To a solution of compound 19 (340 mg, 0.291 mmol, 1 equiv.) and prednisolone (1) (157 mg, 0.436 mmol, 1.5 equiv.) in anhydrous DMF (5 mL) were addedDMAP (71.0 mg, 0.581 mmol, 2 equiv.) and pyridine (68.9 mg, 0.872 mmol, 3 equiv.) and the resulting mixture was stirred at 25 °C for 12 h. Volatiles were removed under reduced pressure and the residue was purified by reverse phase flash chromatography (0.1% FA) to give compound 20 (130 mg) as a colorless solid in 32% yield.1H NMR (400 MHz, DMSO-7e) <510.03 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.72 (t, J = 5.6 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.37 - 7.27 (m, 3H), 6.33 - 6.23 (m, 2H), 6.19 - 6.13 (m, 1H), 5.98 (t, 7 = 5.7 Hz, 1H), 5.91 (s, 1H), 5.51- 5.36 (m, 3H), 5.17 - 5.02 (m, 3H), 4.82 - 4.68 (m, 2H), 4.44 - 4.34 (m, 1H), 4.32 - 4.25 (m, 1H), 4.19 (dd, J = 7.3, 8.4 Hz, 1H), 4.09 - 4.00 (m, 1H), 3.98 - 3.89 (m, 1H), 3.03 - 2.95 (m, 4H), 2.29- 2.10 (m, 5H), 2.03 - 1.81 (m, 7H), 1.70 - 1.56 (m, 7H), 1.48 - 1.33 (m, 40H), 1.30 - 1.16 (m, 8H),I.04 - 0.73 (m, 12H). ESI MS: 1391.9 ([M+H]+).[00524J(2S)-2-[[(lS)-l-Carboxy-5-[[8-[[(1S)-l-[[(1S)-l-[[4-[[2- [(8S,9S,10R,H5,135,14S,17R)-I I,17-dihydroxy-10,13-dimethyl-3-oxo-7.8.9.11,12,14.15,16-octahydro-6H- cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]carbonyloxymethyl]phenyl]carbamoyl]-4- ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-8-oxo- octanoyl]amino]pentyl]carbamoylamino]pentanedioic acid (C5).purified twice by reverse phase flash chromatography (0.1% FA) and prep-HPLC (column: Phenomenex luna C18 150*25mm* 10 μm; mobile phase: [water(FA)-MeCN] gradient: 25%-55% B over 10 min) to give compound C5 (15.0 mg) as a colorless solid in 19% yield.1H NMR ((400 MHz, DMSO-rie) <5 12.98 - 11.74 (m, 3H), 10.04 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.76 - 7.68 (m, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.41 - 7.26 (m, 3H), 6.37 - 6.25 (m, 2H), 6.19 - 6.13 (m, 1H), 5.98 (t, J = 5.2 Hz, 1H), 5.91 (s, 1H), 5.43 (br d, J = 4.8 Hz, 3H), 5.18 - 5.01 (m, 3H), 4.83 - 4.69 (m, 2H), 4.45 - 4.33 (m, 1H), 4.32 - 4.25 (m, 1H), 4.24 - 4.16 (m, 1H), 4.14 - 3.99 (m, 2H), 3.09 - 2.91 (m, 4H), 2.32 - 2.11 (m, 5H), 2.08 - 1.86 (m, 7H), 1.76 - 1.56 (m, 7H), 1.54 - 1.40 (m, 8H), 1.39 (s, 3H), 1.38 - 1.14 (m, 10H), 1.10 - 0.75 (m, 12H). ESI MS: 1223.7 ([M+H]+).

[0526] Scheme 6: Synthesis of intermediate 25.

[0527] Methyl(((4-nitrobenzyl)oxy)methyl)sulfane (22).

[0528] To a solution of (4-nitrophenyl)methanol (21) (20.0 g, 130.6 mmol, 1 equiv.) in anhydrous DMSO (80 mL) were added AC2O (65.2 g, 638 mmol, 4,9 equiv.) and CH3COOH (41.9 g, 698 mmol, 5.3 equiv.) and the mixture wasstirred in the dark at 25 °C for 48 h. The resulting mixture was drop wise added to a vigorously stirred slurry of NaHCO3(200 g, 2.38 mol) in dist. H2O (280 mL) to control the release of carbon dioxide. The reaction mixture was stirred in the dark for an additional 24 h, diluted with dist. water (500 mL) and extracted with EtOAc (3x200 mL). The combined organic fractions were washed with brine (150 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica (PE to PE / EtOAc; 10:1) to give compound 22 (12.0 g) as a colorless oil in 43% yield.1H NMR (400 MHz, CDCI3) <5 8.43 - 8.12 (m, 2H), 7.53 (d, J = 8.8 Hz, 2H), 4.75 (s, 2H), 4.73 (s, 2H), 2.21 (s, 3H).

[0529] l-((Chloromethoxy)methyl)-4-nitrobenzene (23).

[0530] Compound 22 (5.0 g, 23.5 mmol, 1 equiv.) was dissolved in anhydrous DCM (50 mL), cooled to -70 °C and the solution of sulfuryl chloride (3.16 g, 23.5 mmol, 1 equiv.) in anhydrous DCM (3 mL) was dropwise addedduring 10 minutes and the resulting mixture was stirred at same temperature for1 h. Volatiles were removed under reduced pressure to obtain crude product 23 (4.7 g) as a yellow oil and this compound was used to the following step without further purification.1H NMR (400 MHz, CDC13) d 8.24 (d, J= 8.8 Hz, 2H), 7.54 (d, J - 8.8 Hz, 2H), 5.58 (s, 2H), 4.86 (s, 2H).

[0531] ((85, 95, 10R, 115, 135,145, 17R)-ll,17-Dihydroxy-10,13-dimethyl -17-(2-(((4- nitrobenzyl)oxy)methoxy)acetyl)-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-31 / - cyclopenta[a]phenanthren-3-one (24).DMF (100 mL), the resulting mixture was degassed with nitrogen (3 times) and the mixture was stirred at 25 °C for 24 h under N2 atmosphere. The reaction mixture was diluted with dist. water (300 mL) and extracted with EtOAc (3x100 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by reverse phase flash chromatography (0.1% ammonia in CH3CN) to give product 24 (3.50 g) as a colorless solid in 46% yield.1H NMR (400 MHz, DMSO-d6) 8.23 (d, J= 8.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 2H), 7.32 (d, J= 10.0 Hz, 1H), 6.16 (dd, 7= 1.6, 10.0 Hz, 1H), 5.91 (s, 1H), 5.37 - 5.24 (m, 1H), 4.85 - 4.55 (m,6H), 4.43 - 4.18 (m, 2H), 2.33 - 2.23 (m, 1H), 2.02 (d, J = 8.8 Hz, 2H), 1.87 (dd, J = 2.8, 13.2 Hz, 1H), 1.74 - 1.52 (m, 3H), 1.48 - 1.22 (m, 6H), 1.09 - 0.84 (m, 3H), 0.83 - 0.69 (m, 3H). ESI MS: 526.3 ([M+H]+).

[0533] (85, 95,10R,115, 135, 145,17R)-17-(2-(((4-Aminobenzyl)oxy) methoxy)acetyl)-ll,17- dihydroxy-10, 13-dimethyl-6, 7, 8, 9, 10, 11,12, 13,14, 15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one (25).and H2O (50 mL) was degassed with nitrogen (3 times) and themixture was heated to 60 °C for 0.5 h under N2 atmosphere. The mixture was filtered, washed with EtOH and volatiles were removed under reduced pressure. The residue was purified by reversed phase flash chromatography (0.1% ammonia in CH3CN) to give product 25 (1.50 g) as a colorless solid in 64% yield.1H NMR (400 MHz, DMSO-76) 6 7.33 (d, J = 10.0 Hz, 1H), 6.97 (d, J = 8.4 Hz, 2H), 6.52 (d, 7 = 8.4 Hz, 2H), 6.16 (dd, 7 = 1.6, 10.0 Hz, 1H), 5.91 (s, 1H), 5.29 (s, 1H), 5.06 (s, 2H), 4.69 - 4.59 (m, 4H), 4.44 - 4.18 (m, 4H), 2.57 - 2.51 (m, 2H), 2.29 (dd, 7 = 3.2, 13.2 Hz, 1H), 2.11 - 1.96 (m, 2H), 1.87 (dd, 7= 3.2, 13.2 Hz, 1H), 1.70 - 1.50 (m, 3H), 1.48 - 1.34 (m, 4H), 1.32 - 1.21 (m, 1H), 1.08 - 0.96 (m, 1H), 0.93 - 0.86 (m, 1H), 0.81 - 0.73 (m, 3H). ESI MS: 496.2 ([M+H]+).[00536J Allyl (2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9- ylmethoxycarbonylamino)hexanoate (27).

[0537] Compound (25)-6-(Zer / -butoxycarbonylamino)-2-(977- fluoren-9-ylmethoxycarbonylamino) hexanoic acid (26) (10 g, 21.3 mmol, 1 equiv.), 3-bromoprop-l-ene (2.84 g, 23.5 mmol, 1.1 equiv.) and NaiCCh(2.49 g, 23.5 mmol, 1.1 equiv.) were dissolved in the mixture of solvents DMF (100 mL) and H2O (10 mL), degassed nitrogen (3 times) and stirred at 25 °C for 12 h under N2 atmosphere. The mixture was diluted with dist. water (250 mL), and extracted with EtOAc (3x150 mL). The combined organic fractions were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give product 27 (8.70 g) as a yellow oil in 80% yield.1H NMR (400 MHz, DMSO-76) d 7.90 (d, 7= 7.6 Hz, 2H), 7.80 (d, 7 = 7.6 Hz, 1H), 7.72 (d, 7 = 7.6 Hz, 2H), 7.48 - 7.39 (m, 2H), 7.37 - 7.28 (m, 2H), 6.79 (t, 7 = 5.6 Hz, 1H), 5.97 - 5.79 (m, 1H), 5.39 - 5.12 (m, 2H), 4.58 (d, 7 = 5.2 Hz, 2H), 4.37 - 4.17 (m, 3H), 4.11 - 3.95 (m, 1H), 2.90 (d, 7 = 5.6 Hz, 2H), 1.77 - 1.55 (m, 2H), 1.49 - 1.21 (m, 13H). ESI MS: 409.2 ([M- 100]+).

[0538] Allyl (2S)-2-amino-6-(tert-butoxycarbonylamino)hexanoate (28).

[0539] Compound 27 (8.70 g, 17.1 mmol, 1 equiv.) was dissolved in anhydrous DCM (80 mL) and piperidine (13.8 g, 162 mmol, 9.5 equiv.) was added during 5 minutes. The resulting mixture was degassed with nitrogen(3 times) and stirred at 25 °C for 2 h under N2 atmosphere. Volatiles were evaporated under reduced pressure and the residue was purified by column chromatography on silica (PE / EtOAc; 10: 1 to EtOAc) to give product 28 (4.80 g) as a yellow oil in 98% yield.1H NMR (400 MHz, DMSO-t / 6) d 6.74 (d, J = 5.2 Hz, 1H), 6.02 - 5.82 (m, 1H), 5.40 - 5.13 (m, 2H), 4.63 - 4.50(m, 2H), 3.56 (s, 1H), 3.48 (t, 7= 6.8 Hz, 1H), 2.89 - 2.85 (m, 2H), 2.33 (d, J= 1.6 Hz, 1H), 1.62 -1.24 (m, 15H). ESI MS: 287.3 ([M+H]+).

[0540] Diallyl (2S)-2-[[(lS)-l-allyloxycarbonyl-5-(terf- butoxycarbonylamino)pentyl]carbamoylamino]pentanedioate (30).

[0541] To a solution of diallyl (2S)-2-aminopentanedioate hydrochloride (29) (4.00 g, 15.2 mmol, 1 equiv.) and bis(trichloromethyl) carbonate (1.58 g, 5.31 mmol, 0.33 equiv.) in anhydrous DCM (100 mL) was added TEA (4.60 g, 6.33 mL, 45.5 mmol, 3 equiv.) at -70 °C and the mixture was stirred at 25 °C for 2 h. The solution of compound 28 (4.78g, 16.7 mmol, 1.1 equiv.) in anhydrous DCM (10 mL) was added at -70°C and the mixture was stirred at 25 °C for additional 12 h. DCM (1000 mL) was added, and organic phase was washed with water (3x100 3), dried over anhydrous NaiSCU, filtered and concentrated under reduced. The residue was purified by column chromatography on silica (PE / EtOAc; 20:1 to 3:1) to give product 30 (1.70 g) as a yellow oil in 21% yield.1H NMR (400 MHz, DMSO-d6) d 6.76 (t, J = 5.2 Hz, 1H), 6.45 (dd, J = 8.2, 16.4 Hz, 2H), 5.99 - 5.81 (m, 3H), 5.40 - 5.13 (m, 6H), 4.64- 4.50 (m, 6H), 4.29 - 4.07 (m, 2H), 2.88 (q, J = 6.8 Hz, 2H), 2.45 - 2.34 (m, 2H), 2.03 - 1.92 (m, 1H), 1.85 - 1.75 (m, 1H), 1.70 - 1.50 (m, 2H), 1.43 - 1.24 (m, 13H). ESI MS: 540.3 ([M+H]+).

[0542] Diallyl (2S)-2-[[(lS)-l-allyloxycarbonyl-5-amino- pentyl]carbamoylamino]pentanedioate (31).

[0543] Compound 30 (1.00 g, 1.85 mmol, 1 equiv.) was dissolved in anhydrous DCM (10 mL), TFA (4.61 g, 40.4 mmol, 22 equiv.) was added and the resulting mixture was degassed with nitrogen (3 times) and stirred at 25 °C for 1 h under N2 atmosphere. Volatiles were removed under reduced pressure togive product 31 (1.0 g) as a yellow oil as TFA salt. ESI MS: 440.1 ([M+H]+).

[0544] Scheme 8: Synthesis of conjugate C6.

[0545] (25)-2-[[(2S)-2-Amino-3-methyl-butanoyl]amino]-5-ureido-pentanoic acid (33).

[0546] Compound (25)-2-[[(25,)-2-(rerr-butoxycai‘bonylamino)-3-methyl- butanoyl]amino]-5-ureido-pentanoic acid (32) (5.00 g, 13.4 mmol, 1 equiv.) was dissolved in anhydrous DCM (50 mL), TFA (30.5 g, 267 mmol, 20 equiv.) was added during 5 minutes and the resulting mixture was degassed with nitrogen (3times) and the mixture was stirred at 25 °C for 2 h under N2 atmosphere. Volatiles were removed under reduced pressure to give product 33 as a TFA salt (5.10 g) as a colorless oil.XH NMR (400 MHz, DMSO-r / 6) h 8.67 (d, J = 7.6 Hz, 2H), 8.09 (d, J = 4.0 Hz, 3H), 4.31 - 4.13(m, 1H), 3.62 (t, J= 5.6 Hz, 1H), 2.98 (t, J= 6.8 Hz, 2H), 2.09 - 2.07 (m, 1H), 1.86 - 1.68 (m, 1H),1.68 - 1.55 (m, 1H), 1.51 - 1.35 (m, 2H), 1.03 - 0.89 (m, 6H).

[0547] (2S)-2-[[(2S)-2-[[8-(2,5-Dioxopyrrolidin-l-yl)oxy-8-oxo-octanoyl]amino]-3-methyl- butanoyl]amino]-5-ureido-pentanoic acid (34).octanedioate (16) (14.5 g, 39.4 mmol, 3 equiv.) in anhydrousDMF (400 mL) and stirred at 25 °C for 2 h. Petroleum ether (5000 mL) was added, the suspensionwas stirred for additional 30 minutes and filtered to give a residue. The crude product was triturated with DCM (100 mL), stirred at 25 °C for 4 h and filtered to give product 34 (5.0 g) as a colorless solid in 72% yield.1H NMR (400 MHz, DMSO-d6) 8.22 - 8.03 (m, 1H), 7.84 - 7.69 (m, 1H), 5.94 (d, 7= 5.2 Hz, 1H), 5.37 (s, 2H), 4.33 - 4.17 (m, 1H), 4.11 (d, 7= 5.6 Hz, 1H), 2.94 (d, 7= 6.0Hz, 2H), 2.80 (s, 3H), 2.65 - 2.62 (m, 2H), 2.24 - 2.07 (m, 2H), 2.03 - 1.88 (m, 1H), 1.76 - 1.54 (m, 4H), 1.52 - 1.11 (m, 9H), 0.84 - 0.81 (m, 6H). ESI MS: 528.3 ([M+H]+). (2

[0549] S)-2-[[(2S)-2-[[8-[[(5S)-6-Allyloxy-5-[[(lS)-4-allyloxy-l- allyloxycarbonyl-4-oxo- butyl]carbamoylamino]-6-oxo-hexyl]amino]-8-oxo-octanoyl]amino]-3-methyl- butanoyl]amino]-5-ureido-pentanoic acid (35).. concentrated under reduced pressure. The residue was purified by reversed phase flash chromatography (0.1% FA in CH3CN) to give product 35 (1.20 g) as a colorless solid in 78% yield.XH NMR (400 MHz, DMSO-76) d 13.03 -11.82 (m, 1H), 8.14 (d, 7 = 7.2 Hz, 1H), 7.88 - 7.59 (m,2H), 6.46 (dd, J= 8.4, 14.0 Hz, 2H), 6.04 - 5.76 (m, 4H), 5.39 - 5.18 (m, 8H), 4.66 - 4.50 (m, 6H), 4.33 - 3.90 (m, 4H), 3.03 - 2.90 (m, 4H), 2.42 - 2.36 (m, 2H), 2.20 - 1.89 (m, 6H), 1.85 - 1.51 (m, 5H), 1.50 - 1.16 (m, 14H), 0.84 (dd, 7 = 6.8, 14.6 Hz, 6H). ESI MS: 852.4 ([M+H]+).

[0551] Diallyl (2.S’)-2-||(l.S)- l-allyloxycarbonyl-5-| |8-| |( LS’)-1-||( LS’)-1-||4-||2-[(8S,9S,10R ,H5,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo- ethoxy]methoxymethyl]phenyl]carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl- propyl]amino]-8-oxo-octanoyl]amino]pentyl]carbamoylamino]pentanedioate (36).qu v. y ou . m wHOBt (190 mg, 1.41 mmol, 1.5 equiv.) and EDCI (270 mg, 1.41 mmol, 1.5 equiv.) and the mixture was stirred at 25 °C for 12 h. The mixture was poured into dist. water (150 mL) cooled to 0 °C,stirred 5 minutes and the precipitate was filtered. The residue was purified by normal phase HPLC (column: Welch Ultimate XB-CN 250*70*10 μm; mobile phase: [Hexane-EtOH (0.1% ammonia]; gradient: 40%-80% B over 15 min) to give racemic product 36 (750 mg) as a colorless solid in 60% yield.1H NMR (400 MHz, DMSO-d6) <510.12 - 9.72 (m, 1H), 8.59 - 8.03 (m, 1H), 7.98 - 7.79 (m, 1H), 7.76 - 7.54 (m, 3H), 7.36 - 7.22 (m, 3H), 6.46 (J = 8.2, 13.6 Hz, 2H), 6.17 (dd, J = 2.0, 10.2 Hz, 1H), 6.04 - 5.80 (m, 5H), 5.43 - 5.17 (m, 9H), 4.74 - 4.48 (m, 12H), 4.43 - 4.07 (m, 6H), 3.04 - 2.91 (m, 4H), 2.45 - 2.28 (m, 4H), 2.22 - 1.75 (m, 12H), 1.67 - 1.53 (m, 6H), 1.49 - 1.21 (m, 18H), 1.07 - 0.65 (m, 12H). ESI MS: 1330.8 ([M+H]+).

[0553] (2S)-2-[[(lS)-l-Carboxy-5-[[8-[[(lS)-l-[[(lS)-l-[[4-[[2-[(8S,9S,10R,llS,13S,14S,17R)-11.17-dihydroxy- 10.13-dimethyl-3-oxo-7.8.9.11,12, 14,15, 16-octahydro-6 / / - cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methoxymethyl]phenyl]carbamoyl]-4-ureido- butyl] carbamoyl] -2-methyl-propyl] amino] -8-oxo- octanoyl]amino]pentyl]carbamoylamino]pentanedioic acid (C6).morpholine (737 mg, 8.46 mmol, 15 equiv.) and the resulting mixture was stirred at 0 °C for 1 h. The mixture was poured to ice-water bath (200 mL), extracted with EtOAc (3x50 mL) and the aqueous phase was lyophilized to give a residue. The residue was redissolved in methanol (250 mL), filtered and concentrated under reduced pressure. The crude product was triturated with DCM / MeOH (250 mL, 100:1) at 25 °C for 2 h to give racemic product C6 (500 mg) as a colorless solid in 73% yield. The racemic product C6 was separated by preparative-HPLC (column: Waters Xbridgc 150*25mm 10 pm; mobile phase: [watcr( NH4HCO3)-McCN]; gradient: l%-28% B over 10 min) to give compound C6 (180 mg, peak 1) as a colorless solid in 35% yield.1H NMR (400 MHz, DMSO-< / 6) 5 9.86 (s, 1H), 8.47 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.72 - 7.62 (m, 3H), 7.32 (d, J = 10.1 Hz, 1H), 7.25 (d, J = 8.6 Hz, 2H), 6.35 (d, J = 8.0 Hz, 1H), 6.26 (d, 7 = 5.6 Hz, 1H), 6.20 - 6.11 (m, 1H), 6.08 - 5.98 (m, 1H), 5.91 (s, 1H), 5.41 (s, 2H), 4.74 - 4.61 (m, 4H), 4.50 (s, 2H), 4.40 - 4.24 (m, 4H), 4.14 - 3.96 (m, 4H), 3.01 - 2.95 (m, 4H), 2.36 - 2.09 (m, 6H), 2.07 - 1.85 (m, 7H), 1.83 - 1.71 (m, 3H), 1.70 - 1.53 (m, 6H), 1.50 - 1.13 (m, 21H), 1.10 - 0.96 (m, 1H), 0.93 - 0.75 (m, 10H). ESI MS: 1209.6 ([M+H]+).

[0555] Scheme 9: Synthesis of conjugate C7.distilled H2O (40 mL) and tert-butyl N- [4-(bromomethyl)phenyl] carbamate (9.53 g, 33.3 mmol, 2 equiv.) were added. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction mixture was quenched by addition of aq. ammonium chloride (200 mL), diluted with dist. water (300 mL) and extracted with EtOAc (3x500 mL). The combined organic layers were washed with brine (3x200 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by LC on silica (petroleum ether / EtOAc; 2:1 to 1:1) and finally re-purified by reversed phase flash [water (0.1% FA)-MeCN] to give compound 36 (1.20 g, 12.7% yield) as a yellow solid. *H NMR (400 MHz, CDCh) d 7.29 - 7.27 (m, 4H), 7.27 - 7.23 (m, 1H), 6.59 (s, 1H), 6.24 (dd, J = 2.0, 10.0 Hz, 1H), 6.00 (s, 1H), 4.69 - 4.63 (m, 1H), 4.58 - 4.52 (m, 1H), 4.43 - 4.37 (m, 1H), 4.36 - 4.31 (m, 1H), 4.19 (d, J = 17.6 Hz, 1H), 2.76 - 2.66 (m, 1H), 2.55 (dt, J = 4.4, 13.6 Hz, 1H), 2.32 (dd, J =3.6, 13.2 Hz, 1H), 2.16 - 2.03 (m, 2H), 1.96 (d, 7 = 1.2 Hz, 1H), 1.83 - 1.72 (m, 2H), 1.60 - 1.56(m, 1H), 1.53 (s, 9H), 1.50 - 1.45 (m, 1H), 1.45 (s, 3H), 1.43 - 1.36 (m, 1H), 1.19 - 1.07 (m, 2H), 1.04 - 0.97 (m, 1H), 0.88 (s, 3H). ESI MS: 566.3 ([M+H]+).

[0558] (8S,9S,10 / f,llS,13S,14S,17 / ?)-17-[2-[(4-Aminophenyl)methoxy]acetyl]-ll,17- dihydroxy-10, 13-dimethyl-7, 8, 9, 11, 12, 14,15, 16-octahydro-6H-cyclopenta[a]phenanthren-3- one (37).

[0559] Compound 36 (2 g, 3.54 mmol) was dissolved in formic acid (16.3 g, 354 mmol, 100 equiv.) and the mixture wasstirred at room temperature for 1 h. After the reaction was completed, the reaction mixture was quenched with aq. Na2COa (300 mL), and then extracted with EtOAc (3x200 mL). The combined organic layers were washed with brine (3x100 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150*25mm*5um; mobile phase: [H2O (0.05%NH3)-MeCN]; gradient: 20%-50% B over 12.0 min) to give compound 37 (700 mg, 43% yield) as a colorless solid.1H NMR (400 MHz, DMSO-d6) 6 7.31 (d, J = 10.0 Hz, 1H), 6.99 (d, 7 = 8.4 Hz, 2H), 6.52 (d, 7 = 8.4 Hz, 2H), 6.16 (dd, 7= 2.0, 10.0 Hz, 1H), 5.91 (s, 1H), 5.22 (s,1H), 5.05 (s, 2H), 4.63 (s, 1H), 4.49 (d, J= 18.4 Hz, 1H), 4.34 - 4.20 (m, 3H), 4.11 (d, J= 18.4 Hz, 1H), 2.58 - 2.51 (m, 2H), 2.29 (dd, J = 3.2, 13.2 Hz, 1H), 2.08 - 1.98 (m, 2H), 1.84 (dd, J = 3.2, 13.2 Hz, 1H), 1.69 - 1.57 (m, 2H), 1.57 - 1.50 (m, 1H), 1.43 - 1.33 (m, 4H), 1.33 - 1.23 (m, 1H), 1.03 - 0.93 (m, 1H), 0.88 (dd, 7= 3.2, 10.8 Hz, 1H), 0.78 (s, 3H). ESI MS: 488.2 ([M+23]+).

[0560] Diallyl (2.S’)-2-||(l.S)- l-allyloxycarbonyl-5-| |8-| |( LS’)-1-||( l.S’)-l-||4-||2-[(8S,95,10R ,115,13S,145,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6H-cyclopenta[a]phenanthren- 17-yl]-2-oxo-ethoxy ] methyl] phenyl ]carbamoyl ]-4- ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-8-oxo- octanoyl]amino]pentyl]carbamoylamino]pentanedioate (38).

[0561] Compound 35 (540 mg, 0.634 mmol, 1 equiv.) and compound 37 (295 mg, 0.634 mmol, 1 equiv.) were dissolved in anhydrous DMF (1 mL), EDCI (182 mg, 0.951 mmol, 1.5 equiv.),DIPEA (246 mg, 1.90 mmol, 3 equiv.) and HOBt(129 mg, 0.951 mmol, 1.5 equiv.) were added and the mixture was stirred at room temperature for12 h. After the reaction was completed, DCM (20 mL) was added and the suspension was extracted with DCM / zPrOH, 5:1 (3x50 mL). The combined organic layers were washed with brine (3x20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Welch Ultimate XB-SiOH 250*70 mm*10 μm; mobile phase: [hexane-EtOH] ; gradient: 15%-55% B over 15.0 min) to give compound 38 (250 mg, 30% yield) as a yellow solid. NMR showed two set peaks; long time LCMS showed two peaks. The epimerization happened.1H NMR (400 MHz, DMSO-d6) 3 10.01 - 9.79 (m, 1H), 8.46 - 8.06 (m, 1H), 7.99 - 7.78 (m, 1H), 7.75 - 7.70 (m, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.61 - 7.55 (m, 1H), 7.35 - 7.23 (m, 4H), 6.46 (dd, 7= 8.4, 13.2 Hz, 2H), 6.18 - 6.17 (m, 1H), 6.15 (dd, J= 1.6, 10.0 Hz, 1H), 5.91 (s, 5H), 5.41 (d, J = 5.2 Hz, 2H), 5.34 - 5.30 (m, 2H), 5.30 - 5.25 (m, 2H), 5.24 (s, 1H), 5.21 (d, J = 1.2 Hz, 2H), 4.63 (s, 1H), 4.59 - 4.53 (m, 8H), 4.47 - 4.42 (m, 1H), 4.38 (dd, J = 2.8, 12.0 Hz, 2H), 4.30 - 4.23 (m, 2H), 4.21 (d, J = 3.2 Hz, 2H), 4.12 (t, 7 = 7.2 Hz, 2H), 3.03 -2.95 (m, 4H), 2.42 - 2.37 (m, 2H), 2.36 - 2.26 (m, 2H), 2.21 - 2.12 (m, 2H), 2.05 - 1.91 (m, 8H), 1.68 - 1.52 (m, 8H), 1.48 - 1.35 (m, 13H), 1.30 - 1.26 (m, 2H), 0.90 - 0.83 (m, 8H), 0.80 (s, 3H). ESI MS: 1299.8 ([M+H]+).

[0562] (65,9S,24S,28S)-l-Amino-6-((4-((2-((8S,9S, 101?, 11S,13S, 14S,177?)-ll,17-dihydroxy- 10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)methyl)phenyl)carbamoyl)-9-isopropyl- l,8,ll,18,26-pentaoxo-2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylic acid (C7).. mmo , . equ v.) were a e . e m xture was stirred at 0 °C for 1 h under N2 atmosphere. After the reaction was completed, the reaction mixture was added to ice-water phase (100 mL), extracted with EtOAc (3x50 mL), then the aqueous phase was lyophilized. The crude mixture was dissolved in methanol (100 mL), filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge C18 150*50 mm* 10 μm; mobile phase: [H2O (WmM HCOONH4)-MeCN]; gradient:O%- 30% B over 15.0 min) and (column: CD07-Daisogel SP -100-8-ODS-PK 150*25*10 μm;mobile phase: [H2O (lOmM NH4HCO3)-MeCN (0.02% CH3COOH)]; gradient: 12%-42% B over 10.0 min)to give compound C7_l (106 mg, 21 % yield) as a colorless solid, and C7_2 (56 mg, 11 % yield) as a colorless solid.

[0564] C7_l

[0565] 1H NMR (400 MHz, DMSO-d6) h 9.97 (s, 1H), 8.63 (d, J= 7.6 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.74 - 7.69 (m, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 10.0 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 6.35 (d, J = 7.6 Hz, 1H), 6.27 - 6.19 (m, 1H), 6.18 - 6.10 (m, 2H), 5.91 (s, 1H), 5.46 (s, 2H), 4.56 (d, J = 18.4 Hz, 2H), 4.48 - 4.36 (m, 3H), 4.35 - 4.25 (m, 3H), 4.18 (d, J= 18.4 Hz, 2H), 4.11 (t, J = 7.6 Hz, 2H), 3.97 - 3.92 (m, 2H), 3.01 - 2.94 (m, 4H), 2.33 - 2.12 (m, 6H), 2.09 - 1.91 (m, 6H), 1.86 - 1.75 (m, 3H), 1.64 - 1.55 (m, 5H), 1.50 - 1.42 (m, 6H), 1.40 - 1.32 (m, 7H), 1.31 - 1.13 (m, 8H), 1.03 - 0.94 (m, 1H), 0.86 - 0.80 (m, 7H), 0.80 (s, 3H). ESI MS: 1179.6 ([M+H]+).

[0566] C7_2

[0567] 1H NMR (400 MHz, DMSO-d6) h 10.15 (s, 1H), 8.49 - 8.39 (m, 1H), 8.32 - 8.20 (m, 1H), 7.76 - 7.70 (m, 1H), 7.66 - 7.58 (m, 2H), 7.32 (d, J = 10.0 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 6.36 - 6.29 (m, 1H), 6.21 (d, J = 5.2 Hz, 1H), 6.16 (d, J = 1.2 Hz, 1H), 6.14 (d, J = 1.2 Hz, 1H), 5.91 (s, 1H), 5.49 (s, 2H), 4.55 (d, J= 18.4 Hz, 2H), 4.47 - 4.42 (m, 2H), 4.40 - 4.39 (m, 1H), 4.39 (s, 1H), 4.27 (d, J = 2.0 Hz, 2H), 4.15 (s, 2H), 3.88 (s, 1H), 3.04 - 2.96 (m, 4H), 2.34 - 2.13 (m, 6H), 2.07 - 1.93 (m, 6H), 1.89 - 1.75 (m, 3H), 1.66 - 1.57 (m, 5H), 1.49 - 1.42 (m, 6H), 1.40 - 1.33 (m, 7H), 1.30 - 1.15 (m, 8H), 1.04 - 0.97 (m, 1H), 0.90 - 0.83 (m, 7H), 0.80 (s, 3H). ESI MS: 1179.6 ([M+H]+).

[0569] ditert-Butyl (2.S’)-2-||( l.S’)-l-tert-biitoxycarbonyl-5-||8-||( l.S)-l-||(LS’)-l-||4-||(2 / ?)-2- [[ter / -butoxycarbonyl(methyl)amino]methyl]pyrrolidine-l-carbonyl]oxyniethyl]phenyl] carbamoyl]-4-ureido-butyl]carbamoyl]-2-methyl-propyl]amino]-8-oxo-octanoyl]amino] pentyl]carbamoylamino]pentanedioate (39).

[0570] Compound 19 (500 mg, 0.427 mmol, 1 equiv.) was dissolved in anhydrous DMF (10 mL) and DIPEA(110.4 mg, 0.855 mmol, 2 equiv.) followed by tert -Butyl N-methyl-N-[[(27?)-pyrrolidin-2-yl]methyl]carbamate (137 mg, 0.640 mmol, 1.5 equiv.) were added. The mixture was stirred at room temperature for 1 h. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was purified by reverse phase flash (0.1% FA) to give compound 39 (350 mg, 66% yield) as a colorless solid.1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 8.09 (d, J= 7.6 Hz, 1H), 7.80 (d, J= 8.6 Hz,1H), 7.74 - 7.69 (m, 1H), 7.64 - 7.54 (m, 2H), 7.38 - 7.10 (m, 2H), 6.38 - 6.14 (m, 2H), 6.03 - 5.88 (m, 1H), 5.41 (s, 2H), 4.98 (s, 2H), 4.47 - 4.29 (m, 1H), 4.26 - 4.12 (m, 1H), 4.08 - 3.86 (m, 3H), 3.23 - 3.10 (m, 2H), 3.04 - 2.89 (m, 4H), 2.86 - 2.73 (m, 2H), 2.67 (d, J = 1.6 Hz, 2H), 2.27 - 2.08 (m, 4H), 2.04 - 1.92 (m, 3H), 1.90 - 1.76 (m, 4H), 1.53 (d, J = 8.6 Hz, 6H), 1.44 - 1.34 (m, 44H), 1.28 - 1.15 (m, 7H), 0.90 - 0.78 (m, 6H). ESI MS: 1245.8 ([M+H]+).

[0571] (2S)-2-[[(lS)-l-Carboxy-5-[[8-[[(lS)-2-methyl-l-[[(lS)-l-[[4-[[(2R)-2- (methylaminomethyl)pyrrolidine-l-carbonyl]oxymethyl]phenyl]carbamoyl]-4-ureido- butyl]carbamoyl]propyl]amino]-8-oxo-octanoyl]amino]pentyl]carbamoylamino] pentanedioic acid (40).

[0572] Compound 39 (250 mg, 0.201 mmol) was dissolved in DCM (2 mL) and TFA (3.8 g, 33.7 mmol, 168 equiv.) was added. The resulting mixture was degassed with N2and thenstirred at room temperature for 1 h under inert atmosphere. After the reaction was completed, the residue was concentrated in vacuo to give compound 40 (190 mg, 97% yield) as a yellow oil. ESI MS: 977.6 ([M+H]+).

[0573] [2-[(8S,9S,10R ,llS,13S,14S,17 / ?)-ll,17-Dihydroxy-10,13-dimethyl-3-oxo- 7,8,9,H,12,14,15,16-octahydro-6 / 7-cyclopenta[a]phenanthren-17-yl]-2-oxo-ethyl](4- nitrophenyl) carbonate (41).

[0574] To a solution of prednisolone (1.00 g, 2.8 mmol, 1 equiv.) and (4-nitrophenyl) carbonochloridate (838 mg, 4.2 mmol, 1.5 equiv.) in anhydrous DCM (10 mL) was added pyridine(329 mg, 4.2 mmol, 1 .5 equiv.) at 0 °C. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was purified by LC on silica (PE / EtOAc; 10:1 to EtOAc) to give compound 41 (1.20 g, 82% yield) as colorless solid.1H NMR (400 MHz, DMSO- fc) 88.42 - 8.27 (m, 2H), 7.62 - 7.50 (m, 2H), 7.31 (d, J = 10.0 Hz, 1H), 6.22 - 6.09 (m, 1H), 5.92 (s, 1H), 5.51 (s, 1H), 5.28 (d, J = 17.6 Hz, 1H), 4.93 (d, J = 17.6 Hz, 1H), 4.72 (d, J = 4.0 Hz, 1H), 4.28 (s, 1H), 2.59 - 2.51 (m, 2H), 2.36 - 2.23 (m, 1H), 2.13 - 1.99 (m, 2H), 1.97 - 1.86 (m, 1H), 1.76 - 1.58 (m, 3H), 1.56 - 1.44 (m, 1H), 1.43 - 1.25 (m, 4H), 1.10 - 0.95 (m, 1H), 0.93 - 0.87 (m, 1H), 0.80 (s, 3H). ESI MS: 526.3 ([M+H]+).

[0575] (2S)-2-[[(lS)-l-Carboxy-5-[[8-[[(lS)-2-methyl-l-[[(lS)-l-[(4-methyl-2-oxo-chromen-7- yl)carbamoyl]-4-ureido-butyl]carbamoyl]propyl]amino]-8-oxo- octanoyl]amino]pentyl]carbamoylamino]pentanedioic acid (C8).

[0576] Compound 40 (190 mg, 0.194 mmol, 1 equiv.) was dissolved in anhydrous DMF (3 mL) and DIPEA (251mg, 1.94 mmol, 10 equiv.) followed by compound 41 (153 mg, 0.292 mmol, 1.5 equiv.) were added. The resulting mixture was stirred at room temperature for 12 h. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was purified by reversed phase flash (0.1% FA in MeCN) to give a residue, then the residue was purified by prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [H2O(0.225% FA)-MeCN]; gradient: 28%-58% B over 15.0 min) to give compound C8 (166 mg, 63% yield) as a colorless solid.1H NMR (400 MHz, DMSO-d6) 12.83 - 11. δ82 (m, 3H), 9.98 (s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.75 - 7.66 (m, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.40 - 7.21 (m, 3H), 6.42 - 6.24 (m, 2H), 6.21 - 6.11 (m, 1H), 6.04 - 5.96 (m, 1H), 5.91 (s, 1H), 5.43 (s, 2H), 5.34 (d, J = 11.6 Hz, 1H), 5.12 - 4.88 (m, 3H), 4.80 - 4.64 (m, 2H), 4.41 - 4.34 (m, 1H), 4.28 (s, 1H), 4.22 - 4.16 (m, 1H), 4.13 - 4.07 (m, 1H), 4.07 - 3.94 (m, 2H), 3.21 (d, J = 12.0 Hz, 1H), 3.08 - 2.69 (m, 8H), 2.55 (s, 2H), 2.34 - 2.11 (m, 5H), 2.10 - 1.97 (m, 5H), 1.97 -1 .77 (m, 6H), 1 .77 - 1 .57 (m, 8H), 1 .57 - 1 .29 (m, 15H), 1 .29 - 1 .17 (m, 6H), 1 .07 - 0.94 (m, 1 H), 0.92 - 0.71 (m, 10H). ESI MS: 1363.7 ([M+H]+).

[0578] 9H-Fluoren-9-ylmethyl N-[2-[[2-[(8S,9S,10R ,llS,13S,14S,17 / ?)-ll,17-dihydroxy- 10,13-dimethyl-3-oxo-7,8,9,l 1.12.14.15.16-(>ctahydro-6W-cyck>penta|a|phcnanthren- 17-yl ]-2- oxo-ethoxy]methylamino]-2-oxo-ethyl]carbamate (43).mixture was degassed with N2 and stirred at room temperature for12 h under N2 atmosphere. After the reaction was completed, the reaction mixture was concentrated in vacuo, and purified by reversed phase flash chromatography (0.1 % FA in MeCN) to give compound 43 (2.00 g, 53 % yield) as a colorless solid.1H NMR (400 MHz, DMSO-d6) d 8.76 - 8.62 (m, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.63 - 7.57 (m, 1H), 7.47 - 7.39 (m, 2H), 7.36 - 7.25 (m, 3H), 6.20 - 6.06 (m, 1H), 5.91 (s, 1H), 5.26 - 5.10 (m, 1H), 4.62 - 4.49(m, 4H), 4.32 - 4. 11 (m, 5H), 3.63 (d, J = 6.0 Hz, 2H), 2.54 (d, J = 4.4 Hz, 2H), 2.35 - 2.24 (m,1H), 2.06 - 1.94 (m, 2H), 1.88 - 1.80 (m, 1H), 1.70 - 1.50 (m, 3H), 1.48 - 1.34 (m, 4H), 1.32 - 1.18 (m, 1H), 1.06 - 0.91 (m, 1H), 0.89 - 0.82 (m, 1H), 0.76 (s, 3H). ESI MS: 669.3 ([M+H]+).

[0580] 2-Amino-N-[[2-[(8S,9S,10R ,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo- 7,8,9, 11,12, 14, 15, 16-octahydro-6H-cyclopenta[a ]phenanthren- 17-yl ]-2-oxo- ethoxy] methyl] acetamide (44).room empera ure or un er 2 a mosp ere. er e reac on was completed, the reaction mixture was concentrated to give crude compound 44 (667 mg) as a yellow oil. The crude product was used for the next step without further purification. ESI MS: 477.3 ([M+H]+).

[0582] 9H-Fluoren-9-ylmethyl N-[2-[[2-[[(lS)-l-benzyl-2-[[2-[[2-[(8S,9S,10R,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylamino]-2-oxo- ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (46).DMF (10 mL) and finally solution of compound 44 (550 mg, 1.23 mmol, 1 equiv.) in anhydrous DMF ( 1 mL) was added. The resulting mixture was degassed with N2 and stirred at room temperature for 1 h under N2 atmosphere. After the reaction was completed, the residue was concentrated in vacuo. The residue was purified by reversed phase flash chromatography (0.1% FA in MeCN) to give compound 46 (550 mg, 46% yield) as a colorless solid.1H NMR (400 MHz, DMSO-dfe) d 8.71 - 8.51 (m, 1H), 8.41 - 8.31 (m, 1H), 8.14 (d, 7 - 8.0 Hz, 1H), 8.06 - 7.94 (m, 1H), 7.89 (d, 7 - 7.6 Hz, 2H), 7.70 (d, 7 = 7.6 Hz, 2H), 7.61 - 7.56 (m, 1H), 7.48 - 7.38 (m, 2H), 7.37 - 7.28 (m, 3H),7.27 - 7.21 (m, 4H), 7.20 - 7.12 (m, 1H), 6.16 - 6. 10 (m, 1H), 5.91 (s, 1H), 5.23 (s, 1H), 4.64 - 4.43 (m, 5H), 4.38 - 4.11 (m, 5H), 3.83 - 3.55 (m, 6H), 3.13 - 3.02 (m, 1H), 2.85 - 2.73 (m, 1H), 2.55 (d, 7 = 4.4 Hz, 2H), 2.35 - 2.23 (m, 1H), 2.11 - 1.96 (m, 2H), 1.91 - 1.81 (m, 1H), 1.70 - 1.53 (m,3H), 1 .48 - 1 .34 (m, 4H), 1 .32 - 1 .20 (m, 1 H), 1 .07 - 0.94 (m, 1 H), 0.91 - 0.84 (m, 1 H), 0.77 (s, 3H). ESI MS: 930.4 ([M+H]+).

[0584] (25)-2-[[2-[(2-Aminoacetyl)amino]acetyl]amino]-N-[2-[[2-[(85,95,10R,115,135,145,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylamino]-2-oxo-ethyl]-3- phenyl-propanamide (47).degassed with N2 and stirred at room temperature for 1 h under N2 atmosphere. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was purified by reverse phase flash (0.1% FA in MeCN) to give compound 47 (370 mg, 97% yield) as a colorless solid. H NMR (400 MHz, DMSO-d6) <5 8.63 (s, 1H), 8.40 (s, 1H), 8.28 (d, J= 4.0 Hz, 2H), 8.22 - 8.12 (m, 1H), 7.40 - 7.12 (m, 6H), 6.22 - 6.06 (m, 1H), 5.91 (s, 1H), 5.51 - 5.01 (m, 1H), 4.88 - 4.41 (m, 6H), 4.28 (d, J = 2.0 Hz, 1H), 4.17 (d, J = 18.4 Hz, 1H), 3.87 - 3.59 (m, 6H), 3.25 - 3.16 (m, 2H), 3.11 - 3.02 (m, 1H), 2.85 - 2.72 (m, 1H), 2.35 - 2.22 (m, 1H), 2.10 - 1.96 (m, 2H), 1.93 - 1.74 (m,1H), 1.69 - 1.50 (m, 3H), 1.46 -1.34 (m, 4H), 1.33 - 1.19 (m, 1H), 1.09 - 0.94 (m, 1H), 0.91 - 0.84 (m, 1H), 0.77 (s, 3H). ESI MS: 708.4 ([M+H]+).

[0586] (25)-2-[[(lS)-5-[[8-[[2-[[2-[[(lR)-l-Benzyl-2-[[2-[[2-[(85,9S,10R, 115,135, 145, 17R)- 11, 17-dihydroxy-10, 13-dimethyl-3-oxo-7, 8,9,11,12, 14,15, 16-oclahydro-6 / 7- cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylamino]-2-oxo-ethyl]amino]-2-oxo- ethyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]amino]-8-oxo-octanoyl]amino]-l-carboxy- pentyl]carbamoylamino]pentanedioic acid (C9).HOA g, μm , q . yDMF (3 mL). The resulting mixture was degassed with N2 and stirred at room temperature for Ih under N2 atmosphere. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was purified by reversed phase flash (0.1% FA in MeCN) to give compound C9 (142.15 mg, 46% yield) as an off-white solid.XH NMR (400 MHz, DMSO-d6) d 8.78 - 8.69 (m, IH), 8.60 - 8.52 (m, IH), 8.43 (d, J = 5.6 Hz, IH), 8.37 (d, J = 8.0 Hz, IH), 8.34 - 8.25 (m, IH),7.83 - 7.66 (m, 1H), 7.31 (d, J = 10.0 Hz, 1H), 7.28 - 7.22 (m, 4H), 7.21 - 7.14 (m, 1H), 6.31 (d, J = 7.2 Hz, 1H), 6.26 - 6.18 (m, 1H), 6.17 - 6.09 (m, 1H), 5.91 (s, 1H), 4.64 - 4.54 (m, 3H), 4.51 - 4.41 (m, 1H), 4.29 (d, J = 2.8 Hz, 1H), 4.17 (d, J = 18.4 Hz, 1H), 3.96 (d, J = 1.2 Hz, 1H), 3.91 - 3.84 (m, 1H), 3.80 - 3.65 (m, 5H), 3.61 - 3.53 (m, 1H), 3.10 - 2.96 (m, 3H), 2.89 - 2.80 (m, 1H), 2.61 - 2.52 (m, 2H), 2.34 - 2.20 (m, 2H), 2.18 - 2.09 (m, 3H), 2.07 - 1.95 (m, 4H), 1.88 - 1.75 (m, 2H), 1.70 - 1.54 (m, 5H), 1.53 - 1.42 (m, 5H), 1.41 - 1.31 (m, 5H), 1.28 - 1.17 (m, 6H), 1.03 - 0.92 (m, 5H), 0.91 - 0.85 (m, 1H), 0.78 (s, 3H). ESI MS: 1165.6 ([M+H]+).

[0589] ditert -Butyl (2S)-2-[[(lS)-l-tert-butoxycarbonyl-5-[[8-(2,5-dioxopyrrolidin-l-yl)oxy-8- oxo-octanoyl]amino]pentyl]carbamoylamino]pentanedioate (49).y . at room temperature for 2 h. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was purified by prep-HPLC (column: Phenomenex luna Cl 8 150*25mm*10μm; mobile phase: [H2O (0.225% FA)-MeCN]; gradient: 45%-75% B over 10.0 min) to give compound 49 (450 mg, 59% yield) as a yellow gum.NMR (400 MHz, DMSO-cfe) 87.78 - 7.63 (m, 1H), 6.35 - 6.18 (m, 2H), 4.07 - 3.99 (m, 1H), 3.99 - 3.91 (m, 1H), 3.06 - 2.94 (m, 2H), 2.81 (s, 4H), 2.69 - 2.61 (m, 2H), 2.28 - 2.17 (m, 2H), 2.07 - 1.97 (m, 2H), 1.93 - 1.81 (m, 1H), 1.75 - 1.54 (m, 6H), 1.44 - 1.31 (m, 30H), 1.29 - 1.15 (m, 6H). ESI MS: 741.5 ([M+H]+).

[0591] (2S)-2-[[(lS)-l-Carboxy-5-[[8-(2,5-dioxopyrrolidin-l-yl)oxy-8-oxo-octanoyl]amino] pentyl]carbamoylamino]pentanedioic acid (48).the reaction was completed, the reaction mixture was concentrated in vacuo to give compound 48(150 mg, 97% yield) as a yellow oil. The residue was used for next step without further purification.ESI MS: 573.3 ([M+H]+).

[0593] Scheme 13: Synthesis of intermediate 45.

[0594] tert -Butyl (2S)-2-[ |2- [ 12-(9H-fl uoren-9-y Imethoxycarbonylamino )acetyl ]amino] acetyl]amino]-3-phenyl-propanoate (52).

[0595] Compound 51 (13.8 g, 38.8 mmol, 1 equiv.), DIPEA (17.6 g, 135.8 mmol, 3.5 equiv.) and HATU (17.7 g, 46.6 mmol, 1.2 equiv.) were dissolved in anhydrous DMF (100 mL) and finally compound 50 (10.0 g, 38.8 mmol, 1 equiv.; HC1 salt) was added. The resulting mixture was stirred at roomtemperature for 12 h. After the reaction was completed, the reaction mixture was diluted with dist. water (500 mL) and extracted with EtOAc (3x150 mL). The organic layer was washed with brine (100 mL), dried over anhydrous NaiSCL, filtered and concentrated in vacuo. The residue was purified by column chromatography (PE / EtOAc; 10:1 to 5:1) to give compound 52 (20.0 g, 92% yield) as a colorless solid.1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 7.6 Hz, 1H), 8.11 - 8.02 (m, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.63 - 7.57 (m, 1H), 7.46 - 7.38 (m, 2H), 7.35 - 7.26 (m, 4H), 7.23 - 7.19 (m, 3H), 4.42 - 4.20 (m, 4H), 3.73 (d, J = 5.2 Hz, 2H), 3.64 (d, J= 6.0 Hz, 2H), 3.01 - 2.88 (m, 2H), 1.31 (s, 9H). ESI MS: 580.3 ([M+Na]+).

[0596] (25)-2-[[2-[[2-(9H-fluoren-9-ylmethoxycarbonylamino)acetyl ]amino]acetyl ]amino]-3- phenyl-propanoic acid (45).

[0597] Compound 52 (20.0 g, 35.8 mmol, 1 equiv.) was dissolved in DCM (200 mL), TFA (61.4 g, 538 mmol, 15 equiv.) was added, the mixture was degassed and purged with N2, and the mixture was stirred for 2 h at roomtemperature. After the reaction was completed, the reaction mixture was concentrated in vacuo. The residue was triturated with petroleum ether (50 mL) at room temperature to give compound 45 (17.0 g, 95% yield) as a colorless solid.1H NMR (400 MHz, DMSO-rfc) <58.16 (d, J = 8.0 Hz, 1H), 8.10 - 7.97 (m, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.62 - 7.55 (m, 1H), 7.45 - 7.38 (m, 2H), 7.36 - 7.16 (m, 7H), 4.49 - 4.39 (m, 1H), 4.32 - 4.21 (m, 3H), 3.72 - 3.63 (m, 4H), 3.07 - 2.99 (m, 1H), 2.90 - 2.82 (m, 1H). ESI MS: 502.2([M+H]+).

[0598] Scheme 14: Synthesis of conjugate C10.

[0599] tert -Butyl (R)-2-(((2-(dimethylainino)etbyl)amino)methyl)pyrrolidine-1-carboxylate (54). [oo6oo]tert -Butyl (2R ) -2-formy [pyrrolidine- 1 -carboxylate (5.00 g, 25.1 mmol, 1 equiv.) and N',N'-dimethylethane-1,2-diamine (2.21 g, 25.1 mmol, 2.74 mL, 1 equiv.)were dissolved in anhydrous MeOH (50 mL), AcOH (3.01 g, 50.2 mmol, 2.87 mL, 2 equiv. was) was added and the mixture was stirred at room temperature for 0.5 h. Finally, NaBH3;CN (2.37 g, 37.64 mmol, 1.5 equiv.) was added and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to remove MeOe. The residue was diluted with H2O (50 mL) and extracted with EtOAc (3x50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give a residue. The crude product was purified by rcvcrscd-phase HPLC (0.1% FA condition, gradient: 0%-15% MeCN / HiO) to give compound 54 (3.00 g, 44.1% yield) as a yellow oil.1H NMR (400 MHz, DMSO-d6) δ = 3.94 (s, 1H), 3.31 - 3.20 (m, 3H), 2.96 (td, J = 6.0,12.0 Hz, 2H), 2.91 - 2.84 (m, 1H), 2.78 - 2.70 (m, 1H), 2.68 - 2.61 (m, 2H), 2.46 - 2.29 (m, 6H), 1.96 - 1.74 (m, 4H), 1.45 - 1.39 (m, 9H). ESI MS: 272.3 ([M+H]+).

[0601] tert -Butyl (R)-2-((((2-((8S,9S,10R ,11S,13S,14S,17R )-11,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2- oxoethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-l-carboxylate (55).

[0602] Compound 41 (1.00 g, 1.90 mmol, 1 equiv.) and compound 54 (620 mg, 2.28 mmol, 1.2 equiv.) were dissolved in anhydrous DMF (10 mL) and DMAP (23.2 mg, 0.190 mmol, 0.1 equiv.) followed by pyridine (451 mg, 460 pL, 5.71 mmol, 3 equiv.) wereadded and the mixture was stirred at room temperature for 6 h. The reaction mixture was diluted with H2O (10 mL) and extracted with DCM (3x20 mL). The combined organic layers were dried over anhydrous NaaSCL, filtered and concentrated under reduced pressure at 25 °C to give a residue.The residue was purified by LC on silica (ISCO®; 20 g SepaFlash® Silica Flash Column, Eluent of 0-20% methanol / EtOAc gradient 100 mL / min) to provided desired compound 55 (0.24 g, 19.2% yield) as a yellow gum. ESI MS: 658.4 ([M+H]+).

[0603] 2-((8S, 95,101?, llS,13S,14S,17fl)-ll,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11, 12,13, 14, 15, 16,17-dodecahydro-3H -cyclopenta[a]phenanthren-17-yl)-2-oxoethyl(2-(dimethylamino)ethyl)(((R)-pyrrolidin-2-yl)methyl)carbaniate (56).

[0604] Compound 55 (440 mg, 0.669 mmol, 1 equiv.) was dissolved in DCM (4 mL), TFA (0.4 mL) was added, and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentratedunder reduced pressure to provide compound 56 (0.42 g, 93.5% yield, TFA salt) as a yellow oil. ESI MS: 558.4 ([M+H]+).

[0605] tri-terf-Butyl (6S,9S,24S,285)-1-amino -6-((4-((((R)-2-((((2-((85,95,101?,115,13S,145,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta|a]phenanthren-17-yl)-2- oxoethoxy)carbonyl)(2-(dimethylamino)ethyl)amino)methyl)pyrrolidine-l-carbonyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-l,8,11,18,26-pentaoxo-2,7,10,19,25,27- hexaazatriacontane-24,28,30-tricarboxylate (57).

[0606] Compound 56 (0.42 g, 0.625 mmol, 1 equiv., TFA salt) and compound 19 (732 mg, 0.625 mmol, 1 equiv.) were dissolved in anhydrous DMF(4 mL), DIPEA (404 mg, 545 pL, 3.13 mmol, 5 equiv.) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through filter membrane to afford filtrate. The filtrate was purified by prepar ative HPLC (Phenomenex Gemini C18 (150 x 25 mm, 10 μm); flow rate: 25 mL / min; gradient: 35% - 65% B over 10 min; mobile phase A: 0.1% aq. NH4HCO3, mobile phase B: MeCN) to afforded compound 57 (500 mg, 50.3% yield) as a colorless solid.1H NMR (400 MHz, DMSO-d6) δ = 9.99 (s, 1H), 8.15 - 8.05 (m, 1H), 7.80 (br d, J = 8.8 Hz, 1H), 7.72 (brs, 1H), 7.59 (brd, 7 = 7.2 Hz, 2H), 7.36 - 7.25 (m, 3H), 6.32 - 6.23 (m, 2H), 6.19 - 6.13 (m, 1H), 5.97 (brt, J= 5.6 Hz, 1H), 5.91 (s, 1H), 5.41 (s, 2H), 5.36 (brs, 1H), 5.00 (brs, 2H), 4.74 (brs, 2H), 4.38 (brd, J = 5.6 Hz, 1H), 4.28 (brs, 1H), 4.19 (brt, J = 8.0 Hz, 1H), 4.08 - 3.99 (m, 2H), 3.98 - 3.93 (m, 1H), 3.06 - 2.91 (m, 5H), 2.36 - 2.10 (m, 11H), 2.01 (brt, 7= 7.2 Hz, 8H), 1.92 - 1.79 (m, 5H), 1.70 - 1.57 (m, 7H), 1.48 - 1.34 (m, 43H), 1.30 - 1.17 (m, 8H), 0.96 (brd, 7 = 4.0 Hz, 2H), 0.91 - 0.76 (m, 10H). ESI MS: 1590.2 (M+H]+).

[0607] (6.S.9.S.24.S.28S)-l-Amino-6-((4-((((R)-2-((((2-((8S,9S, 10 / C 11S,13S, 14S,17R)-11, 17- dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)(2- (dimethylamino)ethyl)amino)methyl)pyrrolidine-l-carbonyl)oxy)methyl)phenyl)carbamoyl)- 9-isopropyl-l,8,ll,18,26-pentaoxo-2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylic acid (CIO).

[0608] Compound 57 (0.50 g, 315 μmol, 1 equiv.) was dissolved in DCM (6 mL), TFA (3.07 g,26.9 mmol, 2 mL, 85.6 equiv.) was added and themixture was stirred at room temperature for 1 h The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (column: CD24- WePure Biotech XPT C18 150*25*7μm; mobile phase: [ H2O (lOmM NH4HCO3)-MeCN]; gradient: 9%-39% B over 15.0 min) to afford compound CIO (98.1 g, 21.5% yield, 98.1 purity) as a colorlesssolid.1H NMR (400 MHz, DMSO-d6) 10.09 (s, 1H), 8.32 (brd, J= 6.4 Hz, 1H), 8.06 (brd, J= 6.8 Hz, 1H), 7.73 (brt, J = 5.2 Hz, 1H), 7.61 (brd, J = 7.6 Hz, 2H), 7.31 (brt, J = 10.4 Hz, 3H), 6.40 (brd, J = 7.6 Hz, 1H), 6.26 (brd, J = 1.6 Hz, 1H), 6.19 - 6.09 (m, 2H), 5.91 (s, 1H), 5.47 (brs, 2H), 4.99 (brs, 3H), 4.76 (brs, 2H), 4.42 - 4.33 (m, 1H), 4.28 (brs, 1H), 4.16 (brt, J = 7.6 Hz, 1H), 4.07 - 3.88 (m, 4H), 3.47 - 3.35 (m, 2H), 3.23 - 3.10 (m, 2H), 3.06 - 2.89 (m, 5H), 2.38 (brs, 3H), 2.21 - 2.11 (m, 7H), 2.07 - 1.94 (m, 9H), 1.90 - 1.75 (m, 6H), 1.70 - 1.57 (m, 7H), 1.51 - 1.33 (m, 14H), 1.29 - 1.14 (m, 8H), 1.06 - 0.97 (m, 1H), 0.89 - 0.81 (m, 7H), 0.78 (brs, 3H). ESI MS: 1422.0 ([M+H]+).

[0609] Scheme 15: Synthesis of conjugate Cll.

[0610] 9H-Fluoren-9-ylmethyl -[(1S)-l-[[2-[[2-[(8S, 9S,101R, 115,135,145, 17R )-11, 17- dihydroxy-10, 13-dimethyl-3-oxo-7, 8, 9, 11, 12, 14,15, 16-octahydro-6 / / - cyclopentaln |phenanthren-17-yl |-2-oxo-ethoxy |methylamino|-2-oxo-ethyl Icarbamoyl |-3- methyl-butyl]carbamate (58).

[0611] Fmoc-L-Leu-OH (633 mg, 1.79 mmol, 1 equiv.), HATU (1.02 g, 2.69 mmol, 1.5 equiv.) and DIPEA (695 mg, 936 pL, 5.37 mmol, 3 equiv.) were dissolved inanhydrous DMF (10 mL) and finally solution of compound44 (800 mg, 1.79 mmol, 1 equiv.) in anhydrous DMF (5 mL) was added. The mixture was stirred at room temperature for 1 h. The resulting suspension was filtered, and volatiles were removed under reduced pressure. The residue was purified by preparative HPLC (Phenomenex Gemini C18(150x25 mm, 10 μm); flow rate: 75 mL / min; gradient: 0% - 55% B over 20 min; mobile phase A: 0.1% aq. NH4HCO3, mobile phase B: McCN) to afforded compound 58 (1.00 g, 71.4% yield) as a colorless solid. ESI MS: 782.4 ([M+H]+).

[0612] (2S)-2-Amino-^-[2-[[2-[(8S,9S,10R ,llS,13S,14S,17 / ?)-ll,17-dihydroxy-10,13- dimethyl-3-oxo-7,8,9,ll,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo- ethoxy]methylamino]-2-oxo-ethyl]-4-methyl-pentanamide (59).

[0613] Compound 58 (1.00 g, 1.28 mmol, 1 equiv.) was dissolved in anhydrous DMF (6 mL), 1- methylpiperidine (2 mL) was added and the mixture was stirred at room temperature for 1 h. The resultingsuspension was filtered, and volatiles were removed under reduced pressure. The residue was purified by preparative HPLC (Phenomenex Gemini C18 (150x25 mm, 10 μm); flow rate: 50 mL / min; gradient: 0% - 48% B over 20 min; mobile phase A: 0.1% aq. NH4HCO3, mobile phase B: MeCN) to afford compound 59 (600 mg, 84 %) as a colorless solid. ESI MS: 560.5 ([M+H]+).

[0614] 9H-Fluoren-9-ylmethyl A-[2-[[(1S)-l-benzyl-2-[[(1S)-l-[[2-[[2-[(8S,9S,10R ,11S,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylamino]-2-oxo- ethyl]carbamoyl]-3-methyl-butyl]amino]-2-oxo-ethyl]amino]-2-oxo-ethyl]carbamate (60).

[0615] Fmoc-Gly-L-Phe-OH (477 mg, 1.07 mmol, 1 equiv.), HATU (611 mg, 1.61 mmol, 1.5 equiv.) and DIPEA (416 mg, 560 pL, 3.22 mmol, 3 equiv.) weredissolved in anhydrous DMF (8 mL) and finally solution of compound 59 (600 mg, 1.07 mmol, 1 equiv.) in anhydrous DMF (2 mL) was added. The mixture was stirred at room temperature for 1 h, filtered and volatiles evaporated. The residue was purified by preparative HPLC (Phenomenex Gemini C18 (150x25 mm, 10 μm); flow rate: 50 mL / min; gradient: 0% - 55% B over 22 min; mobile phase A: 0.1% aq. NH4HCO3, mobile phase B: MeCN) to afforded compound 60 (0.80 g, 75.7% yield) as a colorless solid. ESI MS: 986.3 ([M+H]+).

[0616] (2S)-2-[[(2S)-2-[(2-aminoacetyl)amino]-3-phenyl-propanoyl]amino]-A-[2-[[2- [(8S,9S,10R ,11S,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6 / / -cydopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylamino]-2-oxo-ethyl]-4-methyl-pentanamide (61).p . reaction mixture was filtered and the volatiles evaporated under reduced pressure. The crude product was purified by preparative HPLC (Phenomenex Gemini C18 (150 x 25 mm, 10 μm); flow rate: 35 mL / min; gradient: 0% -40% B over 10 min; mobile phase A: 0.1% aq. NH4HCO3, mobile phase B: MeCN) to afforded product 61 (500 mg, 80.7% yield) as a colorless solid.1H NMR (400 MHz, DMSO-^6) 6 8.61 (t, 7= 6.8 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.11 (t, J = 6.0 Hz, 1H), 8.07 - 7.98 (m, 1H), 7.30 (d, J = 10.0 Hz, 1H), 7.26 - 7.13 (m, 5H), 6.19 - 6.09 (m, 1H), 5.91 (s, 1H), 5.24 (s, 1H), 4.65 - 4.51 (m, 5H), 4.34 - 4.24 (m, 2H), 4.17 (d, J= 18.4 Hz, 1H), 3.78 - 3.67 (m, 2H), 3.04 (s, 2H), 3.00 (d, J = 4.8 Hz, 1H), 2.87 - 2.78 (m, 1H), 2.37 - 2.10 (m, 3H), 2.02 (brd, J = 8.4 Hz, 2H), 1.88 - 1.80 (m, 1H), 1.74 - 1.43 (m, 7H), 1.38 (s, 4H), 1.32 - 1.21 (m, 1H), 1.06 - 0.95 (m, 1H), 0.93 - 0.80 (m, 8H), 0.77 (s, 3H). ESI MS: 764.4 ([M+H]+).

[0618] (2S)-2-[[(lS)-5-[[8-[[2-[[(lS)-l-benzyl-2-[[(lS)-l-[[2-[[2-[(8S,9S,10R, 11S,13S, 14S,17R)- ll,17-dihydroxy-10,13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16-octahydro-6 I- cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylamino]-2-oxo-ethyl]carbamoyl]-3- methyl-buty 1] amino] -2-oxo-ethyl]amino] -2-oxo-ethyl] amino] -8-oxo-octanoy 1] amino] - 1 - carboxy-pentyl]carbamoylamino]pentanedioic acid (Cll).room temperature for 1 h. The reaction mixture was filtered and volatiles evaporated. The residue was purified by preparative HPLC (CD01 -Phenomenex luna C18 150*25mm*10μm; mobile phase: [H2O (0.225% FA)-ACN]; gradient: 18%-48% B over 10.0 min) to afforded desired product Cll (116 mg, 28.7% yield, 98.4% purity) as a colorless solid.XH NMR (400 MHz, DMSO-t / e) d 12.77 - 12.13 (m, 2H), 8.60 (br t, J= 6.8 Hz, 1H), 8.14 (brd, J = 8.0 Hz, 1H), 8.07 - 7.97 (m, 3H), 7.73 (bit, J = 5.6 Hz, 1H), 7.30 (d, J = 10.0 Hz, 1H), 7.27 - 7.20 (m, 4H), 7.19 - 7.14 (m, 1H), 6.37 - 6.27 (m, 2H), 6.19 - 6.10 (m, 1H), 5.91 (s, 1H), 5.23 (brs, 1H), 4.63 - 4.48 (m, 5H), 4.31 - 4.23 (m,2H), 4.17 (brd, J = 18.4 Hz, 1H), 4.12 - 3.99 (m, 2H), 3.74 - 3.65 (m, 3H), 3.57 - 3.51 (m, 1H), 3.06 - 2.95 (m, 3H), 2.82 - 2.73 (m, 1H), 2.31 - 2.19 (m, 3H), 2.10 - 1.98 (m, 6H), 1.84 (brd, J = 11.2 Hz, 2H), 1.59 (brd, 7= 6.4 Hz, 7H), 1.53 - 1.34 (m, 14H), 1.29 - 1.18 (m, 7H), 1.07 - 0.96 (m, 1H), 0.90 - 0.82 (m, 7H), 0.77 (s, 3H). ESI MS: 1221.9 ([M+H]+).

[0621] (((4-((S)-2-((5)-2-((75,llS)-7,ll-Bis(ferributoxycarbonyl)-2,2-dimethyl-4,9,17-trioxo-3- oxa-8,10,16-triazatetracosan-24-amido)-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)carbonyl)glycyl-L-proline (62)..(27.7 mg, 0.205 mmol, 0.2 equiv.) and the mixture was stirred for 12 h at room temperature. The reaction mixture was diluted with DMSO (10 mL) and filtered to remove insoluble precipitates. The crude mixture was purified by reversed-phase flash chromatography (0.1% FA in water, phase B: MeCN, 45-65% B, flow rate: 80 mL / min) and lyophilized to give desired product 62 (1.10 g, 914 μmol, 89.1% yield) as an off-white solid. ESI MS: 1203.9 ([M+H]+).

[0623] tri / c / 7-Butyl (6S,9S,24S,28S)-l-amino-6-((4-((((2-((S)-2-((2-((8S,9S,101?,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3 / / -cyclopenta[a]phenanthren-17-yl)-2- oxoethoxy)carbonyl)pyrrolidin-l-yl)-2-oxoethyl)carbamoyl)oxy)methyl) phenyl)carbamoyl)- 9-isopropyl-l, 8, 11,18, 26-pentaoxo-2, 7, 10, 19,25, 27-hexaazatriacontane-24, 28,30- tricarboxylate (63).0.3 equiv.) in anhydrous MeCN (10 mL) was added DCC (206 mg, 0.997 mmol, 1.2 equiv.) and the mixture was stirred at 50 °C for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue. The crude mixture was purified by reversed-phase flash chromatography (0.1% FA in water, phase B: MeCN, 45-65% B, flow rate: 80 mL / min) and lyophilized to give desired product 63 (0.90 g, 70.1% yield) as a light-brown solid. ESI MS: 1547.1 ([M+H]+).

[0625] (6S,9S,24S,28S)-l-Amino-6-((4-((((2-((S)-2-((2-((8S,9S,10R,H5,13S,14S,17 / ?)-ll,17- dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)pyrrolidin-l-yl)-2- oxoethyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-l,8,ll,18,26-pentaoxo- 2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylic acid (C12).mixture was stirred at room temperature for 2 h. The reaction mixture was neutralized with NFh’fLO to pH = 7~8 and dissolved in DMSO (6 mL). The mixture was filtered to remove the insoluble solid and concentrated under vacuum. The crude product was purified by reversed-phase flash: (CD45- Waters Xbridge BEH C18 150x40 mmxlO μm); flow rate: 25 mL / min; gradient: 15% - 45% B over 11 min; mobile phase A: 10 mmol / L NH4HCO3, mobile phase B: MeCN) and again by reversed- phase flash chromatography (CDOl-Phenomenex luna C18 150x25 mmxlO μm); flow rate: 25 mL / min; gradient: 12% - 42% B over 16 min; mobile phase A: 0.225% aq. FA, mobile phase B: MeCN) and lyophilized to give desired product C12 (113 mg, 82.0 μmol, 14.1% yield, 100% purity) as a light yellow solid.1H NMR (400 MHz, DMSO-afe) 10.02 (s, 1 δH), 8.18 (brd, J= 7.2 Hz, 1H), 7.91 (brd, J = 8.0 Hz, 1H), 7.73 (brt, J = 5.2 Hz, 1H), 7.60 (brd, J = 8.4 Hz, 2H), 7.39 - 7.21 (m, 4H), 6.41 - 6.25 (m, 2H), 6.16 (dd, J1= 1.6, J2 = 10.0 Hz, 1H), 6.04 (brt, J = 5.6 Hz, 1H), 5.91 (s, 1H), 5.44 (brs, 2H), 5.03 - 4.75 (m, 5H), 4.49 - 4.31 (m, 2H), 4.31 - 4.24 (m, 1H), 4.22 - 4.14 (m, 1H), 4.08 - 3.98 (m, 2H), 3.93 - 3.71 (m, 2H), 3.58 - 3.48 (m, 2H), 3.04 - 2.92 (m, 4H), 2.31 - 2.12 (m, 7H), 2.06 - 1.86 (m, 8H), 1.84 - 1.56 (m, 9H), 1.54 - 1.14 (m, 22H), 1.07 - 0.96 (m, 1H), 0.90 - 0.74 (m, 10H). ESI MS: 1378.1 ([M+H]+).

[0628] 3-bromo-2V-methylpyridin-2-amine (65).■r

[0629] 3-bromo-2-fluoropyridine (64) (3.00 g, 17.0 mmol, 1 equiv.) and methylamine hydrochloride (3.45 g, 51.1 mmol, 3 equiv.) were dissolved in anhydrousNMP (25 mL) and DIPEA (8.81 g, 11.8 mL, 68.2 mmol, 4 equiv.) was added. The resulting mixture was heated to 120 °C for 12 h in sealed tube. The mixture was cooled to room temperature, poured into distilled water (150 mL) and extracted with EtOAc (2x150 mL), combined organic phases were washed with sat. NaCl (100 mL), dried over anhydrous Na2SC>4, filtered and volatiles were removed by reduced pressure. The residue was purified by LC on silica (hexanes / EtOAc; 1 to 10:1) to give the desired product 65 (7.75 g) in 81% yield as a yellow liquid.1H NMR (400 MHz, CDCh) 5 8.10 (dd, J1= 1.2, J2= 4.8 Hz, 1H), 7.60 (dd, J1= 1.6, J2= 7.6 Hz, 1H), 6.45 (dd, J1= 4.8, J = 7.6 Hz, 1H), 5.04 (brs, 1H), 3.04 (d, J = 4.8 Hz, 3H). ESI MS: 187.0 ([M+HD-

[0630] (3-Bromopyridin-2-yl)(methyl)carbamic chloride (66).

[0631] 3-Bromo-A-methyl-pyridin-2-amine (5.00 g, 26.7 mmol, 1 equiv.) was dissolved in anhydrous DCM (80 mL) and DIPEA (6.91 g, 9.31 mL, 53.4 mmol, 2 equiv.) followed by bis(trichloromethyl) carbonate (7.93 g, 26.7 mmol, 1 equiv.) wereadded while cooling to 0° C. The reaction was heated to room temperature and stirred for 1 h. The precipitate was filtered off and volatiles evaporated. The residue was purified by LC on silica (hexanes / EtOAc 1:0 to 4:1) to give the desired product 66 (3.40 g) in 51% yield as a yellow liquid.1!! NMR (400 MHz, CDCh) 5 8.54 (br dd, A = 1.2, J2= 4.4 Hz, 1H), 8.06 (dd, A =1.6, J2=8.0 Hz, 1H), 7.31 - 7.27 (m, 1H), 3.36 (s, 3H). ESI MS: 250.7 ([M+H]+).

[0632] 2-((8S,9S,10R,llS,13S,14S,17R)-11,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15, 16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl(3-bromopyridin-2-yl)(methyl)carbamate (67).

[0633] Prednisolone (6.36 g, 17.6 mmol, 2.2 equiv.) was dissolved in anhydrous DMF (80 mL) and 60% suspension of NaH (384 mg, 9.62 mmol, 1.2 equiv.) was added while cooling to 0 °C. The mixture was stirred for 10 minutes and compound 66 (2.00 g, 8.02 mmol, 1 equiv.) inanhydrous DMF (20 mL) was added at same temperature. The mixture was heated to room temperature and stirred for an additional 1.5 h. Saturated NH4CI (100 mL) was added, and suspension was extracted with EtOAc (5x100 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by preparative HPLC (0.1% FA condition) to give the desired product 67 (4.00 g) in 87% yield as a violet solid. NMR (400 MHz, DMSO-d6) δ 8.51 (dd, J1= 1.2, J2= 4.8 Hz, 1H), 8.21 (dd, J1= 1.2, J2= 8.0 Hz, 1H), 7.35 (dd, J1= 4.8, J2= 8.0 Hz, 1H), 7.31 (d, J = 10.4 Hz, 1H), 6.16 (dd, J1= 1.6, J = 10.4 Hz, 1H), 5.91 (s, 1H), 5.36 (brs, 1H), 5.18 - 4.77 (m, 2H), 4.76 - 4.50 (m, 2H), 4.27 (brs, 1H), 3.19 (brs, 3H), 2.28 (dd, J1= 3.2, J2= 12.8 Hz, 1H), 2.06 - 2.00 (m, 2H), 1.86 (brd, J = 10.4 Hz, 1H), 1.63 (brd, J = 6.4 Hz, 3H), 1.46 - 1.40 (m, 1H), 1.38 (s, 3H), 1.33 - 1.23 (m, 1H), 1.06 - 0.85 (m, 3H), 0.78 (s, 3H). ESI MS: 573.2 ([M+H]+).

[0634] 2-((8S,9S,10R,llS,13S,14S,17R)-11,17-Dihydroxy-10,13-dimethyl-3-oxo- 6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl (3-(((terf-butoxycarbonyl)amino)methyl)pyridin-2-yl)(methyl)carbamate (68).

[0635] Compound 67 (2.00 g, 3.49 mmol, 1 equiv.) and potassium (tert-butoxycarbonylamino)methyl-trifluoro-boranuide (1.65 g, 6.98 mmol, 2 equiv.) were dissolved in the mixture of solvents toluene (30 mL) and H2O (5 mL) and CS2CO3(2.27 g, 6.98 mmol, 2 equiv.)followed by [2-(2-aminophenyl)phenyl]palladium; bis(l-adamantyl)-butyl-phosphane; methanesulfonate (253 mg, 348 μmol, 0.1 equiv.) were added and the resulting mixture was heated to 100 °C for 1 h under N2atmosphere. The reaction was concentrated under reduced pressure. The residue was purified by preparative HPLC (0.1% NH4HCO3condition). The mixture was concentrated to remove acetonitrile and extracted by EtOAc (2x200 mL) and combined organicphase were dried over anhydrous NtoSC , filtered and concentrated under reduced pressure to give the desired compound 68 (1.60 g) in 74% yield as a pink solid. *H NMR (400 MHz, DMSO-d6) 8 8.37 (brd, J = 3.6 Hz, 1H), 7.72 (brd, J = 3.2 Hz, 1H), 7.49 - 7.26 (m, 3H), 6.15 (dd, J1= 1.2, J2= 10.4 Hz, 1H), 5.91 (s, 1H), 5.38 (brd, J = 1.6 Hz, 1H), 5.20 - 4.87 (m, 1H), 4.83 - 4.55 (m, 2H), 4.44 - 4.05 (m, 3H), 3.34 - 3.12 (m, 4H), 2.36 - 2.21 (m, 1H), 2.08 - 1.99 (m, 2H), 1.93 - 1.79 (m, 1H), 1.64 (brs, 3H), 1.39 (brs, 12H), 1.36 - 1.21 (m, 3H), 1.00 (dd, J1= 4.4, h = 12.8 Hz, 1H), 0.88 (dd, J1- 2.8, J2- 10.8 Hz, 1H), 0.80 (brs, 3H). ESI MS: 624.4 ([M+H]+).

[0636] 2-((8S, 95, 10R,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6.7.8.9.10.11.12.13.14.15.16.17-dodecahydro-3H-cyclopenta[a ]phenanthren-17-yl)-2-oxoethyl (3-(aminomethyl)pyridin-2-yl)(methyl)carbamate (69).

[0637] Compound 68 (300 mg, 480 μmol, 1 eq) was dissolved in 4M solution of HC1 in dioxane (6 mL) and stirred at room temperature for 0.5 h. The reaction was concentrated under reduced pressure to give the desired product 69 (270 mg, HC1 salt) in a quantitative yield as a yellowsolid. ESI MS: 524.4 ([M+H]+).

[0638] Tri-tert-butyl (65,95,245,285)-l-amino-6-((4-(((((2-(((2-((85,95,101?,115,135,145,17R)-11.17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)(methyl)amino)pyridin-3- yl)methyl)carbamoyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-l,8,ll,18,26-pentaoxo- 2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylate (70).

[0639] Compound 19 (564 mg, 482 μmol, 1 equiv.) was dissolved in anhydrous DMF (15 mL) and DIPEA (311 mg, 419 pL, 2.41 mmol, 5 equiv.) followed bycompound 69 (270 mg, 482 μmol, 1 equiv.) in anhydrous DMF (5 mL) were added. The reaction mixture was stirred at room temperature for 1 h. The volatiles were removed under vacuo and the residue was purified by preparative HPLC (0.1% NH4HCO3condition). The mixture was concentrated to remove the acetonitrile and extracted with EtOAc (2x100 mL). The combined organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the desired product 70 (300 mg) in 40% yield as a colorless solid. ESI MS: 1555.5 ([M+H]+).

[0640] (65, 95, 245, 285)-l-amino-6-((4-(((((2-(((2-((85, 95,101?, 115, 135, 145, 17R)-11, 17-dihydroxy-10,13-dimethyl-3-oxo-6, 7, 8, 9,10,11 ,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)(methyl)amino)pyridin-3- yl)methyl)carbamoyl)oxy)methyl)phenyI)carbamoyl)-9-isopropyl-l,8,ll,18,26-pentaoxo- 2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylic acid (C13).

[0641] Compound 70 (300 mg, 192 μmol, 1 equiv.) was dissolved in anhydrous DCM (9 mL), TFA (3 mL) was added, and the reaction mixture was stirred at room temperature for 1.5 h. The pH of the reaction was adjusted by ammonia hydrate to 7 and the mixturewas concentrated under reduced pressure. The residue was purified by preparative HPLC (column: CDOl-Phenomenex luna C18 150*25mm*10pm; mobile phase: [H2O (0.225% FA)-MeCN]; gradient: 20%-50% B over 16.0 min) and lyophilized to give the desired product C13 (166 mg) in 40% yield as a colorless solid.1H NMR (400 MHz, DMSO-d6) 1 δ3.48 - 11.49 (m, 2H), 10.08 - 9.89 (m, 1H), 8.37 (brd, J = 3.6 Hz, 1H), 8.11 (brd, J =7.2 Hz, 1H), 7.88 - 7.65 (m, 4H), 7.59 (brd, J = 8.4 Hz, 2H), 7.36 (dd, J1= 4.8, J2= 7.6 Hz, 1H), 7.33 - 7.20 (m, 3H), 6.31 (t, J = 7.6 Hz, 2H), 6.15 (dd, J1=1.6, J2= 10.4 Hz, 1H), 6.00 (brt, J= 5.6 Hz, 1H), 5.91 (s, 1H), 5.43 (s, 2H), 5.40 - 5.23 (m, 1H), 5.20 - 5.01 (m, 1H), 4.97 (brs, 2H), 4.85 - 4.64 (m, 2H), 4.44 - 4.24 (m, 3H), 4.24 - 4.12 (m, 2H), 4.12 - 3.95 (m, 3H), 3.21 - 3.14 (m, 2H), 2.99 (dq, J1= 6.8, J2= 13.2 Hz, 5H), 2.35 - 2.07 (m, 6H), 2.01 (brt, 7= 7.6 Hz, 4H), 1.98 - 1.83 (m, 3H), 1.75 - 1.57 (m, 7H), 1.45 (brd, 7 = 3.6 Hz, 7H), 1.38 (s, 6H), 1.30 - 1.16 (m, 7H), 1.05 - 0.96 (m, 1H), 0.92 - 0.87 (m, 1H), 0.84 (dd, J1= 6.8, J2= 11.6 Hz, 6H), 0.81 - 0.73 (m, 3H). ESI MS: 1386.5 ([M+H]+).

[0642] Scheme 18. Synthesis of conjugate C14.

[0643] 2-[(4-Nitrophenyl)methoxycarbonylamino]acetic acid (72).

[0644] Glycine (3.00 g, 40.0 mmol, 8.62 mmol, 1 equiv.) was dissolved in 2M NaOH (20.0 mL, 8.62 mmol, 1 equiv.) and to the stirring mixture were added simultaneously 4M NaOH (10.0 mL, 8.62 mmol, 1equiv.) and solution of (4-nitrophenyl)methyl carbonochloridate (71) (10.0 g, 46.4 mmol, 5 equiv.) in toluene (100 mL) via two dropping funnels. The resulting mixture was stirred at room temperature for 3 h and extracted with EtOAc (2x30 mL). The aqueous phase was acidified to pH 1 with HC1 (4 M solution), extracted with DCM (3x50 mL) and combined organic fractions were dried over anhydrous NaiSCU, and volatiles were evaporated under vacuum to afford compound 72 (8.00 g) in 68% yield as a colorless solid. ’H NMR (400 MHz, DMSO-t / e) 5 = 12.61 (hr s, 1H), 8.39 - 8.16 (m, 2H), 7.73 (brt, 7- 6.0 Hz, 1H), 7.67 - 7.56 (m, 2H), 5.19 (s, 2H), 3.81 -3.65 (m, 2H).

[0645] [(4-Nitrophenyl)methoxycarbonylamino]methyl acetate (73).

[0646] Compound 72 (4.60 g, 18.1 mmol, 1 equiv.) was dissolved in anhydrous THF (50 mL), triacctoxyplumbyl acetate (12.0 g, 27.1 mmol,1.5 equiv.) and Cu(OAc)2 (329 mg, 1.81 mmol, 0.1 equiv.) were added and the mixture was stirred at 50 °C for 2 h. The reaction mixture was diluted with dist. water (100 mL) and extracted with EtOAc (3x50 mL). The combined organic phases were washed with brine (2x50 mL), dried over anhydrous sodium sulfate, filtered and volatiles were removed under vacuum. The residue was purified by LC on silica (ISCO, 80 g SepaFlash Silica Flash Column, eluent 0-40% EtOAc / hexanes, gradient 50 mL / min). to obtain compound 73 (4.00 g) in 82% yield as a colorless solid.1H NMR (400 MHz, DMSO-rfc) = 8 δ.60 (brt, J = 6.8 Hz, 1H), 8.32 - 8.20 (m, 2H), 7.62 (d, J= 8.8 Hz, 2H), 5.22 (s, 2H), 5.08 (brd, 7 = 7.2 Hz, 2H), 2.00 (s, 3H).

[0647] (4-Nitrophenyl)methyl N-[[2-[(8S,9S,10R,llS,13S,14S,17R)-ll,17-dihydroxy-10,13- dimethyl-3-oxo-7,8, 9,11,12,14, 15, 16-octahydro-6H-cyclopenta[a ]phenanthren- 17-yl ]-2-oxo- ethoxy]methyl]carbamate (74).

[0648] Compound 73 (3.00 g, 11.2 mmol, 1 equiv.) and compound 1 (6.05 g, 16.8 mmol, 1.5 equiv.) were dissolved in anhydrous THF (40 mL) and lithium 2-methylpropan-2-olate (448 mg, 504 pL, 5.59 mmol, 0.5 equiv.) was dropwise added at -65 °C and the resulting mixture was stirredfor 1 h. The reaction mixture was heated to room temperature and volatiles evaporated under vacuo. The residue was purified by preparative HPLC (Phenomenex Gemini C18 (150 x 25 mm, 10 μm); flow rate: 100 mL / min; gradient: 0% - 60% B over 10 min; mobile phase A: 0.1% aqueous ammonium hydrogen carbonate, mobile phase B: acetonitrile). The solution obtained was lyophilized to afford compound 74 (2.60 g) in 39% yield as a colorless solid.1H NMR (400 MHz, DMSO-ifc) = 8 δ.35 (brt, J= 6.8 Hz, 1H), 8.24 (d, J= 8.8 Hz, 2H), 7.61 (brd, J= 8.8 Hz, 2H), 7.32 (d, 7 = 10.0 Hz, 1H), 6.22 - 6.11 (m, 1H), 5.91 (s, 1H), 5.31 - 5.17 (m, 3H), 4.66 - 4.46 (m, 4H), 4.26 (brs, 1H), 4.16 (d, J = 18.4 Hz, 1H), 2.35 - 2.25 (m, 1H), 2.02 (brd, J = 9.2 Hz, 2H), 1.88 - 1.79 (m, 1H), 1.70 - 1.51 (m, 3H), 1.44 - 1.34 (m, 4H), 1.33 - 1.23 (m, 1H), 1.08 - 0.94 (m, 1H), 0.92 - 0.84 (m, 1H), 0.77 (s, 3H). ESI MS: 569.2 ([M+H]+).

[0649] (4-Aminophenyl)methyl N-[[2-[(8S,9S,10R,llS,13S,14S,17R)-ll,17-dihydroxy-10,13- dimethyl-3-oxo-7,8,9,l l,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-17-yl ]-2-oxo- ethoxy] methyl] carbamate (75).Compound 74 (100 mg, 0.176 mmol, 1 equiv.) was dissolved in DMF (1 mL), tetrahydroxydiboron (78.8 mg, 0.879 mmol, 5 equiv.) and 4-(4-pyridyl)pyridine (275 mg, 1.76 mmol, 10 equiv.) were added and the mixture was stirred at room temperature for 1 h. The mixture was quenched by dropwise addition of sat. NaHCO3(20 mL). The resulting mixture was extracted with EtOAc (3x20 mL), combined organic fractions washed with brine (2x20 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure affording compound 75 (50.0 mg) in 52.8% yield as a light-yellow oil. ESI MS: 539.2 ([M+H]+).

[0651] 4-((S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-ureidopentanamido)benzyl ((2-((8S,9S,10R,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11,12,13, 14,15, 16, 17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2- oxoethoxy)methyl)carbamate (76).4 equiv.) were added and the mixture was stirred at room temperature for 1 h. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC: (Phenomenex Gemini C18 (150 x 25 mm, 10 μm); flow rate: 75 mL / min; gradient: 0% - 55% B over 30 min; mobile phase A: 0.1% aqueous ammonium hydrogen carbonate, mobile phase B: McCN). The solution obtained was lyophilized to afford the compound 76 (800 mg) as a colorless solid in 39% yield.1H NMR (400 MHz, DMSO-<76) =10 δ.07 (s, 1H), 8.15 (brt, J = 6.4 Hz, 1H), 7.89 (brd, J = 7.6 Hz, 2H), 7.74 (brs, 1H), 7.70 - 7.65 (m, 1H), 7.61 (brd, J = 8.4 Hz, 2H), 7.41 (s, 2H), 7.37 - 7.25 (m, 5H), 6.21 - 6.11 (m, 1H), 5.99 (brt, J = 5.6 Hz, 1H), 5.91 (s, 1H), 5.42 (s, 2H), 5.23 (s, 1H), 4.99 (brs, 2H), 4.68 - 4.58 (m, 2H), 4.56 - 4.41 (m, 3H), 4.32 - 4.06 (m, 6H), 3.08 - 2.87 (m, 2H), 2.29 (brd, J = 10.4 Hz, 1H), 2.02 (brd, J = 8.8 Hz, 2H), 1.85 (brd, 7 = 11.2 Hz, 1H), 1.73 - 1.53 (m, 6H), 1.50 - 1.33 (m, 7H), 1.31 - 1.21 (m, 1H), 1.05 - 0.96 (m, 1H), 0.92 - 0.85 (m, 1H), 0.77 (s, 3H). ESI MS: 918.3 ([M+HD-

[0653] 4-((S)-2-Amino-5-ureidopentanamido)benzyl ((2-((8S,9S,101?,llS,13S,14S,177?)-11,17- dihydroxy-10, 13-dimethyl-3-oxo-6, 7, 8, 9, 10, 11,12, 13,14, 15,16, 17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)methyl)carbamate (77).

[0654] Compound 76 (800 mg, 0.871 mmol, 1 equiv.) was dissolved in anhydrous DMF (10 mL), N- ethylethanamine (3.79 g, 5.33 mL, 51.8 mmol, 59.4 equiv.) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was filtered and the filtrate waspurified by preparative HPLC: (Phenomenex Gemini Cl 8(150 x 25 mm, 10 m); flow rate: 25 mL / min; gradient: 0% - 46% B over 30 min; mobile phase A: 0.1% aqueous ammonia hydroxide, mobile phase B: acetonitrile). The fractions with product were lyophilized to afford the compound 78 (400 mg) in 66% yield as a colorless solid.1H NMR (400 MHz, DMSO-d6) = δ 8.14 (brt, J= 6.4 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.42 - 7.25 (m, 3H), 6.20- 6.11 (m, 1H), 6.00 - 5.88 (m, 2H), 5.36 (s, 1H), 5.23 (s, 1H), 4.99 (s, 1H), 4.68 - 4.56 (m, 2H), 4.55 - 4.44 (m, 2H), 4.28 (brs, 1H), 4.21 - 4.02 (m, 3H), 3.28 (brs, 1H), 3.17 (d, J = 4.4 Hz, 4H), 3.04 - 2.90 (m, 2H), 2.34 - 2.25 (m, 1H), 2.02 (brd, J = 9.6 Hz, 3H), 1.85 (brd, J = 12.8 Hz, 1H),1.72 - 1.50 (m, 5H), 1.47 - 1.34 (m, 7H), 1.31 - 1.20 (m, 1H), 1.06 - 0.95 (m, 1H), 0.88 (brd, J = 10.8 Hz, 1H), 0.77 (s, 3H). ESI MS: 696.3 ([M+H]+).

[0655] [4-[[(2S)-2-[[(2S)-2-(9H-Fluoren-9-ylmethoxycarbonylamino)-3-methyl- butanoyl]amino]-5-ureido-pentanoyl]amino]phenyl]methyl N -[[2-[(8S,9S,10R ,11S,13S,14S,17R )-ll,17-dihydroxy-10, 13-dimethyl-3-oxo-7,8,9,ll,12,14,15,16- octahydro-6#-cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methyl]carbamate (78).

[0656] Compound 77 (400 mg, 0.575 mmol, 1 equiv.) and (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3- methyl-butanoic acid (215 mg, 0.632 mmol, 1.1 equiv.) were dissolved in anhydrous DMF (5 mL) and HATU (328 mg, 0.862 mmol, 1.5 equiv.) followed by DIPEA(223 mg, 300 pL, 1.72 mmol, 3 equiv.) were added and the mixture was stirred at room temperature for 1 h. The precipitate was filtered off and the filtrate was purified by preparative HPLC: (Phenomenex Gemini C18 (150 x 25 mm, 10 μm); flow rate: 75 mL / min; gradient: 0% - 60% B over 20 min; mobile phase A: 0.1% aqueous ammonium hydrogencarbonate, mobile phase B: MeCN). The fractions with product were lyophilized to afford compound 78 (350 mg, 0.344 mmol, 60% yield) as a colorless solid.1H NMR (400 MHz, DMSO- d6) δ 10.12 - 9.90 (m, 1H), 8.35 (brd, 7 = 7.6 Hz, 1H), 8.19 - 8.07 (m, 1H), 7.96 -7.84 (m, 2H), 7.79 - 7.67 (m, 2H), 7.63 - 7.51 (m, 2H), 7.45 - 7.22 (m, 8H), 6.25 - 6.13 (m, 1H), 6.03 - 5.88 (m, 2H), 5.47 - 5.36 (m, 2H), 5.23 (s, 1H), 4.98 (brd, 7 = 2.0 Hz, 2H), 4.86 - 4.68 (m, 1H), 4.65 - 4.38 (m, 5H), 4.34 - 4.17 (m, 5H), 4.02 - 3.91 (m, 1H), 3.04 - 2.88 (m, 2H), 2.29 (brd, 7 = 11.2 Hz, 1H), 2.05 - 1.94 (m, 3H), 1.85 (brd, 7 = 13.2 Hz, 1H), 1.76 - 1.52 (m, 6H), 1.50 - 1.20 (m, 7H), 1.06 - 0.97 (m, 1H), 0.93 - 0.69 (m, 11H). ESI MS: 1017.4 ([M+H]+).

[0657] [4-[[(2S)-2-[[(2S)-2-Amino-3-methyl-butanoyl]amino] -5-ureido- pentanoyl]amino]phenyl]methyl N-[[2-[(8S,9S,10R ,llS,13S,14S,17 / ?)-ll,17-dihydroxy -10,13- dimethyl-3-oxo-7,8,9,l 1,12, 14,15, 16-octahydro-6H-cyclopenta[a]phenanthren-17-yl]-2-oxo- ethoxy]methyl]carbamate (79).

[0658] Compound 78 (350 mg, 0.344 mmol, 1 equiv.) was dissolved in anhydrous DMF (3 mL), N-ethylethanamine (426 mg, 0.60 mL, 5.82 mmol, 16.9 equiv.) was added and the mixture was stirred at room temperature for 1 h. The precipitate was filtered offand the filtrate was purified by preparative HPLC (Phenomenex Gemini C 18 (150 x 25 mm, 10 μm); flow rate: 25 mL / min; gradient: 0% - 50% B over 10 min; mobile phase A: 0.1% NH4HCO3in H2O, mobile phase B: MeCN). The obtained fractions with product were lyophilized to afford the compound 79 (210 mg) in 77% yield as a colorless solid.1H NMR (400 MHz, DMSO-d6) 8 = 10.13 (s, 1H), 8.21 - 8.07 (m, 2H), 7.59 (d, J= 8.8 Hz, 2H), 7.31 (t, J= 9.6 Hz, 3H), 6.22 - 6.12 (m, 1H), 5.97 (brt, J= 6.0 Hz, 1H), 5.91 (s, 1H), 5.41 (s, 2H), 5.24 (s, 1H), 4.99 (s, 2H), 4.64 — 4.58 (m, 2H), 4.56 - 4.43 (m, 4H), 4.28 (brs, 1H), 4.15 (d, J = 18.4 Hz, 1H), 3.07 - 2.98 (m, 2H), 2.97 - 2.88 (m, 1H), 2.35 - 2.25 (m, 1H), 2.03 (brd, 7= 8.8 Hz, 2H), 1.97 - 1.83 (m, 3H), 1.71 - 1.54 (m, 6H), 1.48 - 1.33 (m, 7H), 1.32 - 1.23 (m, 1H), 1.06 - 0.97 (m, 1H), 0.88 (d, J= 6.8 Hz, 4H), 0.80 - 0.76 (m, 6H). ESI MS: 795.4 ([M+H]+).

[0659] (25)-2-[[(lS)-l-Carboxy-5-[[8-[[(lS)-l-[[(lS)-l-[[4-[[2-[(8S,9S, 10R, 11S,13S,14S,17R)- 11, 17-dihydroxy-10, 13-dimethyl-3-oxo-7, 8,9,11,12, 14,15, 16-octahydro-6H- cyclopenta[a]phenanthren-17-yl]-2-oxo-ethoxy]methylcarbamoyloxymethyl] phenyl]carbamoyI]-4-ureido-butyl]carbamoyI]-2-methyl-propyl]amino]-8-oxo- octanoyl]amino]pentyl]carbamoylamino]pentanedioic acid (C14).mg, 0.793 mmol, 1.38 mL, 3 equiv.) were added and the resulting mixture was stirred at room temperature for 1 h. The precipitate was removed by filtration and the residue was purified by preparative HPLC (column: CD45-Waters Xbridge BEH C18 150*40 mm*10 μm; mobile phase: [H2O (lOmM NH4HCCh)-ACN]; gradient: 2%-32% B over 15.0 min). The obtained fractions with product were lyophilized to afford product C14 (129 mg) in 39% yield as a colorless solid.1H NMR (400 MHz, DMSO-A) = δ 10.19 - 9.93 (m, 1H), 8.64 - 8.32 (m, 1H), 8.27 - 8.07 (m, 2H), 7.78 - 7.58 (m, 3H), 7.39 - 7.24 (m, 3H), 6.44 - 6.35 (m, 1H), 6.27 - 6.07 (m, 3H), 5.91 (s, 1H), 5.46 (brd, J = 9.6 Hz, 2H), 5.03 - 4.95 (m, 2H), 4.61 - 4.46 (m, 4H), 4.28 (brs, 4H), 4.19 - 4.10 (m, 3H), 4.00 - 3.90 (m, 4H), 3.01 - 2.95 (m, 4H), 2.27 (brs, 2H), 2.17 - 2.12 (m, 2H), 2.00 (brd, J= 5.2 Hz, 4H),1.89 - 1.79 (m, 2H), 1.64 - 1.57 (m, 4H), 1.49 - 1.33 (m, 12H), 1.20 (brs, 6H), 1.05 (t, 7 = 7.2 Hz,7H), 0.89 - 0.81 (m, 6H), 0.77 (s, 3H). ESI MS: 1252.5 ([M+H]+).arbonyl)glycyl-D-proline (81).

[0663] D-proline (80) (1.00 g, 8.69 mmol, 1 equiv.) and 2,5- dioxopyrrolidin-l-yl (tert-butoxycarbonyl)glycinate (2.36 g, 8.69 mmol, 1equiv.) were dissolved in anhydrous DCM (40 mL), DIPEA (1.68 g, 2.27 mL, 13.0 mmol, 1.5 equiv.) was added and the mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by prep- HPLC (Phenomenex luna C18 150 x 25 mm x 10 μm); flow rate: 80 mL / min; gradient: 15% - 35%B over 10 min; mobile phase A: 0.1 % aq. formic acid, mobile phase B: MeCN) and lyophilized to provide product 81 (1.20 g) as a light yellow solid in 51% yield. ESI MS: 295.3 ([M+Na]+).

[0664] 2-((85,95,l(M?,115,135,145,17R)-ll,17-Dihydroxy-10,13-diiriethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3Z / -cyclopenta[a]phenanthren-17-yl)-2-oxoethyl(tert-butoxycarbonyl)glycyl-D-prolinate (82).

[0665] Prednisolone (1.00 g, 2.77 mmol, 1 equiv.), compound 81 (755 mg, 2.77 mmol, 1 equiv.) and DMAP (102 mg, 0.832 mmol, 0.3 equiv.) were dissolved in the mixture of solventsDCM (10 mL) and DMF (10 mL) and finally EDCI (638 mg, 3.33 mmol, 1.2 equiv.) was added.The resulting mixture was stirred at room temperature for 12 h. The mixture was diluted with additional DCM (10 mL) and washed with dist. water (10 mL). The aqueous phase was extracted with DCM (2x10 mL). The combined organic fractions were washed with brine (2x20 mL), dried over anhydrous sodium sulfate, filtered and solvents removed under vacuum. The residue was purified by LC on silica (hexanes / EtOAc, 1:0 to 0:1) to give desired compound 82 (900 mg) as a light-yellow solid in 53% yield. ESI MS: 615.3 ([M+H]+).

[0666] 2-((8S, 95,101?, 115,135, 145,17R)-ll,17-Dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11, 12, 13, 14, 15, 16,17-dodecahydro-3 / / -cyclopenta|u ]phenanthren-17-yl)-2-oxoethyl glycyl-D-prolinate (83).

[0667] Compound 82 (900 mg, 1.46 mmol, 1 equiv.) was dissolved in anh. DCM (12 mL), TFA (6.14 g, 4.0 mL, 53.8 mmol, 37 equiv.) was added and the mixture was stirred at roomtemperature for 12 h. The volatiles were concentrated under reduced pressure to give the desired product 83 (1.00 g, TFA salt) as a yellow oil. Compound was used to the next step without any purification. ESI MS: 515.3 ([M+H]+).[00668J Tri- rt-butyl (65,95,245,285)-l-amino-6-((4-((((2-((R)-2-((2-((85,95,101?,115,135,145,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11, 12, 13, 14, 15, 16,17-dodecahydro-3 / 7-cyclopenta[a ]phenanthren-17-yl)-2- oxoethoxy)carbonyl)pyrrolidin-l-yl)-2-oxoethyl)carbamoyl)oxy)methyl)phenyl) carbamoyl)-9-isopropyl-l, 8, 11,18, 26-pentaoxo-2, 7, 10, 19,25, 27-hexaazatriacontane-24, 28,30- tricarboxylate (84).mL), DIPEA (308 mg, 416 pL, 2.39 mmol, 5 equiv.) was added and the mixture was stirred at room temperature for 2 h. The precipitates were removed by filtration, solvent were evaporated under reduced pressure and the residue was purified by prep. HPLC (Phenomenex luna C18 150 x 25 mm x 10 μm); flow rate: 60 mL / min; gradient: 45% - 65% B over 10 min; mobile phase A: 0.1% aq. formic acid, mobile phase B: MeCN), fractions with product lyophilized to obtain compound 84 (550 mg) as an off-white solid in 56% yield. ESI MS: 1545.8 ([M+H]+).

[0670] (65, 95,245,285)-l-Amino-6-((4-((((2-((R)-2-((2-((85, 95, 10 / L 115,135, 145,17^) 11, 17- dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3Z / - cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)pyrrolidin-l-yl)-2- oxoethyl)carbamoyI)oxy)methyI)phenyl)carbamoyI)-9-isopropyl-l,8,ll,18,26-pentaoxo- 2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylic acid (C15).stirred at room temperature for 2 h. The reaction mixture was neutralized with aqueous ammonia to pH = 7~8. The precipitates were removed by filtration and solvents concentrated under vacuum. The residue was purified by prep. HPLC (CD05-Phenomenex Luna C18 100 x 40 mm x 10 μm); flow rate: 75 mL / min; gradient: 20% - 50% B over 13 min; mobile phase A: 0.225% aq. formic acid, mobile phase B: MeCN) and lyophilized to afforded product C15 (148 mg) as a colorless solid in 26% yield. ’H NMR (400 MHz, DMSO-d6) = 12.6 δ6 - 12.22 (m, 2H), 9.98 (s, 1H), 8.09 (brd, J = 7.2 Hz, 1H), 7.80 (brd, J = 8.4 Hz, 1H), 7.71 (brt, J = 5.6 Hz, 1H), 7.59 (brd, J = 8.4 Hz, 2H), 7.36 - 7.22 (m, 4H), 6.30 (brt, J - 8.8 Hz, 2H), 6.16 (dd, Ji - 1.6, J2=10.0 Hz, 1H), 5.98 (brt, J = 5.6 Hz, 1H), 5.91 (s, 1H), 5.42 (brs, 3H), 5.23 - 5.13 (m, 1H), 4.99 - 4.92 (m, 2H), 4.74 - 4.65 (m, 2H), 4.47 - 4.33 (m, 2H), 4.29 (brs, 1H), 4.19 (brt, J = 7.6 Hz, 1H), 4.13 - 4.00 (m, 2H), 3.99 - 3.73 (m,2H), 3.60 - 3.47 (m, 2H), 3.42 (brd, J = 8.0 Hz, 1H), 3.05 - 2.91 (m, 4H), 2.35 - 2.07 (m, 8H), 2.05 - 1.85 (m, 9H), 1.74 - 1.57 (m, 7H), 1.53 - 1.32 (m, 14H), 1.31 - 1.17 (m, 7H), 0.91 - 0.77 (m, 10H). ESI MS: 1377.8 ([M+H]+).

[0673] (terMJutoxycarbonyl)-L-prolyl-D-proline (85).

[0674] D-proline (80) (1.00 g, 8.69 mmol, 1 equiv.) and 1 -(tert-butyl) 2-(2,5- dioxopyrrolidin-l-yl) (S)-pyrrolidine-l,2-dicarboxylate (2.71 g, 8.69 mmol, 1equiv.) were suspended in anh. DCM (50 mL), DIPEA (1.68 g, 13.0 mmol,2.27 mL, 1.5 eq) was added and the resulting mixture was stirred at room temperature for 2 h. The reaction mixture concentrated under reduced pressure and the residue was purified by prep HPLC (Phenomenexluna C18 150 x 25 mm x 10 μm); flow rate: 80 mL / min; gradient: 15% - 35% B over10 min; mobile phase A: 0.1% aq. formic acid, mobile phase B: MeCN) to provided product 85 (1.50 g) as a colorless solid in 55% yield. ESI MS: 335.0 ([M+Na]+).

[0675] tert -Butyl (5)-2-((R)-2-((2-((85,95, 102?, 115, 135,145,17R) -ll,17-dihydroxy-10,13- dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cydopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)pyrrolidine-l-carbonyl)pyrrolidine-l-carboxylate (86).DCM (10 mL) and DMF (10 mL) and finally EDCI (638 mg,3.33 mmol, 1.2 equiv.) was added. The mixture was stirred at room temperature for 12 h. DCM (10 mL) was added, and the organic phase was washed with dist. water (10 mL). The aqueous phase was extracted with DCM (2x10 mL), the combined organic fractions were washed with brine (2x20 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. Theresidue was purified by LC on silica (hexanes / EtOAc 1 :0 to 0: 1 ) to give a desired product 86 (900 mg) as a light-yellow solid in 50% yield. ESI MS: 677.6 ([M+Na]+).

[0677] 2-((8S,9S, 101?, llS,13S,14S,17R)-ll,17-dihydroxy-10, 13- dimethyl-3-oxo-6.7.8.9.10.11.12,13.14.15, 16.17-dodecahydro-3Zl-cyclopenta|r / |phenanthren-17-yl)-2-oxoethylL-prolyl-D-prolinate (87).temperature for 2 h. The volatiles were concentrated under reduced pressure to give the desired product 87 (1.00 g, TFA salt) as a yellow oil and compound was used to the following step without any purification. ESI MS: 555.4 ([M+H]+).

[0679] Tri-tert-butyl (6S,9S,24S,28S)-l-amino-6-((4-((((S)-2-((R)-2-((2-((8S,9S,101?,llS,13S,14S,17R)-ll,17-dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,11, 12, 13, 14, 15, 16,17-dodecahydro-3 / / -cyclopenta|u ]phenanthren-17-yl)-2- oxoethoxy)carbonyl)pyrrolidine-l-carbonyl)pyrrolidine-l- carbonyl)oxy)methyl)phenyl)carbamoyl)-9-isopropyl-l, 8, 11,18, 26-pentaoxo-2, 7,10,19,25,27- hexaazatriacontane-24,28,30-tricarboxylate (88).(20 mL), DIPEA (386 mg, 521 pL, 2.99 mmol, 5 equiv.) was added and the mixture was stirred at room temperature for 1 h. The precipitates were removed by filtration and volatiles were removed by vacuum. The residue was purified by prep. HPLC: (Phenomenex luna C18 150 x 25 mm x 10 μm); flow rate: 60 mL / min; gradient: 45% - 65% B over 10 min; mobile phase A: 0.1% aq. formic acid, mobile phase B: MeCN) and lyophilized to afforded product 88 (550 mg) as an off-white solid in 58% yield. ESI MS: 793.9 ([M+H]+ / 2).

[0681] (6S,9S,24S,28S)-l-Amino-6-((4-((((S)-2-((R)-2-((2-((8S,9S,101?,llS,13S,14S,17R)-ll,17- dihydroxy-10,13-dimethyl-3-oxo-6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)carbonyl)pyrrolidine-l-carbonyl) pyrrolidine-Lcarbony l)oxy)methyl) phony l)carbamoy I )-9-isop ropy 1- 1.8.11.18.26- pent aoxo- 2,7,10,19,25,27-hexaazatriacontane-24,28,30-tricarboxylic acid (C16).resulting mixture was stirred at room temperature for 2 h. The reaction mixture was neutralized with aqueous ammonia to pH = 7~8, precipitates were removed by filtration and volatiles were removed under reduced pressure. The residue was purified by prep. HPLC: (CD05-Phenomenex Luna C18 100 x 40 mm x 10 μm); flow rate: 75 mL / min; gradient: 20% - 50% B over 13 min; mobile phase A: 0.225% aqueous formic acid, mobile phase B: MeCN) and lyophilized to afforded product C16 (150 mg) as a colorless solid in 24% yield. "H NMR (400 MHz, DMSO-d6) 12.88 - 12.04 δ (m, 2H), 10.06 - 9.91 (m, 1H), 8.17 - 8.05 (m, 1H), 7.81 (brd, J = 8.4 Hz, 1H), 7.71 (brt, J = 5.6 Hz, 1H), 7.63 - 7.52 (m, 2H), 7.36 - 7.21 (m, 3H), 6.30 (brt, J = 8.0 Hz, 2H), 6.16 (brd, J = 10.0 Hz, 1H), 5.99 (brs, 1H), 5.91 (s, 1H), 5.48 - 5.34 (m, 3H), 5.19 - 4.79 (m, 4H), 4.76 - 4.64 (m, 2H), 4.44 - 4.14 (m, 5H), 4.12 - 3.98 (m, 2H), 3.77 - 3.53 (m, 1H), 3.48 - 3.37 (m, 4H), 3.24 - 3.15 (m, 1H), 3.05 - 2.92 (m, 4H), 2.33 - 2.11 (m, 7H), 2.06 - 1.85 (m, 10H), 1.78 - 1.55 (m, 10H), 1.50 - 1.32 (m, 13H), 1.30 - 1.16 (m, 7H), 1.06 - 0.96 (m, 1H), 0.90 - 0.74 (m, 10H). ESI MS: 1417.9 ([M+H]+).REFERENCES

[0683] All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0684] Choy, C.J., et al., (177)Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice. Theranostics, 2017. 7(7): p. 1928-1939.

[0685] Hollinger, K.R., et al., Dendrimer-2PMPA selectively blocks upregulated microglial GCPII activity and improves cognition in a mouse model of multiple sclerosis. Nanotheranostics, 2022. 6(2): p. 126-142.

[0686] Su, Y., et al., GCPII Inhibition Promotes Remyelination after Peripheral Nerve Injury in Aged Mice. Int J Mol Sci, 2024. 25(13).

[0687] Rais, R.; Jiang, W.; Zhai, H.; Wozniak, K. M.; Stathis, M.; Hollinger, K. R.; Thomas, A. G.; Rojas, C.; Vornov, J. J.; Marohn, M.; Li, X.; Slusher, B. S., FOLH1 / GCPII is elevated in IBD patients, and its inhibition ameliorates murine IBD abnormalities. JCI Insight 2016, 1 (12).

[0688] Colombel, J.F.; Sandborn, W.J.; Rutgeerts, P.; Enns, R.; Hanauer, S.B.; Panaccione, R.; Schreiber, S.; Byczkowski, D.; Li, J.; Kent, J.D.; Pollack, P.F., Adalimumab for maintenance of clinical response and remission in patients with Crohn’s disease: the CHARM trial. Gastroenterology 2007,132:52-65.

[0689] Hamilton, M.J.; Snapper, S.B.; Blumberg, R.S., Update on biologic pathways in inflammatory bowel disease and their therapeutic relevance. J. Gastroenterol. 2012, 47: 1-8.

[0690] Hanauer, S.B.; Feagan, B.G.; Lichtenstein, G.R.; Mayer, L.F.; Schreiber, S.; Colombel, J.F.; Rachmilewitz, D.; Wolf, D.C.; Olson, A.; Bao, W.; Rutgeerts, P., Maintenance infliximab for Crohn’s disease: the ACCENT I randomised trial. Lancet 2002, 359: 1541-1549.

[0691] Kaser, A.; Zeissig, S.; Blumberg, R.S., Inflammatory bowel disease. Annu. Rev. Immunol. 2010, 28:573-621.

[0692] Kozuch, P.L. and Hanauer, S.B., Treatment of inflammatory bowel disease: A review of medical therapy. World J. Gastroenterol. 2008, 14:354-377.

[0693] Lawrance, I.C. What is left when anti-tumour necrosis factor therapy in inflammatory bowel diseases fails? World J. Gastroenterol. 2014, 20: 1248-1258.

[0694] Regueiro, M.; Siemanowski, B.; Kip, K.E.; Plevy, S., Infliximab dose intensification in Crohn’s disease. Inflamm. Bowel Dis. 2007, 13: 1093-1099.

[0695] Sartor, R.B., Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat. Clin. Pract. Gastroenterol. Hepatol. 2006, 3:390-407.

[0696] Schmidt, C; Giese, T.; Hermann, E.; Zeuzem, S.; Meuer, S.C.; Stallmach, A., Predictive value of mucosal TNF-alpha transcripts in steroid-refractory Crohn’s disease patients receiving intensive immunosuppressive therapy. Inflamm. Bowel Dis. 2007, 13:65-70.

[0697] Schreiber, S.; Khaliq-Kareemi, M.; Lawrance, I.C.; Thomsen, O.O. ; Hanauer, S.B.; McColm, J.; Bloomfield, R.; Sandborn, W.J., Maintenance therapy with certolizumab pegol for Crohn’s disease. N. Engl. J. Med. 2007, 357:239-250.

[0698] Strober, W.; Fuss, I.; and Mannon, P., The fundamental basis of inflammatory bowel disease. J. Clin. Invest. 2007, 1 17:514-521.

[0699] Xavier, R.J. and Podolsky, D.K., Unravelling the pathogenesis of inflammatory bowel disease. Nature 2007, 448:427-434.

[0700] Van, A.G.; Van, R.M.; Sciot, R.; Dubois, B.; Vermeire, S.; Noman, M.; Verbeeck, J.; Geboes, K.; Robberecht, W.; Rutgeerts, P., Progressive multifocal leukoencephalopathy after natalizumab therapy for Crohn’s disease. N. Engl. J. Med. 2005, 353 :362-368.

[0701] International PCT Patent Application Publication No. W02016022809 for Methods for Treating Inflammatory Bowel Disease using Prostate Specific Membrane Antigen (PSMA) Inhibitors, to Slusher et al., published February 11, 2016.

[0702] International PCT Patent Application Publication No. WO2021155167 for Bile Acid- GCPII Inhibitor Conjugates to Treat Inflammatory Diseases, to Slusher et al., published August 5, 2021.

[0703] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

1. THAT WHICH IS CLAIMED:

1. A compound of formula (I):C-L-P (I); wherein:C is a corticosteroid;L is a linker; andP is a Glutamate Carboxy peptidase II (GCPII) / pro state- specific membrane antigen (PSMA) binding moiety; and stereoisomers and pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein the compound of formula (I) is:wherein:— - is a bond that can be present or absent; R1and R2 are each independently H or -L-P, provided that one of R1and R2 is -L-P and the other is H; R3is H or C1-C4alkyl; or R2 and R3can combine to form:wherein n is an integer selected from 0, 1, 2, 3, and 4; and R4 is -C1-C4alkyl; R5is -OH or =0;Re is H or C1-C4alkyl;R7 is H or halogen; and stereoisomers and pharmaceutically acceptable salts thereof.

3. The compound of claim 1 or claim 2, wherein the GCPII / PSMA binding moiety, P, is selected from:wherein:Z is tetrazole or CO2Q;Q is H or a protecting group; m is an integer selected from the group consisting of 1, 2, 3, 4, and 5; and R8is selected from H or -CH2-R9, wherein R9is selected from substituted aryl, substituted pyridine, and unsubstituted isoquinoline; and stereoisomers thereof.

4. The compound of claim 3, wherein R9is selected from:wherein X is a halogen or a radiohalogen.

5. The compound of claim 4, wherein X is selected from Br, I, At, and radioisotopes thereof.

6. The compound of any one of claims 1 to 5, wherein the corticosteroid, C, is selected from:

7. The compound of any one of claims 1 to 6, wherein the linker, L, is:wherein: y is an integer selected from 1, 2, 3, 4, 5, 6, 7, and 8; x is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, and 10;RXcan be the same or different and are each independently an amino acid, wherein the amino acid includes both R- or S- isomers; andR26, R27, R28, R29, R30, R31, and R32are each independently H or C1-C4alkyl, orR29 and R30 together can form a 4- to 7-member nitrogen-containing heterocyclic ring with the nitrogen attached to R29; andstereoisomers thereof.

8. The compound of any one of claims 1 to 7, wherein the linker, L, is selected from:B is selected from -N R24-(CH2)y-C(=O)-, -NR24-(CH2)y-C(=O)-NR25-(CH2)y-, and-NR33-CH(R34)-C(=O)-NR35-(CH2)y-C(=O)-NR36-(CH2)y-; each R10, R13, R14, R15, R16, R17, R21, R22, R23, , R25, R34, R37, and R38arc independently H or C1-C4alkyl; each R11, R12, R18, R19, and R34are independently C1-C4alkyl; andR20is C1-C4alkyl or -(CH2)yN(CH3)2; and stereoisomers thereof.

9. The compound of any one of claims 1 to 8, wherein the linker, L, is selected from:

10. The compound of any one of claims 1 to 9, wherein the compound of formula (I) is selected from:and stereoisomers thereof.

11. The compound of any one of claims 1 to 9, wherein the compound of formula (I) is selected from:and stereoisomers thereof.

12. The compound of any one of claims 1 to 9, wherein the compound of formula (I) is selected from:and stereoisomers thereof.

13. The compound of any one of claims 1 to 9, wherein the compound of formula (I)and stereoisomers thereof.

14. A formulation comprising a compound of any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

15. A method for treating an inflammatory bowel disease, the method comprising administering to a subject in need of treatment thereof, a compound of formula (I) of any one of claims 1 to 13 or a formulation of claim 14.

16. The method of claim 15, wherein the inflammatory bowel disease is selected from Crohn’s disease (CD), ulcerative colitis (UC), indeterminate colitis inflammatory bowel disease, and combinations thereof.

17. The method of claim 15 or claim 16, wherein administering the compound of formula (I) decreases GCPII activity in the subject compared to an untreated control subject or a subject without an IBD.

18. The method of any one of claims 15 to 17, wherein administering the compound of formula (I) reduces or alleviates one or more symptoms associated with IBD, including abdominal pain, diarrhea, fecal incontinence and / or urgency to have a bowel movement, rectal bleeding, swelling or masses due to inflammation in the intestines, weight loss, fever, anemia, malnutrition, delayed growth, anxiety, depression.

19. The method of any one of claims 15 to 18, further comprising administering one or more additional therapeutic agents in combination with the compound of formula (I).

20. The method of claim 19, wherein the one or more additional therapeutic agents is selected from an immunosuppressant drug, a biologic, an anti-inflammatory agent, an orally available agents for treating IBD, a tumor necrosis factor (TNF)-alpha inhibitor, an antibiotic, an anti-diarrheal medication, a fiber supplement, a pain reliever, a vitamin, and a mineral supplement.

21. The method of any one of claims 15 to 20, further comprising one or more of stem cell therapy, surgery, and fecal transplant.

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