RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/792,231, filed on January 14, 2019, which is incorporated herein by reference in its entirety.

FIELD

Disclosed herein are compounds and methods for treatment of cytokine release syndrome and/or a cytokine release syndrome-induced neurotoxicity.

BACKGROUND

Immunotherapy of cancer has recently made great strides with therapies reported for a number of different cancers resulting in new FDA-approved drugs. The general concept behind many of these advances is to augment and activate the patient’s own immune system to combat the patient’s cancer. Examples include engineered cytotoxic T-cells that are directed to the tumor (CAR T-cells) as well as bispecific antibodies that bind both T-cells and tumor cells together to enhance T -cell killing of the tumor cells. These therapies also rely on the activation of the patient’s own immune system to aid in the killing of the tumor cells.

One of the major side effects of these therapies, however, is the release of cytokines from this activation that can result in serious and life-threatening adverse effects known as cytokine release syndrome (CRS) (1-8). This syndrome is being recognized as a major complication currently in immunotherapy with some clinical trials reporting an occurrence of 100% (9).

CRS may be caused by many different stimuli, not only from drugs, but also from bacterial and viral infections. It has also been described as a“cytokine storm” that arises from massive overproduction of cytokines not only from T-cell stimulation but also from stimulation of bystander immune cells such as monocytes and macrophages (10,1 1) as well as non-immune cells such as endothelial cells. In fact, a hallmark of the severe form of CRS has been described as the activation of endothelial cells (1). Cytokines that may be elevated in CRS include: IL-6, TNFa, IFNg, IL-8, IL-10, MCP-1, MIR-Ib, and GM-CSF (12,13). The result of CRS is a massive systemic inflammatory response that can extend into the central nervous system resulting in fatal neurotoxicity (14). Potential treatments for CRS are currently being pursued. For example, the FDA recently approved the use of an antibody directed to IL-6, known as Tocilizumab, for the treatment of CAR-T cell-induced severe or life-threatening CRS (15). While IL-6 is a major cytokine that is elevated, it is only one of many deleterious cytokines expressed during CRS. There is need for a treatment that can inhibit the expression of multiple cytokines and the severe inflammatory response expressed during CRS.

Additionally, a serious complication of CRS is the progression to neurotoxicity, which may lead to mental impairment and death. CRS-induced neurotoxicity is associated with a breakdown of the blood brain barrier through endothelial activation resulting in CAR-T cells and high levels of cytokines entering the brain.

In patients being treated for CRS-induced neurotoxicity, the anti-IL-6 antibody, Tocilizumab, has been shown to have little effect. This suggests that once established, neurotoxicity is less responsive to treatment than suppression of IL-6 or CAR-T cell function (14). In severe CRS leading to neurotoxicity, endothelial activation is elevated as determined by biomarkers Ang2 and von Willebrand Factor (VWF) (14,17,18). Figures 42A-D.

In addition to VWF and Ang2, E-selectin is also a biomarker of endothelial activation (19).

Disclosed herein is the use of one or more antagonists, such as selectin antagonists, for use in treating and/or preventing CRS as well as associated neurotoxicities. The selectin antagonists may be heterobifunctional antagonists that inhibit both a selectin (one or more of E-, L-, and P-selectin) and, for example, CXCR4 chemokine receptors or Galectin-3. The selectin antagonists may be multimeric antagonists that inhibit a selectin (one or more of E-, L-, and P-selectin) and optionally, for example, CXCR4 chemokine receptors and/or Galectin-3.

SUMMARY

In some embodiments, the present disclosure provides methods for treating and/or preventing cytokine release syndrome and/or a cytokine release syndrome-induced neurotoxicity comprising administering to a subject in need thereof an effective amount of at least one antagonist chosen from selectin antagonists and galectin antagonists. In some embodiments, the at least one antagonist is chosen from selectin antagonists. In some embodiments, the selectin antagonist is chosen from E-selectin antagonists. In some embodiments, the at least one antagonist is chosen from galectin antagonists. In some embodiments, the galectin antagonist is chosen from galectin-3 antagonists and galectin-9 antagonists. In some embodiments, the at least one antagonist is a small molecule, nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, glycomimetic, lipid, antibody, or an aptamer. In various embodiments, the selectin antagonist binds at or near the binding site on E-selectin to inhibit E-selectin interaction with sialyl Le ^a and/or sialyl Le ^x .

Also disclosed herein are methods of reducing and/or eliminating cytokine expression comprising administering to a subject in need thereof an effective amount of at least one antagonist chosen from selectin antagonists and galectin antagonists. In some embodiments, the at least one antagonist is chosen from selectin antagonists. In some embodiments, the selectin antagonist is chosen from E-selectin antagonists. In some embodiments, the at least one antagonist is chosen from galectin antagonists. In some embodiments, the galectin antagonist is chosen from galectin-3 antagonists and galectin-9 antagonists. In some embodiments, the at least one antagonist is a small molecule, nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, glycomimetic, lipid, antibody, or an aptamer. In various embodiments, the selectin antagonist binds at or near the binding site on E-selectin to inhibit E-selectin interaction with sialyl Le ^a and/or sialyl Le ^x . In some embodiments, the cytokine is chosen from TNF a , IFN-b, IFN-g, IL-23, IL-Ib, IL-6, IL-8, IL-10, MCP-1, MIP- 1 b, and GM-CSF.

Also disclosed herein are methods of reducing and/or eliminating endothelial activation comprising administering to a subject in need thereof an effective amount of at least one antagonist chosen from selectin antagonists and galectin antagonists. In some embodiments, the at least one antagonist is chosen from selectin antagonists. In some embodiments, the selectin antagonist is chosen from E-selectin antagonists. In some embodiments, the at least one antagonist is chosen from galectin antagonists. In some embodiments, the galectin antagonist is chosen from galectin-3 antagonists and galectin-9 antagonists. In some embodiments, the at least one antagonist is a small molecule, nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, glycomimetic, lipid, antibody, or an aptamer. In various embodiments, the selectin antagonist binds at or near the binding site on E-selectin to inhibit E-selectin interaction with sialyl Le ^a and/or sialyl Le ^x . In some embodiments, the subject has Acute Myeloid Leukemia (AML). In some embodiments, the method further comprises administering to the subject at least one cancer therapy. In some embodiments, the at least one cancer therapy is chemotherapy. In some embodiments, the at least one cancer therapy is administered simultaneously with the administration of the at least one antagonist. In some embodiments, the at least one cancer therapy is administered before the at least one antagonist. In some embodiments, the at least one cancer therapy is administered after the at least one antagonist.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagram illustrating the synthesis of compound A14.

Fig. 2 is a diagram illustrating the synthesis of compound A37.

Fig. 3 is a diagram illustrating the synthesis of compound A44.

Fig. 4 is a diagram illustrating the synthesis of compound A49.

Fig. 5 is a diagram illustrating the synthesis of compound A51.

Fig. 6is a diagram illustrating the synthesis of compound A87.

Fig. 7 is a diagram illustrating the synthesis of compound A83.

Fig. 8 is a diagram illustrating the synthesis of compound A86.

Fig. 9 is a diagram illustrating the synthesis of compound 11.

Fig. 10 is a diagram illustrating the synthesis of compound 14.

Fig. 11 is a diagram illustrating the synthesis of compound 22.

Fig. 12 is a diagram illustrating the synthesis of compound 37.

Fig. 13 is a diagram illustrating the synthesis of compound 46.

Fig. 14 is a diagram illustrating the synthesis of compound 56.

Fig. 15 is a diagram illustrating the synthesis of compound 60. Fig. 16 is a diagram illustrating the synthesis of compound 65.

Fig. 17 is a diagram illustrating the synthesis of compound 68. Fig. 18 is a diagram illustrating the synthesis of compound 73. Fig. 19 is a diagram illustrating the synthesis of compound 78. Fig. 20 is a diagram illustrating the synthesis of compound 87. Fig. 21 is a diagram illustrating the synthesis of compound 95. Fig. 22 is a diagram illustrating the synthesis of compound 146. Fig. 23 is a diagram illustrating the synthesis of compound 197. Fig. 24 is a diagram illustrating the synthesis of compound 205. Fig. 25 is a diagram illustrating the synthesis of compound 206. Fig. 26 is a diagram illustrating the synthesis of compound 214. Fig. 27 is a diagram illustrating the synthesis of compound 220. Fig. 28 is a diagram illustrating the synthesis of compound 224. Fig. 29 is a diagram illustrating the synthesis of compound 237. Fig. 30 is a diagram illustrating the synthesis of compound 241. Fig. 31 is a diagram illustrating the synthesis of compound 245. Fig. 32 is a diagram illustrating the synthesis of compound 257. Fig. 33 is a diagram illustrating the synthesis of compound 263. Fig. 34 is a diagram illustrating the synthesis of compound 276. Fig. 35 is a diagram illustrating the synthesis of compound 291.

Fig. 36 is a diagram illustrating the synthesis of compound 295. Fig. 37 is a diagram illustrating the synthesis of compound 307.

Fig. 38 is a diagram illustrating the synthesis of compound 316.

Fig. 39 is a diagram illustrating the synthesis of compound 318.

Fig. 40 is a diagram illustrating the synthesis of compound 145.

Fig. 41 is a diagram illustrating the synthesis of compound 332.

Figs. 42A-D depict the change in biomarker levels of Ang2 and von Willebrand Factor (VWF) during endothelial activation. Fig. 42A shows the fold change over normal of VWF antigen in Grade 0, Grade 1-3, and Grade 4-5 CRS patients. Fig. 42B shows the Ang-2:Ang- 1 ratio during endothelial activation. Fig. 42C shows the Ang-2:Ang-l ratio during endothelial activation. Fig. 42D shows fold change over normal of VWF antigen during endothelial activation.

Fig. 43 depicts sE-Selectin levels (ng/mL) in plasma of AML patients treated with Compound A.

Figs. 44A-D depict the effects of administration of Compound A on cytokine expression in mice. Fig. 44A shows TNF-a (pg/mL bone marrow fluid) levels in control and G-CSF treated mice with and without Compound A administration. Fig. 44B shows IFN-b (pg/mL bone marrow fluid) levels in control and G-CSF treated mice with and without Compound A administration. Fig. 44C shows IL-23 (pg/mL bone marrow fluid) levels in control and G- CSF treated mice with and without Compound A administration. Fig. 44D shows IL-Ib (pg/mL bone marrow fluid) levels in control and G-CSF treated mice with and without Compound A administration.

DETAILED DESCRIPTION

Definitions

In order to better understand the disclosure, certain definitions are provided first.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All references cited herein are incorporated by reference in their entireties. To the extent terms or discussion in references conflict with this disclosure, the latter shall control.

As used herein, the singular forms of a word also include the plural form of the word, unless the context clearly dictates otherwise; as examples, the terms“a,”“an,” and“the” are understood to be singular or plural. By way of example,“an element” means one or more element. The term“or” shall mean“and/or” unless the specific context indicates otherwise.

The term“E-selectin ligand” as used herein, refers to a carbohydrate structure that contains the epitope shared by sialyl Le ^a and sialyl Le ^x . Carbohydrates are secondary gene products synthesized by enzymes known as glycosyltransferases which are the primary gene products coded for by DNA. Each glycosyltransferase adds a specific monosaccharide in a specific stereochemical linkage to a specific donor carbohydrate chain.

The term“treatment” as used herein, is defined as the application or administration of a therapeutic agent to a subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, one or more symptoms of the disease, or the predisposition toward the disease. Moreover, as long as the compositions of the disclosure either alone or in combination with another therapeutic agent cure, heal, alleviate, relive, alter, remedy, ameliorate, improve or affect at least one symptom of cytokine release syndrome (CRS) and/or a cytokine release syndrome-induced neurotoxicity, as compared to that symptom in the absence of treatment, the result should be considered a treatment of the underlying disorder regardless of whether all the symptoms of the disorder are cured, healed, alleviated, relieved, altered, remedied, ameliorated, improved or affected or not. Treatment may be achieved using an“effective amount” of a therapeutic agent, which shall be understood to embrace partial and complete treatment, e.g., partial or complete curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease, one or more symptoms of the disease, or the predisposition toward the disease. An“effective amount” of may be determined empirically. Likewise, a“therapeutically effective amount” is a concentration which is effective for achieving a stated therapeutic effect.

The term“pharmaceutically acceptable salts” includes sodium, potassium, lithium, ammonium (substituted and unsubstituted), calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts, as well as salts of amines. Pharmaceutically acceptable salts may, for example, be obtained using standard procedures well known in the field of pharmaceuticals.

The term“prodrug” as used herein, is defined to include a compound that when administered to a primate host generates an active compound as a result of a spontaneous reaction under physiological conditions, enzymatic catalysis, metabolic clearance, or combinations thereof.

The terms“antagonist” and“inhibitor” are used interchangeably herein.

The term“one or more” as used herein, is defined to mean“at least one.”

Selectin Antagonists

Disclosed herein are methods comprising the use of at least one antagonist chosen from selectin antagonists and galectin antagonists. In some embodiments, the at least one antagonist is chosen from selectin antagonists.

Selectins are a group of structurally similar cell surface receptors important for mediating leukocyte binding to endothelial cells. These proteins are type 1 membrane proteins and are composed of an amino terminal lectin domain, an epidermal growth factor (EGF)-like domain, a variable number of complement receptor related repeats, a hydrophobic domain spanning region and a cytoplasmic domain. The binding interactions appear to be mediated by contact of the lectin domain of the selectins and various carbohydrate ligands.

There are three known selectins: E-selectin, P-selectin, and L-selectin. E-selectin is found on the surface of activated endothelial cells and binds to the carbohydrate sialyl-Lewis ^x (SLe ^x ) which is presented as a glycoprotein or glycolipid on the surface of certain leukocytes (monocytes and neutrophils) and helps these cells adhere to capillary walls in areas where surrounding tissue is infected or damaged. E-selectin also binds to sialyl-Lewis ^a (SLe ^a ) which is expressed on many tumor cells. P-selectin is expressed on inflamed endothelium and platelets and also recognizes SLe ^x and SLe ^a but also contains a second site that interacts with sulfated tyrosine. The expression of E-selectin and P-selectin is generally increased when the tissue adjacent to a capillary is infected or damaged. L-selectin is expressed on leukocytes.

The selectin antagonists suitable for the disclosed methods include pan selectin antagonists. As disclosed herein, any method of inhibiting E-selectin may be used to treat and/or prevent CRS and/or CRS-related conditions. Inhibition can be by any means, for example, antibody, small molecule, biologic, inhibitors of gene expression, etc.

Non-limiting examples of suitable selectin antagonists include small molecules, such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules. Suitably, the selectin antagonist is selected from antigen-binding molecules that are immuno-interactive with a selectin, peptides that bind to the selectin and that block cell-cell adhesion, and carbohydrate or peptide mimetics of selectin ligands. In some embodiments, the selectin antagonist reduces the expression of a selectin gene or the level or functional activity of an expression product of that gene. For example, the selectin antagonist may antagonize the function of the selectin, including reducing or abrogating the activity of at least one of its ligand-binding sites.

In some embodiments, the antagonist is an E-selectin antagonist, which is an agent that inhibits an activity of E-selectin or inhibits the binding of E-selectin to one or more E-selectin ligands (which in turn may inhibit a biological activity of E-selectin). The term“E-selectin antagonist” includes antagonists of E-selectin only, as well as antagonists of E-selectin and either P-selectin or L-selectin, and antagonists of E-selectin, P-selectin, and L-selectin.

E-selectin antagonists include the glycomimetic compounds described herein. E-selectin antagonists also include antibodies, polypeptides, peptides, peptidomimetics, and aptamers which bind at or near the binding site on E-selectin to inhibit E-selectin interaction with sialyl Le ^a (sLe ^a ) or sialyl Le ^x (sLe ^x ).

In some embodiments, the selectin antagonist is an E-selectin antagonist. Further disclosure regarding E-selectin antagonists suitable for the disclosed methods and compounds may be found in U.S. Patent No. 9,254,322, issued Feb. 9, 2016; U.S. Patent No. 9,486,497, issued Nov. 8, 2016, which are hereby incorporated by reference in their entirety. In some embodiments, the selectin antagonist is chosen from E-selectin antagonists disclosed in U.S. Patent No. 9,109,002, issued Aug. 18, 2015, which is hereby incorporated by reference in its entirety. In some embodiments, the selectin antagonist is chosen from heterobifunctional antagonists disclosed in U.S. Patent No. 8,410,066, issued Apr. 2, 2013, and US Publication No. US2017/0305951, published Oct. 26, 2017, which are hereby incorporated by reference in their entirety. Further disclosure regarding E-selectin antagonists suitable for the disclosed methods and compounds may be found in PCT Publication No. WO2018/068010, published Apr. 12, 2018.

E-selectin Antagonists (Formula I) and Heterobifunctional E-selectin and CXCR4 Antagonists (Formula II)

In some embodiments, the at least one antagonist is chosen from E-selectin antagonists.

In some embodiments, the E-selectin antagonist is chosen from compounds of Formula (I):

isomers of Formula (I), tautomers of Formula (I), and pharmaceutically acceptable salts of any of the foregoing, wherein:

R ¹ is chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups;

R ² is chosen from H, -M, and -L-M;

R ³ is chosen from -OH, -NH ₂ , -OC(=0)Y ¹ , -NHC(=0)Y ¹ , and

-NHC(=0)NHY ¹ groups, wherein Y ¹ is chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, C _6-18 aryl, and C _{1 -13} heteroaryl groups;

R ⁴ is chosen from -OH and -NZ'Z ² groups, wherein Z ¹ and Z ² , which may be identical or different, are each independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups, wherein Z ¹ and Z ² may join together along with the nitrogen atom to which they are attached to form a ring;

R ⁵ is chosen from C _3-8 cycloalkyl groups;

R ⁶ is chosen from -OH, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups;

R ⁷ is chosen from -CH ₂ OH, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups;

R ⁸ is chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups;

L is chosen from linker groups; and

M is a non-glycomimetic moiety chosen from polyethylene glycol, thiazolyl, chromenyl, -C(=0)NH(CH ₂ )1- ₄ NH ₂ , C _1-8 alkyl, and -C(=0)0Y groups, wherein Y is chosen from C _1-4 alkyl, C2- ₄ alkenyl, and C2-4 alkynyl groups.

In some embodiments, the E-selectin antagonist is chosen from compounds of Formula (I), wherein the non-glycomimetic moiety comprises polyethylene glycol.

In some embodiments, the E-selectin antagonist is chosen from compounds of Formula (I), wherein the linker is -C(=0)NH(CH ₂ ) _1-4 NHC(=C))- and the non-glycomimetic moiety comprises polyethylene glycol.

In some embodiments, the E-selectin antagonist is chosen from compounds of Formula (la):

and a pharmaceutically acceptable salt thereof, wherein n is chosen from integers ranging from 1 to 100. In some embodiments, n is chosen from 4, 8, 12, 16, 20, 24, and 28. In some embodiments n is 12.

In some embodiments, the E-selectin antagonist is chosen from Compound A:

and pharmaceutically acceptable salts thereof.

In some embodiments, the E-selectin antagonist is a heterobifunctional antagonist to E- selectin and CXCR4 chosen from compounds of Formula (II):

isomers of Formula (II), tautomers of Formula (II), and pharmaceutically acceptable salts of any of the foregoing, wherein: R ¹ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups;

R ² is chosen from -OH, -NH ₂ , -0C(=0)Y ¹ , -NHC(=0)Y ¹ , and

-NHC(=0)NHY' groups, wherein Y ¹ is chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, C _6-18 aryl, and C _1-13 heteroaryl groups;

R ³ is chosen from -CN, -CH ₂ CN, and -C(=0)Y ² groups, wherein Y ² is chosen from C _1-8 alkyl, ` _2-8 alkenyl, C _2-8 alkynyl, -OZ ¹ , -NHOH, -NHOCH ₃ , -NHCN, and -NZ’Z ² groups, wherein Z ¹ and Z ² , which may be identical or different, are independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups, wherein Z ¹ and Z ² may join together along with the nitrogen atom to which they are attached to form a ring;

R ⁴ is chosen from C _3-8 cycloalkyl groups;

R ⁵ is independently chosen from H, halo, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups; n is chosen from integers ranging from 1 to 4; and

L is chosen from linker groups.

In some embodiments, the E-selectin antagonist is a heterobifunctional antagonist chosen from compounds of Formula (Ila): and pharmaceutically acceptable salts thereof.

In some embodiments, the linker groups of Formula I and/or Formula II are independently chosen from groups comprising spacer groups, such spacer groups as, for example, -(CH ₂ )p- and -0(CH ₂ ) _p -, wherein p is chosen from integers ranging from 1 to 30. In some embodiments, p is chosen from integers ranging from 1 to 20.

Other non-limiting examples of spacer groups include carbonyl groups and carbonyl- containing groups such as, for example, amide groups. A non-limiting example of a spacer group is

In some embodiments, the linker groups are independently chosen from

Other linker groups, such as, for example, polyethylene glycols (PEGs)

and -C(=0)-NH-(CH ₂ ) _P -C(=0)-NH-, wherein p is chosen from integers ranging from 1 to 30, or wherein p is chosen from integers ranging from 1 to 20, will be familiar to those of ordinary skill in the art and/or those in possession of the present disclosure.

In some embodiments, at least one linker group is

In some embodiments, at least one linker group is

In some embodiments, at least one linker group is chosen from

-C(=0)NH(CH ₂ ) ₂ NH-, -CH2NHCH2-, and -€(=0)NHO¾-. In some embodiments, at least one linker group is -C(=0)NH(CH ₂ ) ₂ NH-.

In some embodiments, the E-selectin antagonist is chosen form Compound B:

and pharmaceutically acceptable salts thereof.

Multimeric E-selectin Antagonists (Formulas III and IV)

In some embodiments, the at least one antagonist is chosen from E-selectin antagonists.

In some embodiments, the E-selectin antagonist is chosen from compounds of Formula (III):

prodrugs of Formula (III), isomers of Formula (III), tautomers of Formula (III), and pharmaceutically acceptable salts of any of the foregoing, wherein each R ¹ , which may be identical or different, is independently chosen from H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, and -NHC(=0)R ⁵ groups, wherein each R ⁵ , which may be identical or different, is independently chosen from C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _6-18 aryl, and C _1-13 heteroaryl groups; each R ² , which may be identical or different, is independently chosen from halo, - OY ¹ , -NY'Y ² , -0C(=0)Y', -NHC(=0)Y', and -NHC(=0)NY ¹ Y ² groups, wherein each Y ¹ and each Y ² , which may be identical or different, are independently chosen from H, C1- ₁₂ alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _1-12 haloalkyl, C _2-12 haloalkenyl, C _{2- 12} haloalkynyl, C _6-18 aryl, and C _1-13 heteroaryl groups, wherein Y ¹ and Y ² may join together along with the nitrogen atom to which they are attached to form a ring; each R ³ , which may be identical or different, is independently chosen from

wherein each R ⁶ , which may be identical or different, is independently chosen from H, C _1-12 alkyl and C _1-12 haloalkyl groups, and wherein each R ⁷ , which may be identical or different, is independently chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, -OY ³ , -NHOH, -NHOCH ₃ , -NHCN, and -NY ³ Y ⁴ groups, wherein each Y ³ and each Y ⁴ , which may be identical or different, are independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups, wherein Y ³ and Y ⁴ may join together along with the nitrogen atom to which they are attached to form a ring; each R ⁴ , which may be identical or different, is independently chosen from -CN, C _1-4 alkyl, and C _{1 -4} haloalkyl groups; m is chosen from integers ranging from 2 to 256; and

L is chosen from linker groups; with the proviso that when m is 4, each R ¹ and each R ⁴ is methyl, each R ² is - 0C(=0)Ph, and each R ³ is

then the linker groups are not chosen from

In some embodiments, the E-se lectin antagonist is chosen from compounds of Formula (IV):

prodrugs of Formula (IV), isomers of Formula (IV), tautomers of Formula (IV), and pharmaceutically acceptable salts of any of the foregoing, wherein each R ¹ , which may be identical or different, is independently chosen from H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, and -NHC(=0)R ⁵ groups, wherein each R ⁵ , which may be identical or different, is independently chosen from C1- ₁₂ alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _6-18 aryl, and C _1-13 heteroaryl groups; each R ² , which may be identical or different, is independently chosen from halo, - OY ¹ , -NY'Y ² , -OC(=0)Y’, -NHC(=0)Y\ and -NHC(=0)NY ^, Y ² groups, wherein each Y ¹ and each Y ² , which may be identical or different, are independently chosen from H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _1-12 haloalkyl, C _2-12 haloalkenyl, C2- 1 ₂ haloalkynyl, C _6-18 aryl, and C _1-13 heteroaryl groups, wherein Y ¹ and Y ² may join together along with the nitrogen atom to which they are attached to form a ring; each R ³ , which may be identical or different, is independently chosen from

wherein each R ⁶ , which may be identical or different, is independently chosen from H, C _1-12 alkyl and C _1-12 haloalkyl groups, and wherein each R ⁷ , which may be identical or different, is independently chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, -OY ³ , -NHOH, -NHOCH ₃ , -NHCN, and -NY ³ Y ⁴ groups, wherein each Y ³ and each Y ⁴ , which may be identical or different, are independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups, wherein Y ³ and Y ⁴ may join together along with the nitrogen atom to which they are attached to form a ring; each R ⁴ , which may be identical or different, is independently chosen from -CN, C _1-4 alkyl, and C _{1 -4} haloalkyl groups; m is 2; and

L is chosen from

wherein Q is a chosen from

wherein R ⁸ is chosen from H, C _1-8 alkyl, C _6-18 aryl, C _7-19 arylalkyl, and C _1-13 heteroaryl groups and each p, which may be identical or different, is independently chosen from integers ranging from 0 to 250.

In some embodiments, the E-selectin antagonist of Formula III or Formula IV is chosen from compounds of the following Formula (Illa/IVa) (see definitions of L and m for Formula (III) or (IV) above):

In some embodiments, the E-selectin antagonist of Formula III or Formula IV is chosen from compounds of the following Formula (Illb/IVb) (see definitions of L and m for Formula (III) or (IV) above):

In some embodiments, the E-selectin antagonist is Compound C:

Compound C

Heterobifunctional E-Selectin and Galectin-3 Antagonists (Formulas V and VI)

In some embodiments, the at least one antagonist is chosen from selectin antagonists. In some embodiments, the selectin antagonist is a heterobifunctional inhibitor of E-selectin and Galectin-3, chosen from compounds of Formula (V):

prodrugs of Formula (V), isomers of Formula (V), tautomers of Formula (V), and pharmaceutically acceptable salts of any of the foregoing, wherein

R ¹ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl,

groups, wherein n is chosen from integers ranging from 0 to 2, R ⁶ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _4-16 cycloalkylalkyl, and -C(=0)R ⁷ groups, and each R ⁷ is independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C4-16 cycloalkylalkyl, C _6-18 aryl, and C _1-13 heteroaryl groups;

R ² is chosen from -OH, -OY ¹ , halo, -NH ₂ , -NU'U ² -OC(=0)Y', -NHC(=0)Y', and -NHC(=0)NHY ¹ groups, wherein Y ¹ and Y ² , which may be the same or different, are independently chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _4-16 cycloalkylalkyl, C _2-12 heterocyclyl, C _6-18 aryl, and C1-13 heteroaryl groups, wherein Y ¹ and Y ² may join together along with the nitrogen atom to which they are attached to form a ring;

R ³ is chosen from -CN, -CH ₂ CN, and -C(=0)Y ³ groups, wherein Y ³ is chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, -OZ ¹ , -NHOH, -NHOCH ₃ , -NHCN, and -NZ'Z ² groups, wherein Z ¹ and Z ² , which may be identical or different, are independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, and C ₇ -1 ₂ arylalkyl groups, wherein Z ¹ and Z ² may join together along with the nitrogen atom to which they are attached to form a ring;

R ⁴ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, C ₄ -16 cycloalkylalkyl, and C _6-18 aryl groups;

R ⁵ is chosen from -CN, C _1-8 alkyl, and C _{1 -4} haloalkyl groups;

M is chosen from groups, wherein X is chosen from O and S, and R ⁸ and R ⁹ , which may be identical or different, are independently chosen from C _6-18 aryl, C 1-13 heteroaryl, C7-19 arylalkyl, C _7-19 arylalkoxy, C _2-14 heteroarylalkyl, C2-14 heteroarylalkoxy, and -NHC(=0)Y ⁴ groups, wherein Y ⁴ is chosen from C _1-8 alkyl, C _2-12 heterocyclyl, C6-18 aryl, and C1- ₃₃ heteroaryl groups; and

L is chosen from linker groups.

In some embodiments, the selectin antagonist is chosen from compounds having the following Formulae:

In some embodiments, the selectin antagonist is chosen from compounds having the following Formulae:

In some embodiments, the antagonist is Compound D: d D

In some embodiments, the selectin antagonist is chosen from compounds of Formula (VI):

prodrugs of Formula (VI), isomers of Formula (VI), tautomers of Formula (VI), and pharmaceutically acceptable salts of any of the foregoing, wherein

R ¹ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl,

groups, wherein n is chosen from integers ranging from 0 to 2, R ⁶ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C4- ₁₆ cycloalkylalkyl, and -C(=0)R ⁷ groups, and each R ⁷ is independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C4-16 cycloalkylalkyl, C6- ₁₈ aryl, and C _1-13 heteroaryl groups;

R ² is chosen from -OH, -OY ¹ , halo, -NH ₂ , -NY' Y ² -0C(=0)Y', -NHC(=0)Y ¹ , and -NHC(=0)NHY ^I groups, wherein Y ¹ and Y ² , which may be the same or different, are independently chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C4-16 cycloalkylalkyl, C _2-12 heterocyclyl, C _6-18 aryl, and C _1-13 heteroaryl groups, or Y ¹ and Y ² join together along with the nitrogen atom to which they are attached to form a ring;

R ³ is chosen from -CN, -CH ₂ CN, and -C(=0)Y ³ groups, wherein Y ³ is chosen from C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, -OZ ¹ , -NHOH, -NHOCH ₃ , -NHCN, and -NZ'Z ² groups, wherein Z ¹ and Z ² , which may be identical or different, are independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, and C _7-12 arylalkyl groups, or Z ^! and Z ² join together along with the nitrogen atom to which they are attached to form a ring;

R ⁴ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, C _4-16 cycloalkylalkyl, and C _6-18 aryl groups;

R ⁵ is chosen from -CN, C _1-8 alkyl, and C 1-4 haloalkyl groups;

M is chosen from

wherein

X is chosen from -0-, -S-, -C-, and -N(R ¹⁰ )-, wherein R ¹⁰ is chosen from H, C1 -8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, Cus haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups,

Q is chosen from H, halo, and -OZ ³ groups, wherein Z ³ is chosen from H and C _1-8 alkyl groups,

R ⁸ is chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, C _4-16 cycloalkylalkyl, C _6-18 aryl, C1-13 heteroaryl, C7-19 arylalkyl, and C _2.14 heteroarylalkyl groups, wherein the C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl, C4- ₁₆ cycloalkylalkyl, C6-18 aryl, C1- ₁₃ heteroaryl, C _7-19 arylalkyl, and C2-14 heteroarylalkyl groups are optionally substituted with one or more groups independently chosen from halo, C _1-8 alkyl, C _1-8 hydroxyalkyl, C _1-8 haloalkyl, C _6-18 aryl, -OZ ⁴ , -C(=0)OZ ⁴ , -C(=0)NZ ⁴ Z ⁵ , and - SO ₂ Z ⁴ groups, wherein Z ⁴ and Z ⁵ , which may be identical or different, are independently chosen from H, C _1-8 alkyl, and C _1-8 haloalkyl groups, or Z ⁴ and Z ⁵ join together along with the nitrogen atom to which they are attached to form a ring,

R ⁹ is chosen from C _6-18 aryl and C1-13 heteroaryl groups, wherein the C6- ₁₈ aryl and Ci- ₁₃ heteroaryl groups are optionally substituted with one or more groups independently chosen from R ¹¹ , C _1-8 alkyl, C _1-8 haloalkyl, -C(=0)OZ ⁶ , and -C(=0)NZ ⁶ Z ⁷ groups, wherein R ^{1 1} is independently chosen from C _6-18 aryl groups optionally substituted with one or more groups independently chosen from halo, C _1-8 alkyl, -OZ ⁸ , - C(=0)OZ ⁸ , and -C(=0)NZ ⁸ Z ⁹ groups, wherein Z ⁶ , Z ⁷ , Z ⁸ and Z ⁹ , which may be identical or different, are independently chosen from H and C _1-8 alkyl groups, or Z ⁶ and Z ⁷ join together along with the nitrogen atom to which they are attached to form a ring and/or Z ⁸ and Z ⁹ join together along with the nitrogen atom to which they are attached to form a ring, and wherein each of Z ³ , Z ⁴ , Z ⁵ , Z ⁶ , Z ⁷ , Z ⁸ , and Z ⁹ is optionally substituted with one or more groups independently chosen from halo and -OR ¹² groups, wherein R ¹² is independently chosen from H and C _1-8 alkyl groups; and L is chosen from linker groups.

In some embodiments of Formula (VI), M is chosen from

groups.

In some embodiments of Formula (VI), M is chosen from

groups.

In some embodiments of Formula (VI), linker groups may be chosen from groups comprising spacer groups, such spacer groups as, for example, -(CH ₂ ) _t - and -0(CH ₂ )r, wherein t is chosen from integers ranging from 1 to 20. Other non-limiting examples of spacer groups include carbonyl groups and carbonyl-containing groups such as, for example, amide groups. A non-limiting example of a spacer group is

In some embodiments of Formula (VI), the linker group is chosen from

In some embodiments of Formula (VI), the linker group is chosen from polyethylene glycols (PEGs), -C(=0)NH(CH ₂ ) _v O-, -C(=0)NH(CH ₂ ) _V NHC(=0),

-C(=0)NHC(=0)(CH ₂ )NH-, and -C(=0)NH(CH ₂ ) _v C(=0)NH- groups, wherein v is chosen from integers ranging from 2 to 20. In some embodiments, v is chosen from integers ranging from 2 to 4. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, v is 4.

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

In some embodiments of Formula (VI), the linker group is

Compound A2: Compound A1 (1.5 g, 4.02 mmoles) was dissolved in DCM (30 mL).

Thiophenol (0.9 g, 0.82 mL, 8.04 mmoles) was added followed by dropwise addition of boron trifluoride diethyl etherate (1.79 g, 1.49 mL, 12.06 mmoles). The reaction mixture was stirred at room temperature for 2 days. The reaction quenched by addition of aqueous saturated NaHCCL, transferred to a separatory funnel, and extracted 3 times with DCM. The combined organic phases were dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography to give 2 as an off-white solid (1.1 g, 65% yield) LCMS (ESI): m/z calculated for C18H21N3O7S: 423.4, found 424.1 (M+l); 446.1 (M+Na).

Compound A3: Compound A2 (1.1 g, 2.60 mmoles) was dissolved in methanol (25 mL) at room temperature. Sodium methoxide (0.1 mL, 25% sol. in MeOH) was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture neutralized by the addition of Amberlyst acidic resin, filtered and concentrated to give crude 3, which was used for the next step without further purification. LCMS (ESI): m/z calculated for C12H15N3O4S: 297.3, found 298.1 (M+l); 320.1 (M+Na).

Compound A4: Crude compound A3 (2.60 mmoles), 3,4,5-trifluorophenyl-l-acetylene (2.5 equiv), THPTA (0.11 equiv), and copper (II) sulfate (0.1) were dissolved in methanol (15 mL) at room temperature. Sodium ascorbate (2.4 equiv) dissolved in water was added and the reaction mixture was stirred overnight at room temperature. The resultant precipitate was collected by filtration, washed with hexanes and water, and dried to give compound A4 as a pale yellow solid (1.2 g, 100% yield for 2 steps). LCMS (ESI): m/z calculated for

C20H18F3N3O4S: 453.1, found 454.2 (M+l); 476.2 (M+Na).

Compound A5: Compound A4 (1.2 g, 2.65 mmoles) was dissolved in DMF (15 mL) and cooled on an ice bath. Sodium hydride (60% oil dispersion, 477 mg, 11.93 mmoles) was added and the mixture stirred for 30 minutes. Benzyl bromide (1.42 mL, 1 1.93 mmoles) was added and the reaction was warmed to room temperature and stirred overnight. The reaction mixture was quenched by the addition of aqueous saturated ammonium chloride solution, transferred to a separatory funnel and extracted 3 times with ether. The combined organic phases were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound A5 (1.8 g, 94% yield). LCMS (ESI): m/z calculated for C41H36F3N3O4S: 723.2, found 724.3 (M+l); 746.3 (M+Na).

Compound A6: Compound A5 (1.8 g, 2.49 mmol) was dissolved in acetone (20 mL) and water (2 mL) and cooled on an ice bath. Trichloroisocyanuric acid (637 mg, 2.74 mmoles) was added and the reaction mixture stirred on the ice bath for 3 h. The acetone was removed in vacuo and the residue was diluted with DCM, transferred to a separatory funnel, and washed with saturated aqueous NaHCCb. The organic phase was concentrated and the residue was purified by flash chromatography to afford compound A6 (1.5 g, 95%). LCMS (ESI): m/z calculated for C35H32F3N3O5: 631.2, found 632.2 (M+l); 654.2 (M+Na).

Compound A7: Compound A6 (1.0 g, 1.58 mmoles) was dissolved in DCM (20 mL) and cooled on an ice bath. Dess-Martin periodinane (1.0 g, 2.37 mmoles) was added and mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture quenched by the addition of aqueous saturated NaHCCb, transferred to a separatory funnel, and extracted 2 times with DCM. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound A7 (520 mg, 52% yield). LCMS (ESI): m/z calculated for C35H30F3N3O5: 629.2, found 652.2 (M+Na); 662.2 (M+MeOH+1); 684.2 (M+MeOH+Na).

Compound A8: Methyl bromoacetate (253 mg, 1.65 mmoles) dissolved in 0.5 mL of THF was added dropwise to a solution of lithium bis(trimethylsilyl)amide (1.0 M in THF, 1.65 mL, 1.65 mmoles) cooled at -78 C. The reaction mixture was stirred for 30 minutes at -78 C. Compound A7 (260 mg, 0.41 mmoles) dissolved in THF (2.0 mL) was then added. The reaction mixture was stirred at -78 C for 30 minutes. The reaction was quenched by the addition of aqueous saturated NH4CI and warmed to rt. The reaction mixture was transferred to a separatory funnel and extracted 3 times with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and concentrated. The residue was separated by flash chromatography to afford compound A8 (183 mg, 64% yield). ¹ H NMR (400 MHz, Chloroform-d) δ 7.38 - 7.22 (m, 9H), 7.15 - 7.11 (m, 3H), 7.09 (dd, J= 8.4, 6.6 Hz, 1H), 7.06 - 7.00 (m, 2H), 6.98 - 6.93 (m, 2H), 5.11 (dd, .J= 11.3, 3.2 Hz, 1H), 4.60 (d, J= 11.8 Hz, 1H), 4.57 - 4.49 (m, 2H), 4.49 - 4.42 (m, 2H), 4.35 (d, J= 11.8 Hz,

1H), 4.14 (d, J= 3.2 Hz, 1H), 4.05 (s, 1H), 4.02 (d, J= 7.0 Hz, 1H), 3.84 (d, J= 11.0 Hz,

1H), 3.81 (s, 3H), 3.70 (dd, J= 9.5, 7.7 Hz, 1H), 3.62 (dd, J= 9.4, 6.0 Hz, 1H). LCMS (ESI): m/z calculated for C38H34F3N3O7: 701.2, found 702.3 (M+l); 724.3 (M+Na).

Compound A9: Compound A8 (5.0 g, 7.13 mmol) was azeotroped with toluene two times under reduced pressure, and then dried under high vacuum for 2 hours. It was then dissolved in anhydrous CH2CI2 (125 mL) and cooled on an ice bath while stirring under an atmosphere of argon. Tributyltin hydride (15.1 mL, 56.1 mmol) was added dropwise and the solution was allowed to stir for 25 minutes on the ice bath. Trimethylsilyl triflate (2.1 mL, 11.6 mmol) dissolved in 20 mL of anhydrous CH2CI2 was then added dropwise over the course of 5 minutes. The reaction was slowly warmed to ambient temperature and stirred for 16 hours. The reaction mixture was then diluted with CH2CI2 (50 mL), transferred to a separatory funnel, and washed with saturated aqueous NaHCCL (50 mL). The aqueous phase was separated and extracted with CH2CI2 (50 mL x 2). The combined organic phases were washed with saturated aqueous NaHCO ₃ (50 mL), dried over Na2SO ₄ , filtered, and concentrated. The residue was purified by flash chromatography (hexanes to 40% EtOAc in hexanes, gradient) to afford compound A9 (2.65 g, 48%).

'H-NMR (400 MHz, CDCO ₃ ): d 7.65 (s, 1H), 7.36 - 7.22 (m, 8H), 7.16 - 7.06 (m, 7H), 6.96 - 6.90 (m, 2H), 5.03 (dd, J= 10.7, 3.2 Hz, 1H), 4.72 (d, J= 2.3 Hz, 1H), 4.51 (dt, J= 22.6,

11.4 Hz, 3H), 4.41 (d, J= 10.9 Hz, 1H), 4.32 (dd, J= 10.7, 9.2 Hz, 1H), 4.07 (d, 7= 3.1 Hz, 1H), 3.94 (d, J= 10.9 Hz, 1H), 3.92 - 3.84 (m, 3H), 3.78 - 3.71 (m, 4H), 3.65 (dd, J= 9.1,

5.5 Hz, 1H), 0.24 (s, 9H). LCMS (ESI): m/z calculated for C41H44F3N3O7S1: 775.8, found 798.2 (M+Na).

Compound A10: To a solution of compound A9 (2.65 g, 3.4 mmol) was in anhydrous MeOH (40 mL) was added Pd(OH)2 (0.27 g, 20% by wt). The mixture was cooled on an ice bath and stirred for 30 minutes. Triethylsilane (22 mL, 137 mmol) was added dropwise. The solution was allowed to slowly warm to ambient temperature and stirred for 16 hours. The reaction mixture was filtered through a bed of Celite and concentrated. The residue was purified by flash chromatography (hexanes to 100% EtOAc, gradient) to afford compound A10 (1.09 g, 73%).

^! H-NMR (400 MHz, CDsOD): d 8.57 (s, 1H), 7.77 - 7.53 (m, 2H), 4.91 - 4.82 (m, 1H), 4.66 - 4.59 (m, 1H), 4.55 (dd, J = 10.8, 9.4 Hz, 1H), 4.13 (d, J = 2.8 Hz, 1H), 3.86 (dd, J = 9.4, 2.1 Hz, 1H), 3.81 (s, 3H), 3.77 - 3.74 (m, 1H), 3.71 - 3.68 (m, 2H). LCMS (ESI): m/z calculated for C17H18F3N3O7: 433.3, found 456.0 (M+Na).

Compound All: Compound A10 (1.09 g, 2.5 mmol) and CSA (0.115 g, 0.49 mmol) were suspended in anhydrous MeCN (80 mL) under an argon atmosphere. Benzaldehyde dimethyl acetal (0.45 mL, 2.99 mmol) was added dropwise. The reaction mixture was allowed to stir for 16 hours at ambient temperature, during which time it became a homogenous solution. The reaction mixture was then neutralized with a few drops of Et3N, and concentrated. The residue was purified via flash chromatography (CH2CI2 to 10% MeOH in CH2CI2, gradient) to afford compound All (978 mg, 75%). ^J H NMR (400 MHz, DMSO-d6): d 8.84 (s, 1H), 7.95 - 7.73 (m, 2H), 7.33 (qdt, J = 8.4, 5.6, 2.7 Hz, 5 FI), 5.51 (t, J = 3.8 FIz, 2H), 5.47 (d, J = 6.8 Hz, 1H), 5.14 (dd, J = 10.8, 3.6 Hz, 1H), 4.54 (dd, J = 6.7, 2.2 Hz, 1H), 4.47 (ddd, J = 10.8, 9.3, 7.5 Hz, 1H), 4.40 (d, J = 4.0 FFz, 1H), 4.09 - 3.99 (m, 2H), 3.85 (dd, J = 9.3, 2.2 Hz, 1H), 3.81 - 3.76 (m, 1H), 3.71 (s, 3H). LCMS (ESI): m/z calculated for C24H22F3N3O7: 521.4, found 544.1 (M+Na).

Compound A12: Compound All (25.2 mg, 0.048 mmol) was azeotroped with toluene 2 times under reduced pressure, dried under high vacuum for 2 hours, then dissolved in anhydrous DMF (2 mL) and cooled on an ice bath. Benzyl bromide (6 uL, 0.05 mmol) dissolved in 0.5 mL of anhydrous DMF was added and the reaction and was stirred under an atmosphere of argon for 30 minutes at 0 °C. Sodium hydride (2 mg, 0.05 mmol, 60%) was added and the reaction was allowed to gradually warm to ambient temperature while stirring for 16 hours. The reaction mixture was diluted with EtOAc (20 mL), transferred to a separatory funnel, and washed with FLO (10 mL). The aqueous phase was separated and extracted with EtOAc (10 mL x 3). The combined organic phases were washed with FLO (10 mL x 3), dried over Na ₂ S0 ₄ , filtered, and concentrated. The residue was purified via preparative TLC (5% MeOFl in CFI ₂ CI ₂ ) to afford compound A12 (6.3 mg, 21%). LCMS (ESI): m/z calculated for C ₃₁ H ₂₈ F ₃ N ₃ O ₇ : 611.5, found 634.1 (M+Na).

Compound A13: Compound A12 (6.3 mg, 0.01 mmol) was dissolved in anhydrous MeOH (1 mL) containing CSA (0.26 mg, 0.001 mmol). The reaction mixture was heated to 76 °C in a screw-cap scintillation vial while stirring. After 2 hours, an additional 0.13 mg of CSA in 0.5 mL of MeOH was added. The reaction mixture was stirred at 76 °C for 16 hours. The reaction mixture concentrated under reduced pressure. The residue was purified via preparative TLC (10% MeOH in CH2CI2) to afford compound A13 (4.2 mg, 80%).

¾ NMR (400 MHz, DMSO- e) δ 8.80 (s, 1H), 7.94 - 7.86 (m, 2H), 7.48 - 7.42 (m, 2H),

7.38 (t, J= 7.4 Hz, 2H), 7.36 - 7.28 (m, 1H), 5.46 (d, J= 7.7 Hz, 1H), 5.28 (d, J= 6.0 Hz, 1H), 4.85 (dd, J= 10.7, 2.9 Hz, 1H), 4.67 (d, J= 11.0 Hz, 1H), 4.62 - 4.58 (m, 1H), 4.54 (d, J= 11.1 Hz, 1H), 4.44 (d, J= 2.5 Hz, 1H), 4.36 (q, J= 9.5 Hz, 1H), 3.95 - 3.90 (m, 1H), 3.78 (dd, J= 9.3, 2.5 Hz, 1H), 3.71 (s, 3H), 3.61 - 3.54 (m, 1H), 3.52 - 3.43 (m, 1H), 3.43 - 3.38 (m, 1H). LCMS (ESI): m/z calculated for C24H24F3N3O7: 523.4, found 546.0 (M+Na).

Compound A14: To a solution of compound A13 (3.5 mg, 0.007 mmoles) in methanol (0.5 mL) was added 1.0 M NaOH solution (0.1 mL). The reaction mixture was stirred overnight at room temperature then neutralized with acidic resin, filtered and concentrated. The residue was purified by reverse phase chromatography using a C-8 matrix to afford 3.0 mg compound A14 (90%). LCMS (ESI): m/z calculated for C23H22F3N3O7: 509.1, found 508.2 (M-H).

EXAMPLE A2

SYNTHESIS OF BUILDING BLOCKS A15-A35 AND A76

Prophetic Compound A15: Compound A15 is prepared according to Figure 1 by substituting

2-methyl benzyl bromide for benzyl bromide in step k.

Prophetic Compound A16: Compound A16 is prepared according to Figure 1 by substituting

3-methyl benzyl bromide for benzyl bromide in step k.

Prophetic Compound A17: Compound A17 is prepared according to Figure 1 by substituting

4-methyl benzyl bromide for benzyl bromide in step k.

Prophetic Compound A18: Compound A18 is prepared according to Figure 1 by substituting 2-fluoro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A19: Compound A19 is prepared according to Figure 1 by substituting 3-fluoro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A20: Compound A20 is prepared according to Figure 1 by substituting 4-fluoro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A21: Compound A21 is prepared according to Figure 1 by substituting

2-chloro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A22: Compound A22 is prepared according to Figure 1 by substituting

3-chloro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A23: Compound A23 is prepared according to Figure 1 by substituting

4-chloro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A24: Compound A24 is prepared according to Figure 1 by substituting

2-methoxy benzyl bromide for benzyl bromide in step k.

Prophetic Compound A25: Compound A25 is prepared according to Figure 1 by substituting

3-methoxy benzyl bromide for benzyl bromide in step k.

Prophetic Compound A26: Compound A26 is prepared according to Figure 1 by substituting

4-methoxy benzyl chloride for benzyl bromide in step k.

Prophetic Compound A27: Compound All is prepared according to Figure 1 by substituting

2-picolyl bromide for benzyl bromide in step k.

Prophetic Compound A28: Compound A28 is prepared according to Figure 1 by substituting

3-picolyl bromide for benzyl bromide in step k.

Prophetic Compound A29: Compound A29 is prepared according to Figure 1 by substituting

4-picolyl bromide for benzyl bromide in step k.

Prophetic Compound A30: Compound A30 is prepared according to Figure 1 by substituting 3,4-difluoro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A31: Compound A31 is prepared according to Figure 1 by substituting 3-fluoro, 4-chloro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A32: Compound A32 is prepared according to Figure 1 by substituting 3-chloro, 4-fluoro benzyl bromide for benzyl bromide in step k.

Prophetic Compound A33: Compound A33 is prepared according to Figure 1 by substituting phenyl acetylene for 3, 4, 5 -trifluorophenyl-1 -acetylene in step d.

Prophetic Compound A34: Compound A34 is prepared according to Figure 1 by substituting 3 -fluorophenyl acetylene for 3, 4, 5 -trifluorophenyl-1 -acetylene in step d.

Prophetic Compound A35: Compound A35 is prepared according to Figure 1 by substituting 3, 4-difluorophenyl-l -acetylene for 3, 4, 5 -trifluorophenyl-1 -acetylene in step d.

Prophetic Compound A76: Compound A76 is prepared according to Figure 1 by substituting 2-naphthyl bromide for benzyl bromide in step k.

EXAMPLE A3

SYNTHESIS OF BUILDING BLOCK A37

Prophetic Compound A36: Compound A12 is dissolved in DMF and cooled on an ice bath. Sodium hydride (1.1 eq) is added and the reaction mixture is stirred for 30 minutes. Methyl iodide (1.1 eq) is added and the reaction mixture is stirred until completion. The reaction mixture is quenched by the addition of saturated ammonium chloride, transferred to a separatory funnel, and extracted 3 times with ethyl acetate. The combined organic phases are dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound A36.

Prophetic Compound A37: Compound A36 is dissolved in methanol. Sodium hydroxide (3 eq of a 1M solution) is added and the reaction mixture is stirred at room temperature until completion. The pH is adjusted to 2 with 1 M HC1 and the solvent removed. The residue is purified by flash chromatography to afford compound A37.

EXAMPLE A4

SYNTHESIS OF BUILDING BLOCK A44

Prophetic Compound A38: Compound All is dissolved in DMF and cooled on an ice bath. Imidazole (1.1 eq) is added followed by TBSC1 (1.1 eq). The reaction mixture is stirred until completion. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 3 times with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is separated by flash chromatography to afford compound A38.

Prophetic Compound A39: Compound A38 is dissolved in pyridine and cooled on an ice bath. Acetic anhydride (2 eq) is added and the reaction mixture is stirred until completion. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 3 times with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is separated by flash chromatography to afford compound A39.

Prophetic Compound A40: Compound A39 is dissolved in THF and cooled on an icebath. Tetrabutylammonium fluoride (1.1 eq) is added and the reaction mixture is stirred until completion. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 3 times with water. The organic phase is dried over magnesium sulfate, filtered, and concentrated. The residue is separated by flash chromatography to afford compound A40.

Prophetic Compound A41: Compound A40 is dissolved in DCM and cooled on an ice bath. Thionyl bromide (1.1 eq) is added and the reaction mixture is stirred until completion. The reaction mixture is quenched by the addition of saturated aqueous sodium bicarbonate solution. The reaction mixture is transferred to a separatory funnel and extracted 3 times with ethyl acetate. The combined organic phases are dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound A41.

Prophetic Compound A42: Benzyl thiol is dissolved in DMF and cooled to room temperature. Sodium hydride (1.05 eq) is added and the reaction mixture is stirred for 30 minutes.

Compound A41 (0.9 eq) dissolved in DMF is added. The reaction mixture is stirred until completion. The reaction mixture is quenched by the addition of saturated aqueous ammonium chloride solution. The reaction mixture is transferred to a separatory funnel and extracted 3 times with ethyl acetate. The combined organic phases are dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound A42.

Prophetic Compound A43: Compound A42 is dissolved in methanol. A catalytic amount of CSA is added and the reaction mixture is refluxed overnight. The reaction mixture is concentrated and the residue is purified by flash chromatography to afford compound A43.

A44

Prophetic Compound A44: Compound A43 is dissolved in methanol. Sodium hydroxide (3 eq of a 1M solution) is added and the reaction mixture is stirred at room temperature until completion. The pH is adjusted to 2 with 1 M HC1 and the solvent removed. The residue is purified by flash chromatography to afford compound A44.

EXAMPLE A5

SYNTHESIS OF BUILDING BLOCK A 49

A45

Prophetic Compound A45: Compound A6 is dissolved in pyridine and cooled on an ice bath. Acetic anhydride (2 eq) is added and the reaction mixture is stirred until completion. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 3 times with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is separated by flash chromatography to afford compound A45.

A46

Prophetic Compound A46: Compound A45 is dissolved in DCM and cooled on an ice bath. Trimethylsilyl iodide (1.1 eq) is added and the reaction mixture is stirred until completion. The solvent is removed in vacuo and the residue is dissolved in THF. In a separate flask diethyl malonate is dissolved in THF and NaHMDS (0.9 eq) and 15-crown-5 (0.9 eq) are added. After stirring for 30 minutes the glycosyl iodide solution is added and the reaction mixture is stirred until completion. The solvent is removed and the residue is purified by flash chromatography to afford compound A46.

Prophetic Compound A47: Compound A46 is dissolved in DMF (15 mL) and cooled on an ice bath. Sodium hydride (1.1 eq) is added and the mixture stirred for 30 minutes. Phenethyl bromide (1.1 eq) is added and the reaction was warmed to room temperature and stirred until completion. The reaction mixture is quenched by the addition of aqueous saturated ammonium chloride solution, transferred to a separatory funnel and extracted 3 times with ether. The combined organic phases are dried over magnesium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound A47.

Prophetic Compound A48: Compound A47 is dissolved in THF. Sodium hydroxide (5 eq of a 1M aqueous solution) is added and the reaction mixture is stirred at 50°C until completion. A large excess of acetic acid is added and the reaction mixture is stirred at 100°C until completion. The solvent is removed and the residue is purified by flash chromatography to afford compound A48.

Prophetic Compound A49: To a solution of compound A48 dissolved in methanol is added Pd/C. The reaction mixture is hydrogenated on a Paar shaker until completion. The reaction mixture is filtered through Celite and concentrated to afford compound A49.

EXAMPLE A6

SYNTHESIS OF COMPOUND A51

Prophetic Compound A51: To a solution of compound A14 in anhydrous DMF can be added HATU (1 eq) and DIPEA (1.2 eq). The mixture can be stirred at ambient temperature for 15 minutes followed by addition of compound A50 (1.2 eq) (preparation described in

W02018068010). The reaction mixture can be stirred at ambient temperature until completion. The reaction mixture can be concentrated in vacuo and the residue purified by HPLC to afford compound A51.

EXAMPLE A7

SYNTHESIS OF COMPOUNDS A52-A72

Prophetic Compounds A52-A72: Compounds A52-A72 are prepared from compound A50 using the procedures outlined for compound A51 and in Figures 1, 2, and 3.

EXAMPLE A8

SYNTHESIS OF COMPOUND A73

Prophetic Compound A73: Compound A73 is prepared from compound A50 using the procedures outlined for compound A51 and in Figures 1, 2, and 3.

EXAMPLE A9

SYNTHESIS OF COMPOUND A74

Prophetic Compound A74: Compound A74 is prepared from compound A50 using the procedures outlined for compound A51 and in Figures 1, 2, and 3.

EXAMPLE A10

SYNTHESIS OF COMPOUND A75

Prophetic Compound A75: Compound A75 is prepared from compound A50 using the procedures outlined for compound A51 and in Figures 1, 2, and 3.

EXAMPLE Al l

SYNTHESIS OF COMPOUND All

Prophetic Compound A77: Compound All is prepared from compound A50 using the procedures outlined for compound A51 and in Figures 1, 2, and 3.

EXAMPLE A12

SYNTHESIS OF COMPOUND A87

Prophetic Compound A87: To a solution of compound A14 in anhydrous DMF is added HATU (1 eq) and DIPEA (1.2 eq). The mixture is stirred at ambient temperature for 15 minutes followed by addition of compound A109 (1.2 eq) ( see Figure 6; preparation described in WO2013096926). The reaction mixture is stirred at ambient temperature until completion. The reaction mixture is concentrated in vacuo and the residue purified by HPLC to afford compound A87.

EXAMPLE A13

SYNTHESIS OF COMPOUNDS A88-A108

Prophetic Compounds A88-A108: Compounds A88-A108 are prepared from compound A109 using the procedures outlined for Compound A87 and in Figures 1, 2, and 3.

EXAMPLE A14

SYNTHESIS OF COMPOUND A83

Prophetic Compound A79: To a solution of compound A78 ( see Figure 7; preparation described in WO 2007/028050)) and l-ethynyl-3-fluorobenzene (1.2 equivalents) in methanol is added a stock solution of copper sulfate and tris(3-hydroxypropyltriazolylmethyl) amine in water (0.2 equivalents). The reaction is initiated by addition of an aqueous solution of sodium ascorbate (1.3 equivalents). After stirring at ambient temperature for an appropriate length of time the solvent is removed in vacuo. The product is purified by normal phase column chromatography to give the compound A79.

Prophetic Compound A80: A mixture of compound A79 and a catalytic amount of Pd/C in MeOH is stirred at room temperature under a ¾ atmosphere (balloon). After reaction is complete, the mixture is filtered through Celite ^® and concentrated to afford compound A80.

Prophetic Compound A81: To a solution of compound A80 in MeOH at room temperature is added a catalytic amount of NaOMe in MeOH. The reaction mixture is stirred at room temperature until completion. The pH is adjusted to ~4 by addition of acetic acid. The reaction mixture is concentrated and the residue separated by flash chromatography to afford compound A81.

Compound A82: Compound A81 is dissolved in ethylenediamine (10 equivalents) under an atmosphere of argon and stirred at 70 °C until the reaction is complete. The reaction mixture is cooled to room temperature then co-evaporated with methanol and toluene. The residue is purified by HPLC to give compound A82.

Prophetic Compound A83: To a solution of compound A14 in anhydrous DMF is added HATU (1.1 equivalents) and DIPEA (1.3 equivalents). The mixture is stirred at ambient temperature for 15 minutes followed by addition of compound A82 ( see Figure 7; 1 equivalent). The mixture is stirred at ambient temperature until completion. The solvent is removed in vacuo and the residue is purified by HPLC to afford compound A83.

EXAMPLE A15

SYNTHESIS OF COMPOUND A83

Prophetic Compound A85: Compound A84 ( see Figure 8; preparation described in

WO2018068010) is heated at 70°C overnight with 2-aminoethyl amine (10 eq). After cooling to room temperature the reaction mixture is separated by reverse phase chromatography to afford compound A85.

Prophetic Compound A86: Compound A86 is synthesized from compound A85 and compound A14 following the procedure as described for the synthesis of compound A83.

EXAMPLE A16

E-SELECTIN ACTIVITY - BINDING ASSAY

The inhibition assay to screen for and characterize antagonists of E-selectin is a competitive binding assay, which allows the determination of IC50 values. E-selectin/Ig chimera is immobilized in 96 well microtiter plates by incubation at 37°C for 2 hours. To reduce nonspecific binding, bovine serum albumin is added to each well and incubated at room temperature for 2 hours. The plate is washed and serial dilutions of the test compounds are added to the wells in the presence of conjugates of biotinylated, sLe ^a polyacrylamide with streptavidin/horseradish peroxidase and incubated for 2 hours at room temperature.

To determine the amount of sLe ^a bound to immobilized E-selectin after washing, the peroxidase substrate, 3, 3', 5, 5' tetramethylbenzidine (TMB) is added. After 3 minutes, the enzyme reaction is stopped by the addition of H3PO4, and the absorbance of light at a wavelength of 450 nm is determined. The concentration of test compound required to inhibit binding by 50% is determined.

EXAMPLE A17

GALECTIN-3 ACTIVITY - ELISA ASSAY

Galectin-3 antagonists is evaluated for their ability to inhibit binding of galectin-3 to a Galbl- 3GlcNAc carbohydrate structure. The detailed protocol is as follows. A 1 ug/mL suspension of a Galbl-3GlcNAcpi-3Galbl-4GlcNAcP-PA-biotin polymer (Glycotech, catalog number 01-096) is prepared. A 100 uL aliquot of the polymer is added to the wells of a 96-well streptavidin-coated plate (R&D Systems, catalog number CP004). A 100 uL aliquot of IX Tris Buffered Saline (TBS, Sigma, catalog number T5912 - 10X) is added to control wells. The polymer is allowed to bind to the streptavidin-coated wells for 1.5 hours at room temperature. The contents of the wells is discarded and 200 uL of IX TBS containing 1% bovine serum albumin (BSA) is added to each well as a blocking reagent and the plate is kept at room temperature for 30 minutes. The wells are washed three times with IX TBS containing 0.1% BSA. A serial dilution of test compounds is prepared in a separate V- bottom plate (Corning, catalog number 3897). A 75 uL aliquot of the highest concentration of the compound to be tested is added to the first well in a column of the V-bottom plate then 15 ul are serially transferred into 60 uL IX TBS through the remaining wells in the column to generate a 1 to 5 serial dilution. A 60 uL aliquot of 2 ug/mL galectin-3 (IBL, catalog number IBATGP0414) is added to each well in the V-bottom plate. A 100 uL aliquot of the galectin- 3/test compound mixture is transferred from the V-bottom plate into the assay plate containing the Galbl-3GlcNAc polymer. Four sets of control wells in the assay plate are prepared in duplicate containing 1) both Galbl-3GlcNAc polymer and galectin-3, 2) neither the polymer nor galectin-3, 3) galectin-3 only, no polymer, or 4) polymer only, no galectin-3. The plate is gently rocked for 1.5 hours at room temperature. The wells are washed four times with TBS/0.1%BSA. A 100 uL aliquot of anti-galectin-3 antibody conjugated to horse radish peroxidase (R&D Systems, from DGAL30 kit) is added to each well and the plate is kept at room temperature for 1 hour. The wells are washed four times with TBS/0.1%BSA.

A 100 uL aliquot of TMB substrate solution is added to each well. The TMB substrate solution is prepared by making a 1 :1 mixture of TMB Peroxidase Substrate (KPL, catalog number 5120-0048) and Peroxidase Substrate Solution B (KPL, catalog number 5120-0037). The plate is kept at room temperature for 10 to 20 minutes. The color development is stopped by adding 100 uL 10% phosphoric acid (RICCA Chemical Co., catalog number 5850-16). The absorbance at 450 nm (A450) is measured using a FlexStation 3 plate reader (Molecular Devices). Plots of A450 versus test compound concentration and IC50

determinations are made using GraphPad Prism 6.

Galactose Linked Multimeric Inhibitors of E-selectins, Galectin-3, and/or CXCR4 (Formula VII)

In some embodiments, the selectin antagonist is a multimeric inhibitor of E-selectin, Galectin-3, and/or CXCR4, chosen from compounds of Formula (VII): prodrugs of Formula (VII), and pharmaceutically acceptable salts of any of the foregoing, wherein each R ¹ , which may be identical or different, is independently chosen from H, C1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, C _2-8 haloalkynyl,

groups, wherein each n, which may be identical or different, is chosen from integers ranging from 0 to 2, each R ⁶ , which may be identical or different, is independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _4-16 cycloalkylalkyl, and - C(=0)R ⁷ groups, and each R ⁷ , which may idential or different, is independently chosen from H, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _4-16 cycloalkylalkyl, C _6-18 aryl, and C 1-13 heteroaryl groups; each R ² , which may be identical or different, is independently chosen from H, a non- glycomimetic moiety, and a linker-non-glycomimetic moiety, wherein each non- glycomimetic moiety, which may be identical or different, is independently chosen from galectin-3 inhibitors, CXCR4 chemokine receptor inhibitors, polyethylene glycol, thiazolyl, chromenyl, C _1-8 alkyl, R ⁸ , C ₆ -i8 aryl-R ⁸ , C _1-12 heteroaryl-R ⁸ ,

groups, wherein each Y ¹ , which may be identical or different, is independently chosen from Ci -4 alkyl, C _2-4 alkenyl, and C _2-4 alkynyl groups and wherein each R ⁸ , which may be identical or different, is independently chosen from C _1-12 alkyl groups substituted with at least one substituent chosen from -OH, -OSO3Q, -OPO3Q2, -CO2Q, and -SO3Q groups and C _2-12 alkenyl groups substituted with at least one substituent chosen from -OH, -OSO ₃ Q, -OPO ₃ Q ₂ , -CO2Q, and -SO3Q groups, wherein each Q, which may be identical or different, is independently chosen from H and pharmaceutically acceptable cations;

each R ³ , which may be identical or different, is independently chosen from -CN, -CH ₂ CN, and -C(=0)Y ² groups, wherein each Y ² , which may be identical or different, is independently chosen from C 1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, -OZ ¹ , - NHOH, -NHOCH3, -NFICN, and -NZ'Z ² groups, wherein each Z ¹ and Z ² , which may be identical or different, are independently chosen from H, C1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C1-12 haloalkyl, C _2-12 haloalkenyl, C _2-12 haloalkynyl, and C7-12 arylalkyl groups, wherein Z ¹ and Z ² may join together along with the nitrogen atom to which they are attached to form a ring; each R ⁴ , which may be identical or different, is independently chosen from H, C _1-12 alkyl, C _2-12 alkenyl, C _2-12 alkynyl, C _1-12 haloalkyl, C _2-12 haloalkenyl, C _2-12 haloalkynyl, C4-16 cycloalkylalkyl, and C _6-18 aryl groups; each R ⁵ , which may be identical or different, is independently chosen from -CN, C _1-12 alkyl, and C _1-12 haloalkyl groups; each X, which may be identical or different, is independently chosen from -O- and - N(R ⁹ )-, wherein each R ⁹ , which may be identical or different, is independently chosen from H, C1_ ₈ alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 haloalkyl, C _2-8 haloalkenyl, and C _2-8 haloalkynyl groups; m is chosen from integers ranging from 2 to 256; and

L is independently chosen from linker groups.

In some embodiments of Formula (VII), at least one linker groups is chosen from groups comprising spacer groups, such spacer groups as, for example, -(CFfeV and -0(CH2)z-, wherein z is chosen from integers ranging from 1 to 250. Other non-limiting examples of spacer groups include carbonyl groups and carbonyl-containing groups such as, for example, amide groups. A non-limiting example of a spacer group is

In some embodiments of Formula (VII), at least one linker group is chosen from

Other linker groups for certain embodiments of Formula (VII), such as, for example, polyethylene glycols (PEGs) and -C(=0)-NH-(CH ₂ ) _z -C(=0)-NH-, wherein z is chosen from integers ranging from 1 to 250, will be familiar to those of ordinary skill in the art and/or those in possession of the present disclosure. In some embodiments of Formula (VII), at least one linker group is

In some embodiments of Formula (VII), at least one linker group is

In some embodiments of Formula (VII), at least one linker group is chosen from - C(=0)NH(CH ₂ ) ₂ NH-, -CH2NHCH2-, and -C(=0)NHCH2-. In some embodiments of Formula (VII), at least one linker group is -C(=0)NH(CH ₂ ) ₂ NH-.

In some embodiments of Formula (VII), L is chosen from dendrimers. In some embodiments of Formula (VII), L is chosen from polyamidoamine (“PAMAM”) dendrimers. In some embodiments of Formula (VII), L is chosen from PAMAM dendrimers comprising succinamic. In some embodiments of Formula (VII), L is PAMAM GO generating a tetramer. In some embodiments of Formula (VII), L is PAMAM G1 generating an octamer. In some embodiments of Formula (VII), L is PAMAM G2 generating a 16-mer. In some embodiments of Formula (VII), L is PAMAM G3 generating a 32-mer. In some

embodiments of Formula (VII), L is PAMAM G4 generating a 64-mer. In some

embodiments, L is PAMAM G5 generating a 128-mer.

In some embodiments of Formula (VII), m is 2 and L is chosen from

groups, wherein U is chosen from

groups, wherein R ¹⁴ is chosen from H, C _1-8 alkyl, C _6-18 aryl, C7.19 arylalkyl, and C1-13 heteroaryl groups and each y, which may be identical or different, is independently chosen from integers ranging from 0 to 250. In some embodiments of Formula (VII), R ¹⁴ is chosen from C _1-8 alkyl. In some embodiments of Formula (VII), R ¹⁴ is chosen from C ₇ - ₁₉ arylalkyl. In some embodiments of Formula (VII), R ¹⁴ is H. In some embodiments of Formula (VII), R ¹⁴ is benzyl.

In some embodiments of Formula (VII), L is chosen from

wherein y is chosen from integers ranging from 0 to 250.

In some embodiments of Formula (VII), L is chosen from groups, wherein y is chosen from integers ranging from 0 to 250.

In some embodiments of Formula (VII), L is

In some embodiments of Formula (VII), L is chosen from

wherein y is chosen from integers ranging from 0 to 250.

In some embodiments of Formula (VII), L is chosen from

wherein y is chosen from integers ranging from 0 to 250. In some embodiments of Formula (VII), L is chosen from

In some embodiments of Formula (VII), L is

In some embodiments of Formula (VII), L is chosen from groups, wherein y is chosen from integers ranging from 0 to 250.

In some embodiments of Formula (VII), L is

In some embodiments of Formula (VII), L is

In some embodiments of Formula (VII), L is

In some embodiments of Formula (VII), L is chosen from

In some embodiments of Formula (VII), L is

In some embodiments of Formula (VII), L is chosen from

wherein each y, which may be identical or different, is independently chosen from integers ranging from 0 to 250. In some embodiments of Formula (VII), L is chosen from

wherein each y, which may be identical or different, is independently chosen from integers ranging from 0 to 250.

In some embodiments of Formula (VII), L is chosen from

In some embodiments, at least one compound is chosen from compounds of Formula (VII), wherein each R ¹ is identical, each R ² is identical, each R ³ is identical, each R ⁴ is identical, each R ⁵ is identical, and each X is identical. In some embodiments, at least one compound is chosen from compounds of Formula (VII), wherein said compound is symmetrical.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 21

Compound 3: A mixture of compounds 1 (preparation described in WO 2007/028050) and compound 2 (preparation described in WO 2013/096926) (1.7 eq) is azeotroped 3 times from toluene. The mixture is dissolved in DCM under argon and cooled on an ice bath. To this solution is added boron trifluoride etherate (1.5 eq). The reaction mixture is stirred 12 hours at room temperature. The reaction is quenched by the addition of triethylamine (2 eq). The reaction mixture is transferred to a separatory funnel and washed 1 time with half saturated sodium bicarbonate solution and 1 time with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound 3.

Compound 4: Compound 3 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 2 times with water. The organic phase is dried over magnesium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 4.

Compound 5: To a solution of compound 4 in dichloromethane cooled on an ice bath is added DABCO (1.5 eq) followed by monomethyoxytrityl chloride (1.2 eq). The reaction mixture is stirred overnight allowing to warm to room temperature. The reaction mixture is transferred to a separatory funnel and washed 2 times with water. The organic phase is concentrated and the residue is purified by flash chromatography to afford compound 5.

Compound 7: To a solution of compound 5 in methanol is added dibutyltin oxide (1.1 eq). The reaction mixture is refluxed for 3 hours then concentrated. The residue is suspended in DME. To this suspension is added compound 6 (preparation described in Thoma el. al. J. Med. Chem., 1999, 42, 4909) (1.5 eq) followed by cesium fluoride (1.2 eq). The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel, and washed with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is purified by flash

chromatography to afford compound 7.

Compound 8: To a degassed solution of compound 7 in anhydrous DCM at 0 °C is added Pd(PPfi3)4 (0.1 eq), BusSnH (1.1 eq) and N-trifluoroacetyl glycine anhydride (2.0 eq) (preparation described in Chemische Berichte (1955), 88(1), 26). The resulting solution is stirred for 12 hrs allowing the temperature to increase to room temperature. The reaction mixture is diluted with DCM, transferred to a separatory funnel, and washed with water. The organic phase is dried over Na2SO4, then filtered and concentrated. The residue is purified by flash chromatography to afford compound 8.

Compound 9: To a stirred solution of compound 8 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 9.

Compound 10: Compound 9 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is fdtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 10.

Compound 11: Compound 10 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (1.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 11.

Compound 12: Compound 12 can be prepared in an analogous fashion to Figure 9 by substituting (acetylthio)acetyl chloride for N-trifluoroacetyl glycine anhydride in step e.

Compound 13: Compound 10 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (1.5 eq) is added followed by HATU (1.1 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (2 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 13.

Compound 14: Compound 13 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.3 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 14.

Compound 15: Compound 15 can be prepared in an analogous fashion to Figure 10 by using methylamine in place of azetidine in step a.

Compound 16: Compound 16 can be prepared in an analogous fashion to Figure 10 by using dimethylamine in place of azetidine in step a.

Compound 17: Compound 17 can be prepared in an analogous fashion to Figure 10 by using 2-methoxyethylamine in place of azetidine in step a.

Compound 18: Compound 18 can be prepared in an analogous fashion to Figure 10 by using piperidine in place of azetidine in step a.

Compound 19: Compound 19 can be prepared in an analogous fashion to Figure 10 by using morpholine in place of azetidine in step a.

Compound 21: A solution of compound 20 (0.4 eq) in DMSO is added to a solution of compound 11 (1 eq) and DIPEA (10 eq) in anhydrous DMSO at room temperature. The resulting solution is stirred overnight. The solution is dialyzed against distilled water for 3 days with dialysis tube MWCO 1000 while distilled water is changed every 12 hours. The solution in the tube is lyophilized to give compound 21.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 22

Compound 22: A solution of compound 21 in ethylenediamine is stirred overnight at 70 °C. The reaction mixture is concentrated under reduced pressure and the residue is purified by reverse phase chromatography to give compound 22.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 23

Compound 23: Compound 23 can be prepared in an analogous fashion to Figure 11 by replacing compound 20 with PEG-11 diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 24

Compound 24: Compound 24 can be prepared in an analogous fashion to Figure 11 by replacing compound 20 with PEG- 15 diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 25

Compound 25: Compound 25 can be prepared in an analogous fashion to Figure 11 by replacing compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 26 Compound 26: Compound 26 can be prepared in an analogous fashion to Figure 11 by replacing compound 20 with 3,3'-[[2,2-bis[[3-[(2,5-dioxo-l-pyrrolidinyI)oxy]-3-oxopropo xy] methyl]-l,3-propanediyl]bis(oxy)]bis-, l,l'-bis(2,5-dioxo-l-pyrrolidinyl)-propanoic acid ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 27

Compound 27: Compound 27 can be prepared in an analogous fashion to Figure 1 1 by replacing ethylenediamine with 2-aminoethyl ether in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 28

Compound 28: Compound 28 can be prepared in an analogous fashion to Figure 11 by replacing ethylenediamine with 1,5-diaminopentane in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 29

Compound 29: Compound 29 can be prepared in an analogous fashion to Figure 11 by replacing ethylenediamine with l,2-bis(2-aminoethoxy)ethane in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 30

Compound 30: Compound 30 can be prepared in an analogous fashion to Figure 11 by replacing compound 11 with compound 14 and compound 20 with PEG-1 1 diacetic acid di- NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 31

Compound 31: Compound 31 can be prepared in an analogous fashion to Figure 11 by replacing compound 11 with compound 15 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 32

Compound 32: Compound 32 can be prepared in an analogous fashion to Figure 11 by replacing compound 11 with compound 17 and compound 20 with PEG- 15 diacetic acid di- NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 33 Compound 33: Compound 33 can be prepared in an analogous fashion to Figure 11 by replacing compound 11 with compound 16 and compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 24

Compound 34: Compound 34 can be prepared in an analogous fashion to Figure 11 by replacing compound 11 with compound 18 in step a and replacing ethylenediamine with 2- aminoethyl ether in step b.

34 PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 36

Compound 36: To a solution of compound 12 in MeOH at room temperature is added compound 35 followed by cesium acetate (2.5 eq). The reaction mixture is stirred at room temperature until completion. The solvent is removed under reduced pressure. The product is purified by reverse phase chromatography to give compound 36.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 37

Compound 37: Compound 36 is dissolved in ethylenediamine and the reaction mixture is stirred overnight at 70 °C. The reaction mixture is concentrated under reduced pressure and the residue is purified by reverse phase chromatography to give compound 37.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 38

Compound 38: Compound 38 can be prepared in an analogous fashion to Figure 12 by substituting PEG-6-bis maleimidoylpropionamide for compound 35 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 39

Compound 39: Compound 39 can be prepared in an analogous fashion to Figure 12 by substituting compound 35 for, l,l'-[[2,2-bis[[3-(2,5-dihydro-2,5-dioxo-H-pyrrol-l-yl) propoxy]methyl]-l ,3-propanediyl]bis(oxy-3,l-propanediyl)]bis-l H-pyrrole-2, 5 _' dione in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 40

Compound 40: Compound 40 can be prepared in an analogous fashion to Figure 12 by substituting propylenediamine for ethylenediamine in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 44

Compound 41: To a stirred solution of compound 7 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 41.

Compound 42: To a degassed solution of compound 41 in anhydrous DCM at 0 °C is added Pd(PPh3)4 (0.1 eq), Bu3SnH (1.1 eq) and azidoacetic anhydride (2.0 eq). The ice bath is removed and the solution is stirred for 12 hrs under a N2 atmosphere at room temperature.

The reaction mixture is diluted with DCM, washed with water, dried over Na2SO4, then concentrated. The crude product is purified by column chromatography to give compound 42.

Compound 44: A solution of bispropagyl PEG-5 (compound 43) and compound 42 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSCVTHPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 70 °C. The solution is cooled to room temperature and concentrated under reduced pressure. The crude product is purified by chromatography to give compound 44.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 45

Compound 45: Compound 44 is dissolved in MeOH/i-PrOH (2/1) and hydrogenated in the presence of Pd(OH)2 (20 wt %) at 1 atm of ¾ gas pressure for 24 hrs at room temperature. The solution is filtered through a Celite pad. The filtrate is concentrated to give compound

45.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 46

Compound 46: Compound 45 is dissolved in ethyienediamine and stirred for 12 hrs at 70 °C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to give a compound 46.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 47 Compound 47: Compound 47 can be prepared in an analogous fashion to Figure 13 using 3- azidopropanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 48

Compound 48: Compound 48 can be prepared in an analogous fashion to Figure 13 using 4- azidobutanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 49

Compound 49: Compound 49 can be prepared in an analogous fashion to Figure 13 using 4- azidobutanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step b and using l,2-bis(2-propynyloxy) ethane in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 50

Compound 50: Compound 50 can be prepared in an analogous fashion to Figure 13 using 4,7, 10, 13, 16, 19,22,25,28,31-decaoxatetratriaconta-l, 33-diyne in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 51

Compound 51: Compound 51 can be prepared in an analogous fashion to Figure 13 using 3, 3'-[[2.2-bis[(2-propyn- 1 -yloxy)methyl]- 1 ,3-propanediyl]bis(oxy)]bis- 1 -propyne in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 52

Compound 52: Compound 52 can be prepared in an analogous fashion to Figure 13 using 3, 3'-[oxybis[[2,2-bis[(2-propyn-l-yloxy)methyl] _' 3, l-propanediyl]oxy]]bis-l -propyne in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 53

Compound 53: Compound 53 can be prepared in an analogous fashion to Figure 13 using butylenediamine in place of ethylenediamine in step e.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 54

Compound 54: Compound 54 can be prepared in an analogous fashion to Figure 13 using 4- azidobutanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step b and using l,2-bis(2-propynyloxy) ethane in place of compound 43 in step c and using 2-aminoethyl ether in step e.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 55 Compound 55: Compound 54 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 55.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 56

Compound 56: Compound 55 is dissolved in ethylenediamine and stirred for 12 hrs at 70 °C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to give a compound 56.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 57

Compound 57: Compound 57 can be prepared in an analogous fashion to Figure 14 using ethylamine in place of azetidine in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 58

Compound 58: Compound 58 can be prepared in an analogous fashion to Figure 14 using dimethylamine in place of azetidine in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 59

Compound 59: Compound 59 can be prepared in an analogous fashion to Figure 14 using 1 ,2-bis(2-aminoethoxy)ethane in place of ethylenediamine in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 66 Compound 60: To a stirred solution of compound 1 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 60.

Compound 62: Compound 61 is dissolved in acetonitrile at room temperature.

Benzaldehyde dimethylacetal (1.1 eq) is added followed by camphorsulfonic acid (0.2 eq). The reaction mixture is stirred until completion. Triethylamine is added. The solvent is removed and the residue separated by flash chromatography to afford compound 62.

Compound 63: Compound 62 is dissolved in pyridine at room temperature.

Dimethylaminopyridine (.01 eq) is added followed by chloroacetyl chloride (2 eq). The reaction mixture is stirred until completion. The solvent is removed under educed pressure. The residue is dissolved in ethyl acetate, transferred to a separatory funnel and washed two times with 0.1N HC1 and two times with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is separated by column chromatograph to afford compound 63.

Compound 64: Activated powdered 4A molecular sieves are added to a solution of compound 60 and compound 63 (2 eq) in dry DCM under argon. The mixture is stirred for 2 hours at room temperature. Solid DMTST (1.5 eq) is added in 4 portions over 1.5 hours. The reaction mixture is stirred overnight at room temperature. The reaction mixture is filtered through Celite, transferred to a separatory funnel and washed two times with half saturated sodium bicarbonate and two times with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 64.

Compound 65: Compound 64 is dissolved in DMF. Sodium azide (1.5 eq) is added and the reaction mixture is stirred at 50°C until completion. The reaction mixture is cooled to room temperature, diluted with ethyl acetate and transferred to a separatory funnel. The organic phase is washed 4 times with water then dried over sodium sulfate and concentrated. The residue is separated by column chromatography to afford compound 65.

Compound 66: A solution of bispropagyl PEG-5 (compound 43) and compound 65 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSCVTHPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 50°C. The solution is concentrated under reduced pressure. The crude product is purified by chromatography to give a compound 66.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 67

Compound 67: To a solution of compound 66 in dioxane/water (4/1) is added Pd(OH)2/C. The reaction mixture is stirred vigorously overnight under a hydrogen atmosphere. The reaction mixture is filtered through Celite and concentrated. The residue is purified by C-19 reverse phase column chromatography to afford compound 67.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 68 Compound 68: Compound 67 is dissolved in ethylenediamine and stirred for 12 hrs at 70 °C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to afford compound 68.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 69

Compound 69: Compound 69 can be prepared in an analogous fashion to Figure 17 by replacing compound 43 with PEG-8 bis propargyl ether in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 70

Compound 70: Compound 70 can be prepared in an analogous fashion to Figure 17 by replacing compound 43 with ethylene glycol bis propargyl ether in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 71 Compound 71: Compound 71 can be prepared in an analogous fashion to Figure 17 using 3, 3'-[[2,2-bis[(2-propyn-l-yloxy)methyl]-1,3-propanediyl]bis(o xy)]bis-l-propyne in place of compound 43 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 72

Compound 72: Compound 67 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 72.

I l l

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 73

Compound 73: Compound 72 is dissolved in ethylenediamine and stirred for 12 hrs at 70 °C. The reaction mixture is concentrated under reduced pressure. The crude product is purified by C-18 column chromatography followed by lyophilization to afford compound 73.

SYNTHESIS OF MULTIMERIC COMPOUND 76 Compound 75: To a degassed solution of compound 74 (synthesis described in WO 2013/096926) (0.5 g, 0.36 mmole) in anhydrous DCM (10 mL) at 0 °C was added Pd(PPh3)4 (42 mg, 36.3 mmole, 0.1 eq), Bu3SnH (110 mL, 0.4 mmole, 1.1 eq) and azidoacetic anhydride (0.14 g, 0.73 mmole, 2.0 eq). The resulting solution was stirred for 12 hrs under N2 atmosphere while temperature was gradually increased to room temperature. After the reaction was completed, the solution was diluted with DCM (20 mL), washed with distilled water, dried over Na2SO ₄ , then concentrated. The crude product was purified by combi-flash (EtOAc/Hex, Hex only - 3/2, v/v) to give compound 75 (0.33 g, 67%). MS: Calculated (C81H95N4O16, 1376.6), ES-Positive (1400.4, M+Na)).

Compound 76: A solution of bispropargyl PEG-5 (compound 43, 27 mg, 0.1 mmole) and compound 75 (0.33 g, 0.24 mmole, 2.4 eq) in a mixed solution (MeOH/1,4 dioxane, 2/1, v/v, 12 mL) was degassed at room temperature. A solution of CuSCVTHPTA in distilled water (0.04 M) (0.5 mL, 20 mmole, 0.2 eq) and sodium ascorbate (4.0 mg, 20 mmole, 0.2 eq) were added successively and the resulting solution was stirred 12 hrs at 70 °C. The solution was cooled to room temperature and concentrated under reduced pressure. The crude product was purified by combi-flash (EtOAc/MeOH, EtOAc only - 4/1, v/v) to give a compound 76 as a white foam (0.23 g, 70%).

SYNTHESIS OF MULTIMERIC COMPOUND 77

Compound 77: A solution of compound 76 (0.23 g, 0.76 mmole) in solution of MeOH/i- PrOH (2/1, v/v, 12 mL) was hydrogenated in the presence of Pd(OH)2 (0.2 g) and 1 atm of ¾ gas pressure for 24 hrs at room temperature. The solution was filtered through a Celite pad and the cake was washed with MeOH. The combined filtrate was concentrated under reduced pressure. The crude product was washed with hexane and dried under high vacuum to give compound 77 as a white solid (0.14 g, quantitative). MS: Calculated (C80H130N8O35, 1762.8), ES- positive (1785.4, M+Na), ES - Negative (1761.5, M-l, 879.8).

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 78

Compound 78: Compound 77 (60 mg, 34.0 mmole) was dissolved in ethylenediamine (3 mL) and the homogeneous solution was stirred for 12 hrs at 70 °C. The reaction mixture was concentrated under reduced pressure and the residue was dialyzed against distilled water with MWCO 500 dialysis tube. The crude product was further purified by C-18 column chromatography with water/MeOH (9/1 - 1/9, v/v) followed by lyophilization to give a compound 78 as a white solid (39 mg, 63%).

1H NMR (400 MHz, Deuterium Oxide) δ 8.00 (s, 2H), 5.26 - 5.14 (two d, J = 16.0 Hz, 4H), 4.52 (d, J = 4.0 Hz, 2H), 4.84 (dd, J = 8.0 Hz, J = 4.0 Hz, 2H), 4.66 (s, 4H), 4.54 (broad d, J = 12 Hz, 2H), 3.97 (broad t, 2H), 3.91 - 3.78 (m, 6H), 3.77 - 3.58 (m, 28H), 3.57 - 3.46(m,

4H), 3.42 (t, J = 8.0 Hz, 6H), 3.24 (t, J = 12.0 Hz, 2H),3.02 (t, J = 6.0Hz, 4H), 2.67 (s, 2H), 2.32 (broad t, J = 12 Hz, 2H), 2.22 - 2.06 (m, 2H), 1.96 - 1.74 (m, 4H), 1.73 - 1.39 (m, 18H), 1.38 - 1.21 (m, 6H), 1.20 - 0.99 (m, J = 8.0 Hz, 14H), 0.98 - 0.73 (m, J = 8.0 Hz, 10H).

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 79

Compound 79: Compound 79 can be prepared in an analogous fashion to Figure 19 using 3- azidopropanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 80

Compound 80: Compound 80 can be prepared in an analogous fashion to Figure 19 using 4- azidobutanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 81

Compound 81: Compound 81 can be prepared in an analogous fashion to Figure 19 using 4- azidobutanoic anhydride (Yang, C. et. al. JACS, (2013) 135(21), 7791-7794) in place of azidoacetic anhydride in step a and using l,2-bi(2-propynyloxy) ethane in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 82

Compound 82: Compound 82 can be prepared in an analogous fashion to Figure 19 using 4,7,10,13,16,19,22,25,28,31-decaoxatetratriaconta-l, 33-diyne in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 83

Compound 83: Compound 83 can be prepared in an analogous fashion to Figure 19 using 2- aminoethylether in place of ethylenediamine in step d.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 84

[0001] Compound 84: Compound 84 can be prepared in an analogous fashion to Figure 19 using l,2-bi(2-propynyloxy) ethane in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 85

Compound 85: Compound 85 can be prepared in an analogous fashion to Figure 19 using PEG-8 dipropargyl ether in place of compound 43 in step b and 1,5-diaminopentane in place of ethylenediamine in step d.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 86

Compound 86: Compound 77 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 86.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 87

Compound 87: Compound 86 is dissolved in ethylenediamine stirred for 12 hrs at 70 °C. The reaction mixture was concentrated under reduced pressure. The residue was purified by C-18 column chromatography followed by lyophilization to give a compound 87.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 88

Compound 88: Compound 88 can be prepared in an analogous fashion to Figure 20 using 2- aminoethylether in place of ethylenediamine in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 89

Compound 89: Compound 89 can be prepared in an analogous fashion to Figure 20 using dimethylamine in place of azetidine in step a and 2-aminoethylether in place of ethylenediamine in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 90

Compound 90: Compound 90 can be prepared in an analogous fashion to Figure 20 using piperidine in place of azetidine in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 91

Compound 91: Compound 91 can be prepared in an analogous fashion to Figures 11 and 12 using in PEG-9 bis-propargyl ether in place of compound 43 in step b of Scheme 11.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 92

Compound 92: Compound 92 can be prepared in an analogous fashion to Figures 11 and 12 using l,2-bi(2-propynyloxy) ethane in place of compound 43 in step b in Scheme 11.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 93

Compound 93: Compound 93 can be prepared in an analogous fashion to Figures 11 and 12 using l,2-bi(2-propynyloxy) ethane in place of compound 43 in step b in Scheme 11 and using 2-aminoethyl ether in place of ethylenediamine in step b of Scheme 12.

SYNTHESIS OF MULTIMERIC COMPOUND 95

Compound 95: Compound 22 and compound 94 (5 eq)(preparation described in

WO/2016089872) is co-evaporated 3 times from methanol and stored under vacuum for 1 hour. The mixture is dissolved in methanol under an argon atmosphere and stirred for 1 hour at room temperature. Sodium triacetoxyborohydride (15 eq) is added and the reaction mixture is stirred overnight at room temperature. The solvent is removed and the residue is separated by C-18 reverse phase chromatography.

The purified material is dissolved in methanol at room temperature. The pH is adjusted to 12 with IN NaOH. The reaction mixture is stirred at room temperature until completion. The pH is adjusted to 9. The solvent is removed under vacuum and the residue is separated by C- 18 reverse phase chromatography to afford compound 95.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 96

Compound 96: Compound 96 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 23 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 97

Compound 97: Compound 97 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 24 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 98

Compound 98: Compound 98 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 25 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 99 Compound 99: Compound 99 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 26 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 100

Compound 100: Compound 100 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 27 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 101

Compound 101: Compound 101 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 28 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 102 Compound 102: Compound 102 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 29 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 103

Compound 103: Compound 103 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 30 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 104

Compound 104: Compound 104 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 31 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 105

Compound 105: Compound 105 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 32 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 106 Compound 106: Compound 106 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 33 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 107

Compound 107: Compound 107 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 34 in step a.

107 PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 108

Compound 108: Compound 108 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 37 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 109

Compound 109: Compound 109 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 38 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 110

Compound 110: Compound 110 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 39 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 111

Compound 111: Compound 111 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 40 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 112

Compound 112: Compound 112 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 46 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 113 Compound 113: Compound 113 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 47 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 114

Compound 114: Compound 114 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 48 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 115

Compound 115: Compound 115 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 49 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 116

Compound 116: Compound 116 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 50 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 117

Compound 117: Compound 117 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 51 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 118

Compound 118: Compound 118 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 52 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 119

Compound 119: Compound 119 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 53 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 120

Compound 120: Compound 120 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 54 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 121

Compound 121: Compound 121 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 56 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 122

Compound 122: Compound 122 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 57 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 123 Compound 123: Compound 123 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 58 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 124

Compound 124: Compound 124 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 59 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 125

Compound 125: Compound 125 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 68 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 126

Compound 126: Compound 126 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 69 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 127

Compound 127: Compound 127 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 70 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 128

Compound 128: Compound 128 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 71 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 129

Compound 129: Compound 129 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 73 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 130

Compound 130: Compound 130 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 78 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 131 Compound 131: Compound 131 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 79 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 132

Compound 132: Compound 132 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 80 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 133

Compound 133: Compound 133 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 81 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 134

Compound 134: Compound 134 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 82 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 135

Compound 135: Compound 135 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 83 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 136

Compound 136: Compound 136 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 84 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 137

Compound 137: Compound 137 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 85 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 138

Compound 138: Compound 138 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 87 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 139

Compound 139: Compound 139 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 88 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 140

Compound 140: Compound 140 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 89 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 141

Compound 141: Compound 141 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 90 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 142

Compound 142: Compound 142 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 91 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 143

Compound 143: Compound 143 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 92 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 144

Compound 144: Compound 144 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 93 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 146

Compound 315: To a solution of compound 314 (1 gm, 3.89 mmol) (preparation described in WO 2007/028050) and benzyl trichloroacetaimidate (1.1 ml, 5.83 mmol) in anhydrous dichloromethane (10 ml) was added trimethylsilyl trifluoromethanesulfonate (70 uL, 0.4 mmol). The mixture was stirred at ambient temperature for 12 h. After this period the reaction was diluted with dichloromethane, washed with saturated NaHC0 ₃ , dried over MgS0 ₄ and concentrated. The residue was purified by column chromatography to give compound 315 (0.8 gm, 60 %).

Compound 316: To a solution of compound 315 (800 mg, 2.3 mmol) in anhydrous methanol (1 ml) and anhydrous methyl acetate (5 ml) was added 0.5M sodium methoxide solution in methanol (9.2 ml). The mixture was stirred at 40 °C for 4h. The reaction was quenched with acetic acid and concentrated. The residue was purified by column chromatography to afford compound 316 as mixture of epimers at the methyl ester with 75% equatorial and 25% axial epimer (242 mg, 35 %).

¹ H NMR (400 MHz, Chloroform-d) δ 7.48 - 7.32 (m, 6H), 4.97 (d, J = 11.1 Hz, 1H), 4.72 (dd, J = 11.1, 5.7 Hz, 1H), 3.77 - 3.65 (m, 6H), 3.22 - 3.15 (m, 1H), 2.92 - 2.82 (m, 1H), 2.39 (dddd, J = 15.7, 10.6, 5.1, 2.7 Hz, 2H), 1.60 (dtd, J = 13.9, 1 1.2, 5.4 Hz, 3H). MS: Calculated for C15H19N3O4 = 305.3, Found ES- positive m/z =306.1 (M+Na ⁺ ).

Compound 318: A solution of compound 317 (5 gm, 11.8 mmol) (preparation described in WO 2009/139719) in anhydrous methanol (20 ml) was treated with 0.5 M solution of sodium methoxide in methanol (5 ml) for 3h. Solvent was removed in vacuo and the residue was co- evaporated with toluene (20 ml) three times. The residue was dissolved in pyridine (20 ml) followed by addition of benzoyl chloride (4.1 ml, 35.4 mmol) over 10 minutes. The reaction mixture was stirred at ambient temperature under an atmosphere of argon for 22h. The reaction mixture was concentrated to dryness, dissolved in dichloromethane, washed with cold IN hydrochloric acid and cold water, dried over MgSO ₄ , filtered, and concentrated. The residue was purified by column chromatography to give compound 318. MS: Calculated for C33H27N3O7S = 609.2, Found ES- positive m/z = 610.2 (M+Na ⁺ ).

Compound 319: A mixture of compound 318 (2.4 gm, 3.93 mmol), diphenyl sulfoxide (1.5 gm, 7.3 mmol) and 2,6-di-tert-butyl pyridine (1.8 gm, 7.8 mmol) was dissolved in anhydrous dichloromethane (10 ml) at room temperature. The reaction mixture was cooled to -60 °C. Triflic anhydride (0.62 ml, 3.67 mmol) was added dropwise and the mixture was stirred for 15 minutes at the same temperature. A solution of compound 316 (0.8 gm, 2.6 mmol) in anhydrous dichloromethane (10 ml) was added dropwise to the reaction mixture. The mixture was allowed to warm to 0 °C over 2h. The reaction mixture was diluted with

dichloromethane, transferred to a separatory tunnel and washed with saturated sodium bicarbonate solution followed by brine. The organic phase was dried over MgSO ₄ , filtered, and concentrated. The residue was separated by column chromatography to afford compound 319 as a white solid (1.2 gm, 57%). MS: Calculated for C42H40N6O11 = 804.3, Found ES- positive m/z = 805.3 (M+Na ⁺ ).

Compound 320: To a solution of compound 319 (1.2 gm 2.067 mmol) and 2-fluorophenyl acetylene (1.2 ml, 10.3 mmol) in methanol (30 ml) was added a stock solution of copper sulfate and tris(3-hydroxypropyltriazolylmethyl) amine in water (2.58 ml). The reaction was initiated by addition of an aqueous solution of sodium ascorbate (0.9 gm, 4.5 mmol) and the mixture was stirred at ambient temperature for 16 hours. The mixture was co-evaporated with dry silica gel and purified by column chromatography to afford compound 320 as a white solid (1.2 gm, 77%). Stock solution of Copper Sulfate/THPTA - (100 mg of copper sulfate pentahydrate and 200 mg of tris(3-hydroxypropyltriazolylmethyl)amine were dissolved in 10 ml of water).

^! H NMR (400 MHz, Chloroform-d) δ 8.07 - 8.00 (m, 2H), 7.96 (ddd, J = 9.8, 8.2, 1.3 Hz, 4H), 7.79 (d, J = 5.4 Hz, 2H), 7.65 - 7.53 (m, 5H), 7.43 (ddt, J = 22.4, 10.7, 5.0 Hz, 7H), 7.25 - 7.01 (m, 9H), 6.92 (td, J = 7.6, 7.1, 2.2 Hz, 1H), 6.13 - 6.02 (m, 2H), 5.58 (dd, J = 1 1.6, 3.2 Hz, 1H), 5.15 (d, J = 7.5 Hz, 1H), 4.98 (d, J = 10.3 Hz, 1H), 4.68 (dd, J = 11.2, 5.7 Hz, 1H), 4.52 (dq, J = 22.1, 6.6, 5.6 Hz, 2H), 4.35 (dd, J = 11.1, 7.6 Hz, 1H), 4.28 - 4.18 (m, 1H), 4.11 (d, J = 10.3 Hz, 1H), 3.87 (t, J = 9.1 Hz, 1H), 3.71 (s, 3H), 2.95 (s, 1H), 2.62 - 2.43 (m, 3H), 1.55 (dt, J = 12.7, 6.1 Hz, 1H). MS: Calculated for C58H50N6O11 = 1044.4, Found ES- positive m/z = 1045.5 (M+Na ⁺ ).

Compound 145: To a solution of compound 320 (1.2 gm, 1.1 mmol) in Ao-propanol (40 ml) was added Na-metal (80 mg, 3.4 mmol) at ambient temperature and the mixture was stirred for 12 hours at 50 °C. 10% aqueous sodium hydroxide (2 ml) was added to the reaction mixture and stirring continued for another 6 hours at 50 °C. The reaction mixture was cooled to room temperature and neutralized with 50% aqueous hydrochloric acid. To the mixture was added 10% Pd(OH)2 on carbon (0.6 gm) and the reaction mixture was stirred under an atmosphere of hydrogen for 12 hours. The reaction mixture was filtered through a Celite pad and concentrated. The residue was separated by HPLC to give compound 145 as a white solid (0.5 gm, 70%). HPLC Conditions - Waters preparative HPLC system was used with ELSD & PDA detectors. Kinetex XB- C18, 100 A, 5 uM, 250 x 21.2 mm column (from Phenomenex) was used with 0.2% formic acid in water as solvent A and acetonitrile as solvent B at a flow rate of 20 mL/min.

¹ H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.68 (s, 1H), 7.77 - 7.60 (m, 5H), 7.49 (tdd, J = 8.3, 6.1, 2.6 Hz, 3H), 7.15 (tt, J = 8.6, 3.2 Hz, 3H), 4.83 (dd, J = 10.9, 3.1 Hz, 1H), 4.63 (d, J = 7.5 Hz, 1H), 4.53 - 4.41 (m, 1H), 4.10 (dd, J = 10.9, 7.5 Hz, 1H), 3.92 (d, J = 3.2 Hz, 1H), 3.74 (h, J = 6.0, 5.6 Hz, 3H), 3.65 - 3.24 (m, 5H), 2.37 (d, J = 13.4 Hz, 1H), 2.24 - 2.04 (m, 2H), 1.93 (q, J = 12.5 Hz, 1H), 1.46 (t, J = 12.1 Hz, 1H). MS: Calculated for C29H30F2N6O8 = 628.2, Found ES- positive m/z - 629.2 (M+Na ⁺ )

Compound 146: To a solution of compound 145 (3 eq) in anhydrous DMF was added HATU (3.3 eq) and DIPEA (5 eq). The mixture was stirred at ambient temperature for 15 minutes followed by addition of compound 22 (1 eq). The mixture was stirred at ambient temperature for 12h. The solvent was removed in vacuo and the residue was purified by HPLC to afford compound 146.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 147

Compound 147: Compound 147 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 23.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 148

Compound 148: Compound 148 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 24.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 149

Compound 149: Compound 149 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 25.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 150

Compound 150: Compound 150 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 26.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 151

Compound 151: Compound 151 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 27.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 152

Compound 152: Compound 152 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 28.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 153 Compound 153: Compound 153 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 29.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 154

Compound 154: Compound 154 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 30.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 155 Compound 155: Compound 155 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 31.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 156

Compound 156: Compound 156 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 32.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 157

Compound 157: Compound 157 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 33.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 158

Compound 158: Compound 158 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 34.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 159

Compound 159: Compound 159 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 37.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 160 Compound 160: Compound 160 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 38.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 161

Compound 161: Compound 161 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 39.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 162

Compound 162: Compound 162 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 40.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 163

Compound 163: Compound 163 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 46.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 164 Compound 164: Compound 164 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 47.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 165

Compound 165: Compound 165 can be prepared in an analogous fashion to Figure 21 by replacing compound 22 with compound 48.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 166

Compound 166: Compound 166 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 49.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 167

Compound 167: Compound 167 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 50. PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 168

Compound 168: Compound 168 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 51.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 169

Compound 169: Compound 169 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 52.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 170

Compound 170: Compound 170 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 53.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 171

Compound 171: Compound 171 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 54.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 172 Compound 172: Compound 172 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 56.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 173

Compound 173: Compound 173 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 57.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 174

Compound 174: Compound 174 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 58.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 175

Compound 175: Compound 175 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 59.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 176

Compound 176: Compound 176 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 68.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 177

Compound 177: Compound 177 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 69.

177

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 178

Compound 178: Compound 178 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 70.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 179

Compound 179: Compound 179 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 71.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 180

Compound 180: Compound 180 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 73.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 181

Compound 181: Compound 181 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 182 Compound 182: Compound 182 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 79.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 183

Compound 183: Compound 183 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 80.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 184

Compound 184: Compound 184 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 81.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 185

Compound 185: Compound 185 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 82.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 186

Compound 186: Compound 186 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 83.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 187

Compound 187: Compound 187 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 84.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 188

Compound 188: Compound 188 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 85.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 189

Compound 189: Compound 189 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 87.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 190

Compound 190: Compound 190 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 88.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 191

Compound 191: Compound 191 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 89.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 192

Compound 192: Compound 192 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 90.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 193

Compound 193: Compound 193 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 91.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 194

Compound 194: Compound 194 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 92.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 195

Compound 195: Compound 195 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 93.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 197

Compound 197: To a solution of compound 22 (1 eq) in anhydrous DMSO was acetic acid NHS ester (compound 196) (5 eq). The mixture was stirred at ambient temperature for 12 hours. The solvent was removed in vacuo and the residue was purified by HPLC to afford compound 197.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 198 Compound 198: Compound 198 can be prepared in an analogous fashion to Figure 23 by replacing compound 196 with NHS-methoxyacetate.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 199

Compound 199: Compound 199 can be prepared in an analogous fashion to Figure 23 by replacing compound 196 with PEG- 12 propionic acid NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 200 Compound 200: Compound 200 can be prepared in an analogous fashion to Figure 23 by replacing compound 22 with compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 201

Compound 201: Compound 201 can be prepared in an analogous fashion to Figure 23 by replacing compound 22 with compound 78 and replacing compound 196 with NHS- methoxyacetate.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 202 Compound 202: Compound 202 can be prepared in an analogous fashion to Figure 23 by replacing compound 22 with compound 78 and replacing compound 196 with PEGG2 propionic acid NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 203

Compound 203: Compound 203 can be prepared in an analogous fashion to Figure 23 by replacing compound 22 with compound 78.

SYNTHESIS OF MULTIMERIC COMPOUND 206

Compound 205: A solution of compound 204 (synthesis described in Mead, G. et. al, Bioconj. Chem., 2015, 25, 1444 - 1452) (0.25 g, 0.53 mmole) and propiolic acid (0.33 mL, 5.30 mmole, 10 eq) in distilled water (1.5 mL) was degassed. A solution of CUSO4/THPTA in distilled water (0.04 M) (1.3 mL, 53 mmole, 0.1 eq) and sodium ascorbate (21 mg, 0.11 mmole, 0.2 eq) were added successively and the resulting solution was stirred 3 hrs at room temperature. The reaction mixture was concentrated under reduced pressure and partially purified by C-18 column chromatography (water/MeOH, water only - 5/5, v/v). The resulting material was further purified by C-18 column chromatography eluting with water to afford compound 205 (0.16 g, 0.34 mmole, 64%). MS: (Calculated for CsHio3N3Na30i4S3, 537.34), ES-Negative (513.5, M-Na-1).

Compound 206: To a solution of compound 205 (7.5 mg, 14 mmole), DIPEA (2.4 pL, 14 mmole) and a catalytic amount of DMAP in DMF/DMSO (3/1, v/v, 0.15 mL) at 0 °C was added EDCI (1.6 mg, 8.22 pinole). The solution was stirred for 20 min. This solution was slowly added to a solution of compound 78 (5.0 mg, 2.7 mmole) in DMF/DMSO (3/1, v/v, 0.2 mL) cooled at 0 °C. The resulting solution was stirred 12 hrs allowing the reaction temperature to increase to room temperature. The reaction mixture was purified directly by HPLC. The product portions were collected, concentrated under reduced pressure, then lyophilized to give compound 206 as a white solid (0.4 mg, 1.15 mmole, 1.1%). MS:

Calculated ^ ES-Negative (907.7, M/3; 881.0, M-lS0 ₃ /3; 854.1 M-2SO3/3; 685.8 M+lNa/4; 680.5 M/4); Fraction ofRJ= 10.65 min, 1399.4, M+7Na- ISO3/2; 959.3 M+7Na/3; M+7Na-lS0 ₃ /3; 724.8, M+8Na/4; 549.M+lNa/5; 460.9 M+2Na/6; 401.M+4Na/7).

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 207

Compound 207: Compound 207 can be prepared in an analogous fashion to Figure 25 by replacing compound 78 with compound 22.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 208

Compound 208: Compound 208 can be prepared in an analogous fashion to Figure 25 using compound 83 in place of compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 209

Compound 209: Compound 209 can be prepared in an analogous fashion to Figure 25 using compound 87 in place of compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 210

Compound 210: Compound 210 can be prepared in an analogous fashion to Figure 25 using compound 93 in place of compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 211

Compound 211: Compound 211 can be prepared in an analogous fashion to Figure 25 using compound 37 in place of compound 78.

SYNTHESIS OF MULTIMERIC COMPOUND 218

Compound 213: Prepared according to Bioorg. Med Chem. Lett. 1995, 5, 2321-2324 starting with D-threonolactone.

Compound 214: Compound 213 (500 mg, 1 mmol) was dissolved in 9 mL acetonitrile. Potassium hydroxide (1 mL of a 2M solution) was added and the reaction mixture was stirred at 50°C for 12 hours. The reaction mixture was partitioned between dichloromethane and water. The phases were separated and the aqueous phase was extracted 3 times with dichloromethane. The aqueous phase was acidified with IN HC1 until pH ~ 1 and extracted 3 times with dichloromethane. The combined dichloromethane extracts from after acidification of the aqueous phase were concentrated in vacuo to give compound 214 as a yellow oil (406 mg). LCMS (C-18; 5-95 H ₂ O/MeCN): UV (peak at 4.973 min), positive mode: m/z= 407 [M+H] ⁺ ; negative mode: m/z= 405 [M-H] C25H26O ₅ (406).

Compound 215: Prepared in an analogous fashion to compound 214 using L- erythronolactone as the starting material. LCMS (C-18; 5-95 H ₂ 0/MeCN): ELSD (5.08 min), UV (peak at 4.958 min), positive mode: m/z= 407 [M+H] ⁺ ; negative mode: m/z= 405 [M-H] C25H26O5 (406).

Compound 216: Prepared in an analogous fashion to compound 214 using L-threonolactone as the starting material. LCMS (C-18; 5-95 H ₂ O/MeCN): ELSD (5.08 min), UV (peak at 4.958 min), positive mode: m/z= 407 [M+H] ⁺ ; negative mode: m/z= 405 [M-H] ^' C ₂₅ H ₂₆ O ₅ (406).

Compound 217: Prepared in an analogous fashion to compound 214 using D- erythronolactone as the starting material. LCMS (C-18; 5-95 PhO/MeCN): ELSD (5.08 min), UV (peak at 4.958 min), positive mode: m/z= 407 [M+H] ⁺ ; negative mode: m/z= 405 [M-H] C25H26O5 (406).

Compound 218: To a solution of compound 214 (3 eq) in anhydrous DMF was added HATU (3.3 eq) and DIPEA (5 eq). The mixture was stirred at ambient temperature for 15 minutes followed by addition of compound 78 (1 eq). The mixture was stirred at ambient temperature for 12h. The solvent was removed in vacuo and the residue was purified by HPLC to afford compound 218.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 219

Compound 219: Compound 218 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 219.

SYNTHESIS OF MULTIMERIC COMPOUND 220

Compound 220: A solution of the sulfur trioxide pyridine complex (100 eq) and compound 219 (1 eq) in pyridine was stirred at 67 °C for lh. The reaction mixture was concentrated under vacuum. The resulting solid was dissolved in water and cooled to 0 °C. A IN solution of NaOH was then added slowly until pH~10 and the latter was freeze dried. The resulting residue was purified by Gel Permeation (water as eluent). The collected fractions were lyophilised to give compound 220.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 221

Compound 221: Compound 221 can be prepared in an analogous fashion to Figure 27 by replacing compound 214 with compound 215.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 222

Compound 222: Compound 222 can be prepared in an analogous fashion to Figure 27 by replacing compound 214 with compound 216.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 223

Compound 223: Compound 223 can be prepared in an analogous fashion to Figure 27 by replacing compound 214 with compound 217.

SYNTHESIS OF MULTIMERIC COMPOUND 224

Compound 224: To a solution of compound 78 in anhydrous DMSO was added a drop of DIPEA and the solution was stirred at room temperature until a homogeneous solution was obtained. A solution of succinic anhydride (2.2 eq) in anhydrous DMSO was added and the resulting solution was stirred at room temperature overnight. The solution was lyophilized to dryness and the crude product was purified by HPLC to give compound 224.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 225

Compound 225: Compound 225 can be prepared in an analogous fashion to Figure 28 substituting glutaric anhydride for succinic anhydride.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 226

Compound 226: Compound 226 can be prepared in an analogous fashion to Figure 28 substituting compound 87 for compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 227

Compound 227: Compound 227 can be prepared in an analogous fashion to Figure 28 substituting phthalic anhydride for succinic anhydride.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 228

Compound 228: Compound 228 can be prepared in an analogous fashion to Figure 28 using compound 83 in place of compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 229

Compound 229: Compound 229 can be prepared in an analogous fashion to Figure 28 using compound 87 in place of compound 78.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 245

Compound 231: A mixture of compounds 230 (preparation described in Schwizer, et. ah, Chem. Eur J, 2012, 18, 1342) and compound 2 (preparation described in WO 2013/096926) (1.7 eq) is azeotroped 3 times from toluene. The mixture is dissolved in DCM under argon and cooled on an ice bath. To this solution is added boron trifluoride etherate (1.5 eq). The reaction mixture is stirred 12 hours at room temperature. The reaction is quenched by the addition of triethylamine (2 eq). The reaction mixture is transferred to a separatory funnel and washed 1 time with half saturated sodium bicarbonate solution and 1 time with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound 231.

Compound 232: Compound 231 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 2 times with water. The organic phase is dried over magnesium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 232.

Compound 233: To a solution of compound 232 in dichloromethane cooled on an ice bath is added DABCO (1.5 eq) followed by monomethyoxytrityl chloride (1.2 eq). The reaction mixture is stirred overnight allowing to warm to room temperature. The reaction mixture is concentrated and the residue is purified by flash chromatography to afford compound 233.

Compound 234: To a solution of compound 233 in methanol is added dibutyltin oxide (1.1 eq). The reaction mixture is refluxed for 3 hours then concentrated. The residue is suspended in DME. To this suspension is added compound 6 (preparation described in Thoma et. al. J. Med. Chem., 1999, 42, 4909) (1.5 eq) followed by cesium fluoride (1.2 eq). The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel, and washed with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is purified by flash

chromatography to afford compound 234.

4

Compound 235: To a degassed solution of compound 234 in anhydrous DCM at 0 °C is added Pd(PPh3)4 (0.1 eq), BusSnH (1.1 eq) and N-trifluoroacetyl glycine anhydride (2.0 eq) (preparation described in Chemische Berichte (1955), 88(1), 26). The resulting solution is stirred for 12 hrs allowing the temperature to increase to room temperature. The reaction mixture is diluted with DCM, transferred to a separatory funnel, and washed with water. The organic phase is dried over Na ₂ S0 ₄ , then filtered and concentrated. The residue is purified by flash chromatography to afford compound 235.

Compound 236: Compound 235 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 236.

Compound 237: Compound 236 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (1.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-l 8 reverse phase chromatography to afford compound 237.

237

Compound 238: Compound 238 can be prepared in an analogous fashion to Figure 29 by substituting (acetylthio)acetyl chloride for N-trifluoroacetyl glycine anhydride in step e.

Compound 239: Compound 239 can be prepared in an analogous fashion to Figure 29 by substituting the vinylcyclohexyl analog of compound 230 (preparation described in Schwizer, et. al, Chem. Eur. 2012, 18, 1342) for compound 230 in step a.

Compound 240: Compound 236 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (1.5 eq) is added followed by HATU (1.1 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (2 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by flash chromatography to afford compound 240.

240

Compound 241: Compound 240 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.3 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase

chromatography to afford compound 241.

Compound 242: Compound 242 can be prepared in an analogous fashion to Figure 30 by using methylamine in place of azetidine in step a.

Compound 243: Compound 243 can be prepared in an analogous fashion to Figure 30 by using dimethylamine in place of azetidine in step a.

Compound 244: Compound 244 can be prepared in an analogous fashion to Figure 30 by using the ethylcyclohexyl analog of compound 236 in place of compound 236 in step a.

Compound 245: A solution of compound 20 (0.4 eq) in DMSO is added to a solution of compound 237 (1 eq) and DIPEA (10 eq) in anhydrous DMSO at room temperature. The resulting solution is stirred overnight. The reaction mixture is separated by reverse phase chromatography and the product lyophilized to give compound 245.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 246

Compound 246: Compound 246 can be prepared in an analogous fashion to Figure 31 by replacing compound 20 with PEG-11 diacetic acid di-NHS ester.

2

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 247 Compound 247: Compound 247 can be prepared in an analogous fashion to Figure 31 by replacing compound 20 with PEG- 15 diacetic acid di-NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 248

Compound 248: Compound 248 can be prepared in an analogous fashion to Figure 31 by replacing compound 20 with ethylene glycol diacetic acid di-NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 249 Compound 249: Compound 249 can be prepared in an analogous fashion to Figure 31 by replacing compound 20 with 3,3'-[[2,2-bis[[3-[(2,5-dioxo-l-pyrrolidinyl)oxy]-3-oxopropo xy] methyl]-l,3-propanediyl]bis(oxy)]bis-, l,l'-bis(2,5-dioxo-l-pyrrolidinyl)-propanoic acid ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 250

Compound 250: Compound 250 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 239.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 251 Compound 251: Compound 251 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 241 and compound 20 with PEG-11 diacetic acid di-NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 252

Compound 252: Compound 252 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 242.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 253

Compound 253: Compound 253 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 243 and compound 20 with ethylene glycol diacetic acid di-NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 254

Compound 254: Compound 254 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 244 and compound 20 with PEG-11 diacetic acid di-NHS ester.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 255

Compound 255: Compound 255 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 241 and compound 20 with l,l'-[oxybis[(l-oxo-2,l- ethanediyl)oxy]]bis-2,5-pyrrolidinedione.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 256

Compound 256: Compound 256 can be prepared in an analogous fashion to Figure 31 by replacing compound 237 with compound 244 and compound 20 with l,l'-[oxybis[(l-oxo-2,l- ethanediyl)oxy] ]bis-2, 5 -pyrrolidinedione .

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 257

Compound 257: To a solution of compound 238 in MeOH at room temperature is added compound 35 followed by cesium acetate (2.5 eq). The reaction mixture is stirred at room temperature until completion. The solvent is removed under reduced pressure. The product is purified by reverse phase chromatography to give compound 257.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 258

Compound 258: Compound 258 can be prepared in an analogous fashion to Figure 32 by substituting PEG-6-bis maleimidoylpropionamide for compound 35.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 259 Compound 259: Compound 259 can be prepared in an analogous fashion to Figure 32 by substituting compound 35 for, 1 , 1 '-[[2,2-bis[[3-(2,5-dihydro-2,5-dioxo-l H-pyrrol- 1 -yl) propoxy]methyl]- l ,3-propanediyl]bis(oxy-3,l -propanediyl)]bis-l H-pyrrole-2, 5-dione.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 261

Compound 260: To a degassed solution of compound 234 in anhydrous DCM at 0 °C is added Pd(PPh3)4 (0.1 eq), BusSnH (1.1 eq) and azidoacetic anhydride (2.0 eq). The ice bath is removed and the solution is stirred for 12 hrs under a N2 atmosphere at room temperature.

The reaction mixture is diluted with DCM, washed with water, dried over Na ₂ SQ ₄ , then concentrated. The crude product is purified by column chromatography to give compound

260.

Compound 261: A solution of bis-propagyl PEG-5 (compound 43) and compound 260 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSCVTHPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 70 °C. The solution is cooled to room temperature and concentrated under reduced pressure. The crude product is purified by chromatography to give compound 261.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 262

Compound 262: Compound 261 is dissolved in MeOH and hydrogenated in the presence of Pd(OH)2 (20 wt %) at 1 atm of ¾ gas pressure for 24 hrs at room temperature. The solution is filtered through a Celite pad. The filtrate is concentrated to give compound 262.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 263

Compound 263: Compound 262 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 263.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 264

Compound 264: Compound 264 can be prepared in an analogous fashion to Figure 33 using 4,7,10,13,16,19,22,25,28,31-decaoxatetratriaconta-l, 33-diyne in place of compound 43 in step b.

4

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 265

Compound 265: Compound 265 can be prepared in an analogous fashion to Figure 33 using

3,3'-[[2,2-bis[(2-propyn-l-yloxy)methyl]-l,3-prOpanediyl] bis(oxy)]bis-l-propyne in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 266

Compound 266: Compound 266 can be prepared in an analogous fashion to Figure 33 using 3,3'-[oxybis[[2,2-bis[(2-propyn-l-yloxy)methyl]-3,l-propaned iyl]oxy]]bis-1 -propyne in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 267

Compound 267: Compound 267 can be prepared in an analogous fashion to Figure 33 using ethylamine in place of azetidine in step d.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 268

Compound 268: Compound 268 can be prepared in an analogous fashion to Figure 33 using dimethylamine in place of azetidine in step d.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 269

Compound 269: Compound 269 can be prepared in an analogous fashion to Figure 33 using the analog of compound 234 prepared from vinylcyclohexane in place of compound 234 in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 270 Compound 270: Compound 270 can be prepared in an analogous fashion to Figure 33 using propargyl ether in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 271

Compound 271: Compound 271 can be prepared in an analogous fashion to Figure 33 using propargyl ether in place of compound 43 in step b.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 274

Compound 272: Activated powdered 4Ά molecular sieves are added to a solution of compound 230 and compound 63 (2 eq) in dry DCM under argon. The mixture is stirred for 2 hours at room temperature. Solid DMTST (1.5 eq) is added in 4 portions over 1.5 hours. The reaction mixture is stirred overnight at room temperature. The reaction mixture is filtered through Celite, transferred to a separatory funnel and washed two times with half saturated sodium bicarbonate and two times with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 272.

Compound 273: Compound 272 is dissolved in DMF. Sodium azide (1.5 eq) is added and the reaction mixture is stirred at 50°C until completion. The reaction mixture is cooled to room temperature, diluted with ethyl acetate and transferred to a separatory funnel. The organic phase is washed 4 times with water then dried over sodium sulfate and concentrated. The residue is separated by column chromatography to afford compound 273.

3

Compound 274: A solution of bispropagyl PEG-5 (compound 43) and compound 273 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSCVTHPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 50°C. The solution is concentrated under reduced pressure. The crude product is purified by chromatography to give a compound 274.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 275

Compound 275: To a solution of compound 274 in dioxane/water (4/1) is added Pd(OH)2/C. The reaction mixture is stirred vigorously overnight under a hydrogen atmosphere. The reaction mixture is filtered through Celite and concentrated. The residue is purified by C-18 reverse phase column chromatography to afford compound 275.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 276 Compound 276: Compound 275 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by FIATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 276.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 111

Compound 277: Compound 277 can be prepared in an analogous fashion to Figure 34 by replacing compound 43 with PEG-8 bis propargyl ether in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 278

Compound 278: Compound 278 can be prepared in an analogous fashion to Figure 34 by replacing compound 43 with ethylene glycol bis propargyl ether in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 279

Compound 279: Compound 279 can be prepared in an analogous fashion to Figure 34 using 3,3'-[[2,2-bis[(2-propyn-l-yloxy)methyl]-l,3-propanediyl]bis (oxy)]bis-l -propyne in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 280

Compound 280: Compound 280 can be prepared in an analogous fashion to Figure 34 using propargyl ether in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 281

Compound 281: Compound 281 can be prepared in an analogous fashion to Figure 34 using propargyl ether in place of compound 36 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 282

Compound 282: Compound 282 can be prepared in an analogous fashion to Figure 34 by replacing compound 43 with ethylene glycol bis propargyl ether in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 294

Compound 284: A mixture of compounds 283 (preparation described in WO 2007/028050) and compound 2 (preparation described in WO 2013/096926) (1.7 eq) is azeotroped 3 times from toluene. The mixture is dissolved in DCM under argon and cooled on an ice bath. To this solution is added boron trifluoride etherate (1.5 eq). The reaction mixture is stirred 12 hours at room temperature. The reaction is quenched by the addition of triethylamine (2 eq). The reaction mixture is transferred to a separatory funnel and washed 1 time with half saturated sodium bicarbonate solution and 1 time with water. The organic phase is dried over sodium sulfate, filtered, and concentrated. The residue is purified by flash chromatography to afford compound 284.

Compound 285: Compound 284 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (0.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel and washed 2 times with water. The organic phase is dried over magnesium sulfate, filtered and concentrated. The residue is separated by flash chromatography to afford compound 285.

Compound 286: To a solution of compound 285 in dichloromethane cooled on an ice bath is added DABCO (1.5 eq) followed by monomethyoxytrityl chloride (1.2 eq). The reaction mixture is stirred overnight allowing to warm to room temperature. The reaction mixture is transferred to a separatory funnel and washed 2 times with water. The organic phase is concentrated and the residue is purified by flash chromatography to afford compound 286.

2

Compound 287: To a solution of compound 286 in methanol is added dibutyltin oxide (1.1 eq). The reaction mixture is refluxed for 3 hours then concentrated. The residue is suspended in DME. To this suspension is added compound 6 (preparation described in Thoma et. al J. Med. Chem., 1999, 42, 4909) (1.5 eq) followed by cesium fluoride (1.2 eq). The reaction mixture is stirred at room temperature overnight. The reaction mixture is diluted with ethyl acetate, transferred to a separatory funnel, and washed with water. The organic phase is dried over sodium sulfate, filtered and concentrated. The residue is purified by flash

chromatography to afford compound 287.

Compound 288: To a degassed solution of compound 287 in anhydrous DCM at 0 °C is added Pd(PPli3)4 (0.1 eq), BuaSnH (1.1 eq) and N-trifluoroacetyl glycine anhydride (2.0 eq) (preparation described in Chemische Berichte (1955), 88(1), 26). The resulting solution is stirred for 12 hrs allowing the temperature to increase to room temperature. The reaction mixture is diluted with DCM, transferred to a separatory funnel, and washed with water. The organic phase is dried over Na ₂ SC>4, then filtered and concentrated. The residue is purified by flash chromatography to afford compound 288.

Compound 289: To a stirred solution of compound 288 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 289.

Compound 290: Compound 289 is dissolved in methanol and degassed. To this solution is added Pd(OH)2/C. The reaction mixture is vigorously stirred under a hydrogen atmosphere for 12 hours. The reaction mixture is filtered through a Celite pad. The filtrate is concentrated under reduced pressure to give compound 290.

Compound 291: Compound 290 is dissolved in methanol at room temperature. A solution of sodium methoxide in methanol (1.1 eq) is added and the reaction mixture stirred overnight at room temperature. The reaction mixture is quenched by the addition of acetic acid. The reaction mixture is concentrated. The residue is separated by C-18 reverse phase chromatography to afford compound 291.

Compound 292: Compound 292 can be prepared in an analogous fashion to Figure 35 by replacing orotic acid chloride with acetyl chloride in step f.

Compound 293: Compound 293 can be prepared in an analogous fashion to Figure 35 by replacing orotic acid chloride with benzoyl chloride in step f.

Compound 294: A solution of compound 291 (0.4 eq) in DMSO is added to a solution of compound 20 (1 eq) and DIPEA (10 eq) in anhydrous DMSO at room temperature. The resulting solution is stirred overnight. The reaction mixture is separated by reverse phase chromatography and the product lyophilized to give compound 294.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 295

Compound 295: Compound 294 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 295.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 296

Compound 296: Compound 296 can be prepared in an analogous fashion to Figure 36 by replacing compound 20 with ethylene glycol diacetic acid di-NFIS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 297

Compound 297: Compound 297 can be prepared in an analogous fashion to Figure 36 by replacing compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 298

Compound 298: Compound 298 can be prepared in an analogous fashion to Figure 36 by replacing compound 291 with compound 292 and compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 299

Compound 299: Compound 299 can be prepared in an analogous fashion to Figure 36 by replacing compound 291 with compound 292 and compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 300

Compound 300: Compound 300 can be prepared in an analogous fashion to Figure 36 by replacing compound 291 with compound 293 and compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 301

Compound 301: Compound 301 can be prepared in an analogous fashion to Figure 36 by replacing compound 291 with compound 293 and compound 20 with ethylene glycol diacetic acid di-NHS ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 302

Compound 302: Compound 302 can be prepared in an analogous fashion to Figure 28 by replacing compound 20 with 3,3'-[[2,2-bis[[3-[(2,5-dioxo-l-pyrrolidinyl)oxy]-3-oxopropo xy] methyl]- l,3-propanediyl]bis(oxy)]bis-, l,l'-bis(2,5-dioxo-l-pyrrolidinyl)-propanoic acid ester in step a.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 305

Compound 303: To a stirred solution of compound 287 in DCM/MeOH (25/1) at room temperature is added orotic acid chloride (5 eq) and triphenylphosphine (5 eq). The reaction mixture is stirred 24 hours. The solvent is removed and the residue is separated by column chromatography to afford compound 303.

303

Compound 304: To a degassed solution of compound 303 in anhydrous DCM at 0 °C is added Pd(PPh3)4 (0.1 eq), BusSnH (1.1 eq) and azidoacetic anhydride (2.0 eq). The ice bath is removed and the solution is stirred for 12 hrs under a N2 atmosphere at room temperature.

The reaction mixture is diluted with DCM, washed with water, dried over Na ₂ S0 ₄ , then concentrated. The crude product is purified by column chromatography to give compound

304.

Compound 305: A solution of bispropagyl PEG-5 (compound 43) and compound 304 (2.4 eq) in MeOH is degassed at room temperature. A solution of CuSCVTHPTA in distilled water (0.04 M) (0.2 eq) and sodium ascorbate (0.2 eq) are added successively and the resulting solution is stirred 12 hrs at 50 °C. The solution is cooled to room temperature and concentrated under reduced pressure. The crude product is purified by chromatography to give compound 305.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 306

Compound 306: Compound 305 is dissolved in MeOH and hydrogenated in the presence ofPd(OH) ₂ (20 wt %) at 1 atm of ¾ gas pressure for 24 hrs at room temperature. The solution is filtered through a Celite pad. The filtrate is concentrated to give compound 306.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 307

Compound 307: Compound 306 is dissolved in DMF and cooled on an ice bath.

Diisopropylethylamine (2.5 eq) is added followed by HATU (2.2 eq). The reaction mixture is stirred 15 minutes on the ice bath then azetidine (10 eq) is added. The ice bath is removed and the reaction mixture is stirred overnight at room temperature. The solvent is removed under reduced pressure and the residue is separated by reverse phase chromatography to afford compound 307.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 308

Compound 308: Compound 308 can be prepared in an analogous fashion to Figure 37 using 3,3'-[[2,2-bis[(2-propyn-l-yloxy)methyl]-l,3-propanediyl]bis (oxy)]bis-l-propyne in place of compound 43 in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 309

Compound 309: Compound 309 can be prepared in an analogous fashion to Figure 37 using 3,3'-[[2,2-bis[(2-propyn-l-yloxy)methyl]-l,3-propanediyl]bis (oxy)]bis-l-propyne in place of compound 43 in step c.

09

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 310

Compound 310: Compound 310 can be prepared in an analogous fashion to Figure 37 by replacing compound 43 with bis-propargyl ethylene glycol in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 311 Compound 311: Compound 311 can be prepared in an analogous fashion to Figure 37 by replacing compound 43 with bis-propargyl ethylene glycol in step c.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 312

Compound 312: Compound 312 can be prepared in an analogous fashion to Figure 37 by replacing compound 43 with propargyl ether in step c.

2

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 313

Compound 313: Compound 313 can be prepared in an analogous fashion to Figure 37 by replacing compound 43 with propargyl ether in step c.

3

SYNTHESIS OF BUILDING BLOCK 332

Compound 321: Compound 317 (1.1 g, 2.60 mmoles) was dissolved in methanol (25 mL) at room temperature. Sodium methoxide (0.1 mL, 25% sol. in MeOH) was added and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture neutralized by the addition of Amberlyst acidic resin, filtered and concentrated to give crude 321, which was used for the next step without further purification. LCMS (ESI): m/z calculated for C12H15N3O4S: 297.3, found 298.1 (M+l); 320.1 (M+Na).

Compound 322: Crude compound 321 (2.60 mmoles), 3 ,4,5 -trifluorophenyl- 1 -acetylene (2.5 equiv), THPTA (0.11 equiv), and copper (II) sulfate (0.1) were dissolved in methanol (15 mL) at room temperature. Sodium ^ascorbate (2.4 equiv) dissolved in water was added and the reaction mixture was stirred overnight at room temperature. The resultant precipitate was collected by filtration, washed with hexanes and water, and dried to give compound 322 as a pale yellow solid (1.2 g, 100% yield for 2 steps). LCMS (ESI): m/z calculated for

C20H18F3N3O4S: 453.1, found 454.2 (M+l); 476.2 (M+Na).

Compound 323: Compound 322 (1.2 g, 2.65 mmoles) was dissolved in DMF (15 mL) and cooled on an ice bath. Sodium hydride (60% oil dispersion, 477 mg, 11.93 mmoles) was added and the mixture stirred for 30 minutes. Benzyl bromide (1.42 mL, 11.93 mmoles) was added and the reaction was warmed to room temperature and stirred overnight. The reaction mixture was quenched by the addition of aqueous saturated ammonium chloride solution, transferred to a separatory funnel and extracted 3 times with ether. The combined organic phases were dried over magnesium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 323 (1.8 g, 94% yield). LCMS (ESI): m/z calculated for C41H36F3N3O4S: 723.2, found 724.3 (M+l); 746.3 (M+Na).

Compound 324: Compound 323 (1.8 g, 2.49 mmol) was dissolved in acetone (20 mL) and water (2 mL) and cooled on an ice bath. Trichloroisocyanuric acid (637 mg, 2.74 mmoles) was added and the reaction mixture stirred on the ice bath for 3 h. The acetone was removed in vacuo and the residue was diluted with DCM, transferred to a separatory funnel, and washed with saturated aqueous NaFICCb. The organic phase was concentrated and the residue was purified by flash chromatography to afford compound 324 (1.5 g, 95%). LCMS (ESI): m/z calculated for C35H32F3N3O5: 631.2, found 632.2 (M+l); 654.2 (M+Na).

Compound 325: Compound 324 (1.0 g, 1.58 mmoles) was dissolved in DCM (20 mL) and cooled on an ice bath. Dess-Martin periodinane (1.0 g, 2.37 mmoles) was added and mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture quenched by the addition of aqueous saturated NaHCCh, transferred to a separatory funnel, and extracted 2 times with DCM. The combined organic phases were dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 325 (520 mg, 52% yield). LCMS (ESI): m/z calculated for C35H30F3N3O5: 629.2, found 652.2 (M+Na); 662.2 (M+MeOH+1); 684.2 (M+MeOH+Na).

Compound 326: Methyl bromoacetate (253 mg, 1.65 mmoles) dissolved in 0.5 mL ofTHF was added dropwise to a solution of lithium bis(trimethylsilyl)amide (1.0 M in THF, 1.65 mL, 1.65 mmoles) cooled at -78 C. The reaction mixture was stirred for 30 minutes at -78 C. Compound 325 (260 mg, 0.41 mmoles) dissolved in THF (2.0 mL) was then added. The reaction mixture was stirred at -78 C for 30 minutes. The reaction was quenched by the addition of aqueous saturated NH4CI and warmed to rt. The reaction mixture was transferred to a separatory funnel and extracted 3 times with ethyl acetate. The combined organic phases were dried over sodium sulfate, filtered and concentrated. The residue was separated by flash chromatography to afford compound 326 (183 mg, 64% yield).

¾ NMR (400 MHz, Chloroform- ) d 7.38 - 7.22 (m, 9H), 7.15 - 7.11 (m, 3H), 7.09 (dd, J= 8.4, 6.6 Hz, 1H), 7.06 - 7.00 (m, 2H), 6.98 - 6.93 (m, 2H), 5.11 (dd, J= 11.3, 3.2 Hz, 1H), 4.60 (d, J= 11.8 Hz, 1H), 4.57 - 4.49 (m, 2H), 4.49 - 4.42 (m, 2H), 4.35 (d, J= 11.8 Hz,

1H), 4.14 (d, J= 3.2 Hz, 1H), 4.05 (s, 1H), 4.02 (d, J= 7.0 Hz, 1H), 3.84 (d, J= 11.0 Hz, 1H), 3.81 (s, 3H), 3.70 (dd, .J= 9.5, 7.7 Hz, 1H), 3.62 (dd, J= 9.4, 6.0 Hz, 1H). LCMS (ESI): m/z calculated for C38H34F3N3O7: 701.2, found 702.3 (M+l); 724.3 (M+Na).

Compound 327: Compound 326 (5.0 g, 7.13 mmol) was azeotroped with toluene two times under reduced pressure, and then dried under high vacuum for 2 hours. It was then dissolved in anhydrous CH2CI2 (125 mL) and cooled on an ice bath while stirring under an atmosphere of argon. Tributyltin hydride (15.1 mL, 56.1 mmol) was added dropwise and the solution was allowed to stir for 25 minutes on the ice bath. Trimethylsilyl triflate (2.1 mL, 11.6 mmol) dissolved in 20 mL of anhydrous CH2CI2 was then added dropwise over the course of 5 minutes. The reaction was slowly warmed to ambient temperature and stirred for 16 hours. The reaction mixture was then diluted with CH2CI2 (50 mL), transferred to a separatory funnel, and washed with saturated aqueous NaHCCb (50 mL). The aqueous phase was separated and extracted with CH2CI2 (50 mL x 2). The combined organic phases were washed with saturated aqueous NaHCCL (50 mL), dried over Na ₂ SO ₄ , filtered, and concentrated. The residue was purified by flash chromatography (hexanes to 40% EtOAc in hexanes, gradient) to afford compound 327 (2.65 g, 48%).

'H-NMR (400 MHz, CDCI3): d 7.65 (s, 1H), 7.36 - 7.22 (m, 8H), 7.16 - 7.06 (m, 7H), 6.96 - 6.90 (m, 2H), 5.03 (dd, J= 10.7, 3.2 Hz, 1H), 4.72 (d, J= 2.3 Hz, 1H), 4.51 (dt, J= 22.6,

11.4 Hz, 3H), 4.41 (d, 10.9 Hz, 1H), 4.32 (dd, J= 10.7, 9.2 Hz, 1H), 4.07 (d, J= 3.1 Hz,

1H), 3.94 (d, J= 10.9 Hz, 1H), 3.92 - 3.84 (m, 3H), 3.78 - 3.71 (m, 4H), 3.65 (dd, J= 9.1,

5.5 Hz, 1H), 0.24 (s, 9H). LCMS (ESI): m/z (M+Na) calculated for C _4i H ₄₄ F3N ₃ 0 ₇ SiNa: 798.87, found 798.2.

Compound 328: To a solution of compound 327 (2.65 g, 3.4 mmol) in anhydrous MeOH (40 mL) was added Pd(OH)2 (0.27 g, 20% by wt). The mixture was cooled on an ice bath and stirred for 30 minutes. Triethylsilane (22 mL, 137 mmol) was added dropwise. The solution was allowed to slowly warm to ambient temperature and stirred for 16 hours. The reaction mixture was filtered through a bed of Celite and concentrated. The residue was purified by flash chromatography (hexanes to 100% EtOAc, gradient) to afford compound 328 (1.09 g, 73%).

¹ H-NMR (400 MHz, CD3OD): d 8.57 (s, 1H), 7.77 - 7.53 (m, 2H), 4.91 - 4.82 (m, 1H), 4.66 - 4.59 (m, 1H), 4.55 (dd, J = 10.8, 9.4 Hz, 1H), 4.13 (d, J = 2.8 Hz, 1H), 3.86 (dd, J = 9.4, 2.1 Hz, 1H), 3.81 (s, 3H), 3.77 - 3.74 (m, 1H), 3.71 - 3.68 (m, 2H). LCMS (ESI): m/z (M+Na) calculated for C17H18F3N3O7Na: 456.33, found 456.0.

Compound 329: Compound 328 (1.09 g, 2.5 mmol) and CSA (0.115 g, 0.49 mmol) were suspended in anhydrous MeCN (80 mL) under an argon atmosphere. Benzaldehyde dimethyl acetal (0.45 mL, 2.99 mmol) was ^added dropwise. The reaction mixture was allowed to stir for 16 hours at ambient temperature, during which time it became a homogenous solution. The reaction mixture was then neutralized with a few drops of Et3N, and concentrated. The residue was purified via flash chromatography (CH2CI2 to 10% MeOH in CH2CI2, gradient) to afford compound 329 (978 mg, 75%). 1H NMR (400 MHz, DMSO-d6): d 8.84 (s, 1H), 7.95 - 7.73 (m, 2H), 7.33 (qdt, J = 8.4, 5.6, 2.7 Hz, 5H), 5.51 (t, J = 3.8 Hz, 2H), 5.47 (d, J = 6.8 Hz, 1H), 5.14 (dd, J = 10.8, 3.6 Hz, 1H), 4.54 (dd, J = 6.7, 2.2 Hz, 1H), 4.47 (ddd, J = 10.8, 9.3, 7.5 Hz, 1H), 4.40 (d, J = 4.0 Hz, 1H), 4.09 - 3.99 (m, 2H), 3.85 (dd, J = 9.3, 2.2 Hz, 1H), 3.81 - 3.76 (m, 1H), 3.71 (s, 3H). LCMS (ESI): m/z (M+Na) calculated for C ₂₄ H ₂₂ F ₃ N ₃ 0 ₇ Na: 544.43, found 544.1.

Compound 330: Compound 329 (25.2 mg, 0.048 mmol) was azeotroped with toluene 2 times under reduced pressure, dried under high vacuum for 2 hours, then dissolved in anhydrous DMF (2 mL) and cooled on an ice bath. Benzyl bromide (6 uL, 0.05 mmol) dissolved in 0.5 mL of anhydrous DMF was added and the reaction and was stirred under an atmosphere of argon for 30 minutes at 0 °C. Sodium hydride (2 mg, 0.05 mmol, 60%) was added and the reaction was allowed to gradually warm to ambient temperature while stirring for 16 hours. The reaction mixture was diluted with EtOAc (20 mL), transferred to a separatory funnel, and washed with ¾0 (10 mL). The aqueous phase was separated and extracted with EtOAc (10 mL x 3). The combined organic phases were washed with ¾0 (10 mL x 3), dried over Na ₂ S0 ₄ , filtered, and concentrated. The residue was purified via preparative TLC (5% MeOH in CH2CI2) to afford compound 330 (6.3 mg, 21%). LCMS (ESI): m/z (M+Na) calculated for C ₃₁ H ₂ 8F ₃ N ₃ 0 ₇ Na: 634.55, found 634.1.

Compound 331: Compound 330 (6.3 mg, 0.01 mmol) was dissolved in anhydrous MeOH (1 mL) containing CSA (0.26 mg, 0.001 mmol). The reaction mixture was heated to 76 °C in a screw-cap scintillation vial while stirring. After 2 hours, an additional 0.13 mg of CSA in 0.5 mL of MeOH was added. The reaction mixture was stirred at 76 °C for 16 hours. The reaction mixture concentrated under reduced pressure. The residue was purified via preparative TLC (10% MeOH in CH2CI2) to afford compound 331 (4.2 mg, 80%).

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.80 (s, 1H), 7.94 - 7.86 (m, 2H), 7.48 - 7.42 (m, 2H),

7.38 (t, J= 7.4 Hz, 2H), 7.36 - 7.28 (m, 1H), 5.46 (d, J= 7.7 Hz, 1H), 5.28 (d, J= 6.0 Hz, 1H), 4.85 (dd, J= 10.7, 2.9 Hz, 1H), 4.67 (d, J= ¹¹ .O Hz, 1H), 4.62 - 4.58 (m, 1H), 4.54 (d, J = 11.1 Hz, 1H), 4.44 (d, J= 2.5 Hz, 1H), 4.36 (q, , J=9.5 Hz, 1H), 3.95 - 3.90 (m, 1H), 3.78 (dd, J= 9.3, 2.5 Hz, 1H), 3.71 (s, 3H), 3.61 - 3.54 (m, 1H), 3.52 - 3.43 (m, 1H), 3.43 - 3.38 (m, 1H). LCMS (ESI): m/z (M+Na) calculated for C ₂₄ H2 ₄ F ₃ N ₃ O ₇ Na: 546.45, found 546.0.

Compound 332: To a solution of compound 331 (3.5 mg, 0.007 mmoles) in methanol (0.5 mL) was added 1.0 M NaOH solution (0.1 mL). The reaction mixture was stirred overnight at room temperature then neutralized with acidic resin, filtered and concentrated. The residue was purified by reverse phase chromatography using a C-8 matrix to afford 3.0 mg compound 332 (90%).

¹ H NMR (400 MHz, Deuterium Oxide) δ 8.39 (s, 1H), 8.37 (s, 2H), 7.54 - 7.45 (m, 1H), 7.43 (d, J= 7.4 Hz, 2H), 7.35 (dt, J= 14.3, 7.2 Hz, 3H), 4.86 (dd, J= 11.0, 2.9 Hz, 1H), 4.76 (d, J = 11.0 Hz, 1H), 4.40 - 4.30 (m, 2H), 4.16 (d, J= 1.9 Hz, 1H), 4.04 (d, J= 3.0 Hz, 1H), 3.81 (d, J= 9.6 Hz, 1H), 3.73 (d, J= 3.9 Hz, OH), 3.67 (d, J= 7.6 Hz, 1H), 3.56 (dd, J= 11.7, 3.9 Hz, 1H). LCMS (ESI): m/z (M+Na) calculated for C ₂₃ H ₂₂ F ₃ N ₃ 07: 509.1, found 508.2 (M-H).

PROPHETIC SYNTHESIS OF BUILDING BLOCK 333

Compound 333: Compound 333 can be prepared in an analogous fashion to Figure 41 by replacing benzyl bromide with 4-chlorobenzyl bromide in step j.

PROPHETIC SYNTHESIS OF BUILDING BLOCK 334

Compound 334: Compound 334 can be prepared in an analogous fashion to Figure 41 by replacing benzyl bromide with 4-methanesulfonylbenzyl bromide in step j.

PROPHETIC SYNTHESIS OF BUILDING BLOCK 335

Compound 335: Compound 335 can be prepared in an analogous fashion to Figure 41 by replacing benzyl bromide with 3-picolyl bromide in step j.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 336

Compound 336: Compound 336 can be prepared in an analogous fashion to Figure 22 by replacing compound 145 with compound 332.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 337

Compound 337: Compound 337 can be prepared in an analogous fashion to Figure 22 by replacing compound 145 with compound 333.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 338

Compound 338: Compound 338 can be prepared in an analogous fashion to Figure 22 by replacing compound 145 with compound 334.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 339

Compound 339: Compound 339 can be prepared in an analogous fashion to Figure 22 by replacing compound 145 with compound 335.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 340

Compound 340: Compound 340 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 40 and replacing compound 145 with compound 333.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 341

[0002] Compound 341: Compound 341 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 78 and replacing compound 145 with compound 333.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 342

Compound 342: Compound 342 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 87 and replacing compound 145 with compound 333.

PROPHETIC SYNTHESIS OF MULTIMERIC COMPOUND 343

Compound 343: Compound 342 can be prepared in an analogous fashion to Figure 22 by replacing compound 22 with compound 88 and replacing compound 145 with compound 333.

E-SELECTIN ACTIVITY - BINDING ASSAY

The inhibition assay to screen and characterize antagonists of E-selectin is a competitive binding assay, from which IC50 values may be determined. E-selectin/Ig chimera are immobilized in 96 well microtiter plates by incubation at 37 °C for 2 hours. To reduce nonspecific binding, bovine serum albumin is added to each well and incubated at room temperature for 2 hours. The plate is washed and serial dilutions of the test compounds are added to the wells in the presence of conjugates of biotinylated, sLe ^a polyacrylamide with streptavidin/horseradish peroxidase and incubated for 2 hours at room temperature.

To determine the amount of sLe ^a bound to immobilized E-selectin after washing, the peroxidase substrate, 3, 3’, 5, 5’ tetramethylbenzidine (TMB) is added. After 3 minutes, the enzyme reaction is stopped by the addition of H3PO4, and the absorbance of light at a wavelength of 450 nm is determined. The concentration of test compound required to inhibit binding by 50% is determined.

E-Selectin Antagonist Activity

GALECTIN-3 ACTIVITY - ELISA ASSAY

Galectin-3 antagonists can be evaluated for their ability to inhibit binding of galectin-3 to a Galbl-3GlcNAc carbohydrate structure. The detailed protocol is as follows. A 1 ug/mL suspension of a Galbl-3GlcNAcbi -3Galbl-4GlcNAcb-PAA-biotin polymer (Glycotech, catalog number 01-096) is prepared. A 100 uL aliquot of the polymer is added to the wells of a 96-well streptavidin-coated plate (R&D Systems, catalog number CP004). A 100 uL aliquot of IX Tris Buffered Saline (TBS, Sigma, catalog number T5912 - 10X) is added to control wells. The polymer is allowed to bind to the streptavidin-coated wells for 1.5 hours at room temperature. The contents of the wells are discarded and 200 uL of IX TBS containing 1% bovine serum albumin (BSA) is added to each well as a blocking reagent and the plate is kept at room temperature for 30 minutes. The wells are washed three times with IX TBS containing 0.1% BSA. A serial dilution of test compounds is prepared in a separate V- bottom plate (Corning, catalog number 3897). A 75 uL aliquot of the highest concentration of the compound to be tested is added to the first well in a column of the V-bottom plate then 15 ul is serially transferred into 60 uL IX TBS through the remaining wells in the column to generate a 1 to 5 serial dilution. A 60 uL aliquot of 2 ug/mL galectin-3 (IBL, catalog number IBATGP0414) is added to each well in the V-bottom plate. A 100 uL aliquot of the galectin- 3/test compound mixture is transferred from the V-bottom plate into the assay plate containing the Galbl-3GlcNAc polymer. Four sets of control wells in the assay plate are prepared in duplicate containing 1) both Galpl-3GlcNAc polymer and galectin-3, 2) neither the polymer nor galectin-3, 3) galectin-3 only, no polymer, or 4) polymer only, no galectin-3. The plate is gently rocked for 1.5 hours at room temperature. The wells are washed four times with TBS/0.1%BSA. A 100 uL aliquot of anti-galectin-3 antibody conjugated to horse radish peroxidase (R&D Systems, from DGAL30 kit) is added to each well and the plate is kept at room temperature for 1 hour. The wells are washed four times with TBS/0.1%BSA.

A 100 uL aliquot of TMB substrate solution is added to each well. The TMB substrate solution is prepared by making a 1 :1 mixture of TMB Peroxidase Substrate (KPL, catalog number 5120-0048) and Peroxidase Substrate Solution B (KPL, catalog number 5120-0037). The plate is kept at room temperature for 10 to 20 minutes. The color development is stopped by adding 100 uL 10% phosphoric acid (RICCA Chemical Co., catalog number 5850-16). The absorbance at 450 nm (A450) is measured using a FlexStation 3 plate reader (Molecular Devices). Plots of A450 versus test compound concentration and IC50 determinations are made using GraphPad Prism 6.

CXCR4 ASSAY - INHIBITION OF CYCLIC AMP

The CXCR4-cAMP assay measures the ability of a glycomimetic CXCR4 antagonist to inhibit the binding of CXCL12 (SDF-Ia) to CHO cells that have been genetically engineered to express CXCR4 on the cell surface. Assay kits may be purchased from DiscoveRx (95- 0081E2CP2M; cAMP Hunter eXpress CXCR4 CHO-K1). The Gi-coupled receptor antagonist response protocol described in the kit instruction manual can be followed.

GPCRs, such as CXCR4, are typically coupled to one of the 3 G-proteins: Gs, Gi or Gq. In the CHO cells supplied with the kit, CXCR4 is coupled to Gi. After activation of CXCR4 by ligand binding (CXCL12), Gi dissociates from the CXCR4 complex, becomes activated, and binds to adenylyl cyclase, thus inactivating it, resulting in decreased levels of intracellular cAMP. Intracellular cAMP is usually low, so the decrease of the low level of cAMP by a Gi- coupled receptor will be hard to detect. Forskolin is added to the CHO cells to directly activate adenylyl cyclase (bypassing all GPCRs), thus raising the level of cAMP in the cell, so that a Gi response can be easily observed. CXCL12 interaction with CXCR4 decreases the intracellular level of cAMP and inhibition of CXCL12 interaction with CXCR4 by a CXCR4 antagonist increases the intracellular cAMP level, which is measured by luminescence.

Also provided are pharmaceutical compositions comprising at least one compound of Formula (I), (la), (II), (Ila), (III), (IV), (Illa/IVa), (Illb/IVb), (V), (VI), or (VII). These compounds and compositions may be used in the methods described herein.

EXAMPLE 1

Reducing endothelial activation (AML patients)

As noted above, E-selectin antagonists include small molecule glycomimetic drugs. For example, Compound A is a potent glycomimetic antagonist of E-selectin and is in Phase III clinical trials for treatment of AML.

The soluble form of E-selectin (sE-selectin) is a marker of endothelial activation and is elevated in the blood of AML patients.

As shown in Figure 43, treatment of AML patients with Compound A significantly reduced sE-selectin in the blood and the levels stay down longer than the half-life of Compound A in the bloodstream (20). Cohorts 1, 2, and 3 were dosed with 5, 10, and 20 mg/kg of Compound A respectively. A single dose was given prior to dosing twice a day (BID) in combination with MEC (mitoxantrone 10mg/m ² /d, etoposide 100mg/m2/d, and cytarabine 1000mg/m2/d) IV for 5 days. For two days after combination treatment, Compound A was dosed alone BID.

One possible explanation for the suppression of the biomarker of endothelial activation (sE- selectin) for a longer time than its serum half-life (PD > PK) is the suppression of cytokines that induce endothelial cell activation and E-selectin expression.

EXAMPLE 2

Reducing endothelial activation (mice)

In support of the mechanism proposed above, Compound A was found to suppress the expression of multiple cytokines that in turn would induce the expression of E-selectin and endothelial activation (21).

Cohorts of mice were administered saline or G-CSF 3d ± Compound A for 24hrs, then euthanized. The femurs were then flushed in lmL PBS. Bone marrow cell-free fluids were analyzed for inflammatory cytokines using LegendPlex beads. The resulting data is shown below as pg/mL of diluted femoral flush. Statistical significance is indicated below (* = 050; ** = .01). Each dot represents one individual mouse. Figures 44A-D. This experiment shows that E-selectin blockade reverses G-CSF-induced release of inflammatory mediators in bone marrow.

EXAMPLE 3

Reduction of endothelial activation by suppression of E-selectin

As shown in Figures 44A-D, inhibiting E-selectin with the glycomimetic Compound A suppresses expression of cytokines that play central roles in CRS. Some of these cytokines such as TNFa are known to be strong stimulators of endothelial activation and the expression of E-selectin.

By inhibiting E-selectin this feedback loop is broken and will result in blocking CRS, reducing the inflammatory response and inhibiting endothelial activation and the breakdown of the blood brain barrier together with associated neurotoxicities.

Administering any of the antagonists disclosed above would be effective in the treatment and/or prevention of CRS and/or CRS-induced neurotoxicities. Administering combinations of the antagonists disclosed above (e.g., separately, concurrently, or sequentially) would also be effective. One of ordinary skill in the art would recognize further examples of effective combinations of the disclosed antagonists for the treatment and/or prevention of CRS and/or CRS-induced neurotoxicities.

The present invention also contemplates combination therapy and, accordingly, the methods may comprise exposing the subject to an ancillary treatment or prophylactic that treats or prevents CRS and/or CRS-related conditions such as CRS-induced neurotoxicities (e.g., the separate, concurrent, or sequential administration of the treatments disclosed herein with other treatments).

References

The following references are hereby incorporated by reference in their respective entireties.

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