WO2017066668A1 - Drug-linker conjugate pharmaceutical compositions - Google Patents

Abstract

Compositions are disclosed having a cytotoxic and/or vascular disrupting agent (VDA) payload attached to a linker. The linker can be a cathepsin B protease cleavable linker or a non-cleavable linker that may degrade intracellularly. Methods for making and using the compositions are also provided. The compositions can be provided to a patient in need thereof with the composition coming into contact with a cancer cell to activate or release the cytotoxic and/or vascular disrupting agent payload.

Description

DRUG-LINKER CONJUGATE PHARMACEUTICAL COMPOSITIONS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 1 19 to U.S. Provisional Application No. 62/242,220 filed on October 15, 2015 titled "Drug-Linker Conjugates Pharmaceutical Compositions", each of which is herein incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

FIELD

[0003] The present application relates to small molecules for treating cancer that can include a cytotoxic payload and a linker.

BACKGROUND

[0004] Improved methods and therapies are desired for treating cancer. Improved uses for highly cytotoxic compounds are also desired. For example, some compounds with a cytotoxicity in the sub- nanomolar to picomolar range can be too cytotoxic for general cancer treatments because of the chance of killing or damaging healthy cells as a result of the high cytotoxicity of the compounds. The present application describes compositions including an antibody linker bound to a highly cytotoxic compound. The antibody linker can be cleaved in-vivo to deliver the highly cytotoxic portion of the compound to the targeted cancerous tissue. The antibody linker can be selected based on the type of cancer being treated and the location of the cancer such that the composition interacts with the targeted cancer such that the linker is cleaved off to deliver the cytotoxic portion of the compound to the targeted cancer tissue.

SUMMARY OF THE DISCLOSURE

[0005] The present invention relates to novel compounds including a vascular targeting or disrupting agent and a linker along with methods of use. Examples of the linker included a cathepsin B protease cleavable linker and a non-cleavable linker. Non-cleavable linkers can be removed intracellularly within the targeted cell type, such as through lysosomal degradation or other processes.

[0006] In general, in one embodiment, a composition including a compound having the formula la:

or a pharmaceutically acceptable salt thereof, wherein the phenyl ring "Z" is bonded to either carbon "a" or "b"; and R₃, are each, independently, selected from the group consisting of H, lower alkoxy, phosphate, hydroxyl, and a linker (Y), wherein at least one of R, and R₃ is the linker (Y); R4 and R5 are each, independently, selected from the group consisting of H, lower alkoxy, phosphate, and hydroxyl; X is selected from the group consisting of a single bond and C(O); and n is 1 , 2, 3 or 4.

[0007] This and other embodiments can include one or more of the following features. The linker (Y) can include a cathepsm B protease cleavable compound. The linker (Y) can include a non-cleavable linker adapted to be cleaved or degraded intracellularly or not cleaved. In some embodiments R₃ is H; R4 and R₅ are OCH₃; X can be a single bond; n can be 2. The compound can have the formula:

Y

, R] can be the linker (Y).

[0008] In some embodiments R₃ is H; R₄ and R₅ can be OCH₃; X can be a single bond; n can be 1 , 3, or 4. The composition can have the formula:

Y

, R| can be the linker (Y).

[0009] A composition can include a compound having the formula:

, Y can be a linker and R' can be hydrogen or hydroxyl. The linker (Y) can include a cathepsin B protease cleavable compound. The linker (Y) can include a non-cleavable linker adapted to be cleaved degraded intracellularly or not cleaved.

[00010] A composition can include a compound having the formula:

, Y can be a linker. The linker (Y) can include a cathepsin B protease cleavable compound. The linker (Y) can include a non-cleavable linker adapted to be cleaved or degraded intracellularly or not cleaved.

[00011] Y ean include:

[00012] Y can include:

[00017] Y can include:

[00018] The composition can have the formula:

[00019] The composition can have the formula:

[00020] The composition can have the formula:

[00021 ] The composition can have the formula:

[00022] The composition can have the formula:

[00023] The composition can have the formula:

[00024] The composition can have the formula:

[00025] The composition can have the formula:

[00026] The composition can have the formula:

[00027] The composition can have the formula:

[00029] The composition can have the formula:

[00030] The composition can have the formula:

[00031] The composition can have the formula:

[00032] The composition can have the formula:

[00033] The composition can have the formula:

[00034] The composition can have the formula:

[00035] The composition can have the formula:

[00036] The composition can have the formula:

[00037] A pharmaceutical formulation can include any of the above compounds. A method of treating cancer can include administering to a mammal in need thereof according to any of the above compounds. The method can further include contacting the compound with a cathepsin B protease such that all or a portion of the cathepsin B protease cleavable portion of the compound can be cleaved by the cathepsin B protease or such that the linker is not cleaved. The method can further include contacting the compound with a cancer cell such that all or a portion of the linker can be cleaved intracellularly. The method can further include contacting the compound with a cancer cell such that the linker is not cleaved. A method can include contacting in vivo a cathepsin B protease with a compound such that all or a portion of the cathepsin B protease cleavable portion of the compound can be cleaved by the cathepsin B protease. A method can include contacting in a cell a cathepsin B protease with a compound such that all or a portion of the cathepsin B protease cleavable portion of the compound can be cleaved by the cathepsin B protease. A method can include contacting in a cell a lysosome or peptidase with a compound such that all or a portion of the linker of the compound can be cleaved.

[00038] In general, in one embodiment, a method for making a Val-Cit cleavable cathepsin B linker, including the following steps: (a) Fmoc protection of L-citrulline followed by reaction with para-amino benzyl alcohol (PABOH) followed by Fmoc deprotection (b) dipeptide formation by reacting the product of step (a) with Fmoc-Val-OSu followed by Fmoc deprotection; (c) reacting the product of step (b) with activated maleimido caproic acid (MC-OSu) to form the Val-Cit cleavable cathepsin B linker.

BRIEF DESCRIPTION OF THE DRAWINGS

[00039] The novel features of the invention are set forth with particularity in the claims that follow. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[00040] FIGS. 1 A-1 B illustrate examples of synthetic schemes in accordance with some

embodiments.

[00041] FIG. 2 illustrates two synthetic schemes in accordance with some embodiments.

[00042] FIG. 3 illustrates a synthetic scheme in accordance with some embodiments.

[00043] FIG. 4 illustrates a synthetic scheme in accordance with some embodiments.

[00044] FIG. 5 shows a High Resolution Mass Spectroscopy (HRMS) of an isocyanate intermediate compound in accordance with some embodiments.

[00045] FIG. 6A illustrates a synthetic scheme in accordance with some embodiments. FIG. 6B illustrates HRMS of a compound in accordance with some embodiments.

[00046] FIG. 7A illustrates a synthetic scheme in accordance with some embodiments. FIG. 7B illustrates HRMS of a compound in accordance with some embodiments.

[00047] FIGS. 8A-8B illustrate synthetic schemes in accordance with some embodiments.

[00048] FIG. 9 illustrates a synthetic scheme in accordance with some embodiments.

[00049] FIG. 10 illustrates a synthetic scheme in accordance with some embodiments.

[00050] FIG. 1 1 illustrates a synthetic scheme in accordance with some embodiments.

[00051] FIG. 12 illustrates a synthetic scheme in accordance with some embodiments. [00052] FIG. 13 illustrates a synthetic scheme in accordance with some embodiments.

[00053] FIG. 14 illustrates a synthetic scheme in accordance with some embodiments.

[00054] FIG. 15 is a schematic illustration of examples of a cathepsin B protease cleaving a portion of a linker attached to an effective drug.

DETAILED DESCRIPTION

[00055] Antibody-drug conjugates (ADCs) are designed as a novel, effective, and selective therapies for the treatment of cancer. Synthetic approaches toward conjugation of a cleavable or non-cleavable linker with a small-molecule payload are described herein. The payloads described herein can possess dual modes of biological mechanisms of action functioning both as highly cytotoxic agents (GI₅₀ = sub- nanomolar to picomolar range against human cancer cell lines) and also as potent vascular disrupting agents (VDAs). The high cytotoxicity of some of the payloads can make it undesirable to directly provide the payload to a patient, biological tissue, or healthy biological tissue because of the cytotoxic effects. The linkers include cleavable or non-cleavable linkers. Examples of a cleavable linker include a cathepsin B protease cleavable linker. Examples of a non-cleavable linker include a linker that is removed or degraded intracellulariy or not cleaved. The linkers attached to the payloads described herein allow for the molecule to be provided to the patient while reducing cytotoxic effects on health tissue and cells. Cathepsin B can be used as a biomarker for a variety of cancers. In addition excessive presence of cathepsin B is related to metastatic and invasive types of cancer. The compounds described herein can be used to directly target cancer cells because the payload is essentially activated when the cathepsin B protease cleavable linker is cleaved from the compound to provide the active payload or when the non- cleavable linker is degraded or removed intracellulariy by the targeted cell type. The cathepsin B protease is present in and around certain types of cancer cells and diseased tissue. The non-cleavable linker can be designed such that it is degraded or removed intracellulariy by the targeted cell type. The compound with the linker and payload is inactive until the compound is around cancerous, targeted, or diseased tissue and the linker is cleaved or degraded to deliver the active payload to the cancerous, targeted, or diseased tissue. The payload can then treat the cancerous tissue through the cytotoxic effects and vascular disrupting behavior. The ability to selectively treat the cancer can provide large benefits to the patient with the cancer by maximizing the payload action against the cancerous tissue while minimizing undesirable interactions with healthy tissue.

[00056] The payloads can incorporate amino or hydroxyl moieties that can be suitable for covalent attachment to the appropriate linker. In some embodiments the payloads attached to the cleavable or non- cleavable linker have a cytotoxicity in the sub-nanomolar range. In some embodiments the IC₅₀ value can be less than 1 x 10^"9 M. In some embodiments the payloads attached to the linker can have a high VDA activity alone or in combination with a high cytotoxicity.

[00057] As used herein, the following definitions shall apply unless otherwise indicated. [00058] Cathepsin B protease cleavable linker refers to a linker that can be all or partially cleaved by a cathepsin B protease. Cathepsin B protease cleavable linkers can be referred to as a Val-Cit linker or VC linker in some cases.

[00059] Non-cleavable linkers refer to linkers that are not cleavable by a cathepsin B protease but can be all or partially removed intracellularly within the targeted cell type, such as through lysosomal degradation by peptidases or other processes, or not cleaved.

[00060] The terms "cysteine protease" or "cysteine proteinase" or "cysteine peptidase" intend any enzyme of the sub-subclass EC 3.4.22, which consists of proteinases characterized by having a cysteine residue at the active site and by being irreversibly inhibited by sulfhydryl reagents such as iodoacetate. Mechanistically, in catalyzing the cleavage of a peptide amide bond, cysteine proteases form a covalent intermediate, called an acyl enzyme, which involves a cysteine and a histidine residue in the active site (Cys25 and His 159 according to papain numbering, for example). Representative cysteine protease targets for the present disclosure include papain and cathepsin B (EC 3.4.22.1 ).

[00061] Cysteine proteases that can cleave the linkers of the compounds of the present disclosure can be "cathepsin B-like." A cathepsin B-like cysteine protease shares structural and functional similarity with a mammalian cathepsin B, and part of the enzyme can comprise an "occluding loop". Cathepsin B- like cysteine proteases cleave as a substrate the sequences -Arg-Arg-|-Xaa- and -Phe-Arg-|-Xaa-.

Representative cathepsin B-like proteases include cathepsin B, T. cruzi-B, and L. mexicana-B.

[00062] The term "IC₅₀" refers to the concentration of compound that results in half-maximal inhibition of enzyme.

[00063] "Alkyl" refers to monovalent saturated aliphatic hydrocarbon groups having from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms. This term includes, by way of example, linear and branched hydrocarbon groups such as methyl (CH₃-), ethyl (CH₃CH₂-), n-propyl (CH₃CH₂CH₂-), isopropyl ((CH₃)₂CH-), n-butyl (CH₃CH₂CH₂CH₂-), isobutyl ((CH₃)₂CHCH₂-), sec-butyl

((CH₃)(CH₃CH₂)CH-), t-butyl ((CH₃)₃C-), n-pentyl (CH₃CH₂CH₂CH₂CH₂-), and neopentyl ((CH₃)₃CCH₂- )·

[00064] "Alkoxy" refers to the group -O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like.

[00065] "Acyl" refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl-C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclic-C(O)-, and substituted heterocyclic-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Acyl includes the

"acetyl" group CH₃C(0)-.

[00066] "Amino" refers to the group -NH₂. [00067] " AryJ" or "Ar" refers to a monovalent aromatic carbocyclic group of from 6 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl) which condensed rings may or may not be aromatic (e.g., 2-benzoxazolinone, 2H-l ,4-benzoxazin-3(4H)-one-7- yl, and the like), provided that the point of attachment is through an atom of the aromatic aryl group. Preferred aryl groups include phenyl and naphthyl.

[00068] "Alkenyl" refers to straight chain or branched hydrocarbon groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. Such groups are exemplified, for example, bi-vinyl, allyl, and but-3-en-l -yl. Included within this term are the cis and trans isomers or mixtures of these isomers.

[00069] "Alkynyl" refers to straight or branched monovalent hydrocarbon groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH₂C≡CH).

[00070] "Cycloalkyl" refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.

[00071] "Halo" or "halogen" refers to fluoro, chloro, bromo, and iodo and is preferably fluoro, bromo, or chloro.

[00072] "Hydroxy" or "hydroxyl" refers to the group -OH.

[00073] "Heteroaryl" refers to an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl, imidazolyl or furyl) or multiple condensed rings (e.g., indolizinyl, quinolinyl, benzimidazolyl or benzothienyl), wherein the condensed rings may or may not be aromatic and/or contain a heteroatom, provided that the point of attachment is through an atom of the aromatic heteroaryl group. In one implementation, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→0), sulfmyl, or sulfonyl moieties. Preferred heteroaryls include pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl.

[00074] "Heterocycle," "heterocyclic," "heterocycloalkvk" and "heterocyclyl" refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 hetero atoms. These ring atoms are selected from the group consisting of nitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In one implementation, the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -S0₂- moieties.

[00075] Examples of heterocycle and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1 ,2,3,4-tetrahydroisoquinoline, 4,5,6,7- tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, mo holinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1 ,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[00076] "Nitro" refers to the group -N0₂.

[00077] "Nitroso" refers to the group -NO.

[00078] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent "arylalkyloxycarbonyl" refers to the group (aryl)-(alkyl)-0-C(0)-.

[00079] The term "substituted," when used to modify a specified group or radical, means that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[00080] In a preferred implementation, a group that is substituted has 1 , 2, 3, or 4 substituents, 1 , 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[00081] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups are limited to - substituted aryl-(substituted aryl)-substituted aryl.

[00082] "Stereoisomer" and "stereoisomers" refer to compounds that have same atomic connectivity but different atomic arrangement in space. Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers, and diastereomers.

[00083] "Tautomer" refers to alternate forms of a molecule that differ only in electronic bonding of atoms and/or in the position of a proton, such as enol-keto and imine-enamine tautomers, or the tautomeric forms of heteroaryl groups containing a -N=C(H)-NH- ring atom arrangement, such as pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. A person of ordinary skill in the art would recognize that other tautomeric ring atom arrangements are possible.

[00084] "Patient" refers to human and non-human animals, especially mammals.

[00085] "Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium,

tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, and the like. [00086] "Pharmaceutically effective amount" and "therapeutically effective amount" refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and/or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders and neoplasms, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[00087] "Solvate" refers to a complex formed by combination of solvent molecules with molecules or ions of the solute. The solvent can be an organic compound, an inorganic compound, or a mixture of both. Some examples of solvents include, but are not limited to, methanol, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.

[00088] Similarly, it is understood that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 fluoro groups). Such impermissible substitution patterns are easily recognized by a person having ordinary skill in the art.

[00089] In some embodiments the compound including the payload and cathepsin B protease

eral formula la:

or a pharmaceutically acceptable salt thereof,

wherein the phenyl ring "Z" is bonded to either carbon "a" or "b";

R] and R₃, are each, independently, selected from the group consisting of H, lower alkoxy, phosphate, hydroxyl, and a linker (Y), wherein at least one of R] and R₃ is the linker (Y);

R4 and R₅ are each, independently, selected from the group consisting of H, lower alkoxy, phosphate, and hydroxyl;

X is selected from the group consisting of a single bond and C(O); and

n is 1 , 2, 3 or 4. The linker (Y) can be any of the linkers described herein including cleavable linkers and non-cleavable linkers.

[00090] In one embodiment of formula la, R4 and R₅ are OCH₃. In another embodiment of formula la, n is 1. In another embodiment of formula la, n is 2. In another embodiment of formula la, n is 3. In another embodiment of formula la, n is 4.

[00091] In some embodiments of formula la R₃ is H, R₄ and R₅ are OCH₃, X is a single bond, and n is

2. The linker (Y) can be linked through a hydroxyl or amino group on R] .

[00092] In some embodiments of formula la R₃ is H, R₄ and R₅ are OCH₃, X is a single bond, and n is

3. The linker (Y) can be linked through a hydroxyl or amino group on R,. [00093] In particular embodiments of formula la, Ri and R₃ are H or OH. In other embodiments of formula la, R, is OH or NH₂ and R₃ is H. In yet other embodiments of formula la, R, and R₃ are OCH₃. In some embodiments the linker can be linked through a hydroxyl or amino group on R, or R₃.

[00094] In embodiments of formula la either of Ri or R₃ can be linked to the linker. For example, any of the cathepsin B protease cleavable linker compounds and non-cleavable linker compounds, described herein can be attached to compound la through either R, or R₃.

[00095] In some embodiments the compound including the payload and linker includes a compound of the following general Formula lb:

or a pharmaceutically acceptable salt thereof, wherein the dashed line indicates a single or double bond; X is selected from the group consisting of a single bond, CH₂, O, S, N(H), and C(O); X,_, X₂, X₃, X4 and X₅ are each, independently, selected from the group consisting of C, C(H), N, N(H), O and S; Ri, R₂, R₃, R₄, R₅, R6 and R₇ are each, independently, selected from the group consisting of H, halogen, lower alkyl, lower alkoxy, hydroxyl, amine, phosphate, phosphoramidate, amino acid acyl group and a linker (Y), wherein at least one of Ri, R₂, and R₃ is or is attached to the linker (Y), and ring "Z" is bonded to either carbon "a" or "b." In one embodiment of Formula lb, X is a single bond, and Ri, R₂ and R₃, are each, independently, selected from the group consisting of H, OCH₃, phosphate, amino, and OH. In another embodiment of Formula lb, X is a single bond, and R4, R₅ and ¾, are each, independently, selected from the group consisting of H, OCH₃, phosphate, amino, and OH. The linker (Y) can be any of the linkers described herein including cleavable linkers and non-cleavable linkers.

[00096] In embodiments of formula lb either of R R₂, or R₃ can be linked to the cathepsin B protease cleavable linker or non-cleavable linkers. For example any of the cathepsin B protease cleavable compounds described herein can be attached to compound lb through either R R₂, or R₃.

[00097] A number of specific examples of the permutations of Formulas la and lb are discussed in detail below along with various linkers including cathepsin B protease cleavable linkers and non- cleavable linkers that can be attached to the payloads. [00098] In one example, the payload can be 2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-7,8-dihydro- naphthalen- l -ol, also known as KGP03 (OXi6196), which is shown below. This compound has a cytotoxicity in the sub nano-molar range.

[00099] In one example, the payload can be 2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-7,8-dihydro- naphthalen- l -ylamine, also known as KGP05, which is shown below:

[000100] In one example, the payload can be 2-Methoxy-5-(3,4,5-trimethoxy-phenyl)-8,9-dihydro-7H- benzocyclohepten- l -ol, also known as KGP 18, which is shown below. This compound has a cytotoxicity in the nano-molar to sub nano-molar to pico-molar range.

[000101 ] In one example, the payload can be 3-Methoxy-5-(3,4,5-trimethoxy phenyl)-8.9-dihydro-7H- benzocyclo hepten- l -ylamine), also known as KGP156, which is shown below:

[000102] In one example, the payload can be (Z)- l -[3',4',5'-trimethoxyphenyl]-2-[2",3"-dihydroxy-4"- methoxyphenyl]ethane, which is a combretastatin analogue that is also known as combretastatin A-l ( is shown below:

[000103] In one example, the payload can be (Z)-2-methoxy-5-(3,4,5-trimethoxystyryl)phenol, which is a combretastatin analogue that is also known as combretastatin A-4 (CA4). The formula for CA4 is shown below.

[000104] In one example, the payload can be 2-(3-Hydroxy-4-methoxy-phenyl)-6-methoxy-l H-indol- 3-yl]-(3,4,5-trimethoxy-phenyl)-methanone, which is an indole VDA that is also known as KGP01 or r ΟΧΪ8006 is shown below:

[000105] The linker can be attached to the payload through the hydroxyl or amino group on the payload. The payload with the linker can be less cytotoxic than the payload alone. In some examples, the payload plus linker can have a low cytotoxicity. The linker can be selected with an antibody designed to target a desired type of cancer cell. In some cases the linker can include the antibody or can be conjugated to the desired antibody. The payload and linker can be provided to the patient such that the compound can be used to selectively target cancer cells. The compound can interact with the target cancer cell. The compound can be inactive or have a low activity until the compound encounters a cathepsin B protease or lysosomes intracellularly. The cathepsin B protease can be encountered intracellular^ or extracellularly (e.g. secreted). The cathepsin B protease can cleave the cathepsin B protease cleavable linker to provide the payload to the targeted cancer cells. For the non-cleavable linker examples the compound can be inactive or have a low activity until the compound is within a targeted cell where lysosomal degradation or other processes remove or cleave the non-cleavable linker to provide the payload to the targeted cells. In some cases the non-cleavable linker and payload may have activity after being only partially cleaved or not substantially cleaved after antibody targeting.

[000106] In some embodiments any of the compounds described herein can be attached to an antibody that can be used to target the specific cancer cells.

[000107] Several examples of specific pay loads that can be used with any of the cleavable and non- cleavable linkers are described herein. In the formulas below, Y is any of the linkers described herein.

[000108] In some embodiments the payload can have the formula below, with Y as any of the linkers describe herein:

[000109] In some embodiments the payload can have the formula below, with Y as any of the linkers

[000110] In some embodiments the compound is a combretastatin analogue that has the following formula with Y being a linker and with R' as OH (CA-1 payload) or H (CA-4 payload):

[000111] In some embodiments the pay load can have the formula below, with Y as any of the linkers described herein:

[000112] A variety of different cathepsin B protease cleavable linkers can be attached to the payloads as described herein. The cathepsin B protease cleavable linker can be contacted in-vivo by a cathepsin B protease to cleave all or a portion of the linker to convert the compound to a more active form. The highly cytotoxic payload can then contact the cancer cells.

[000113] In one example the linker is a cathepsin B protease cleavable linker with the formula of:

[000114] In one example the linker is a cathepsin B protease cleavable linker with the formula of:

[000115] In one example the linker is a cathepsin B protease cleavable linker with the formula of:

[000116] In one example the linker is a cathepsin B protease cleavable linker with the formula of:

[000117] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000118] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000120] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000121] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000122] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

CH3 o

[000123] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000124] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000125] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000126] In one example the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:

[000127] A number of the linkers include a maleimido portion illustrated with a chain with six carbons. The number of carbons in the chain can be adjusted based on the desired properties of the linker, such that more or less carbons can be used in the chain.

[000128] A number of specific examples of payloads combined with the linkers are described in the following non-limiting examples.

[000129] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula (also referred to as KGP477):

[000130] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000131] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula:

[000132] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000133] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000134] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000135] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000136] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000137] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula:

[000138] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula:

[000139] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000140] In one example the composition includes a payload and a linker that may be cleaved ula:

[000141 ] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000142] In one example the composition includes a payload and a linker that may be intracellularly with the following formula:

[000143] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000144] In one example the composition includes a payload and a linker that may be cleaved intracellularly with the following formula:

[000145] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula (also referred to as KGP475):

[000146] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula (also referred to as GP474):

[000147] In one example the composition includes a payload and a cathepsin B protease cleavable linker with the following formula:

[000148] The biological data has been observed for some of the payload and linker compounds described herein. A 48 hour assay was used for cell lines DU145 and NCI-H460 for each of KGP474, KGPP475, and KGP477. The IC₅₀ values are reported along with standard deviations. For KGP474 an IC₅₀ of 0.203±0.0467 and 0.712±0.21 1 for DU 145 and NCI-H460, respectively, was observed. For KGP475 an ICso of 1.94±0.491 and 1 ,35±0.21 5 for DU145 and NCI-H460, respectively, was observed. For KGP477 an IC₅₀ of 0.130±0.0290 and 0.1 13±0.0143 for DU145 and NCI-H460, respectively, was observed. The IC₅₀ values indicate that the linker can significantly reduce the cytotoxicity of the compound versus the payload alone.

[000149] FIG. 15 shows schematic example of a cathepsin B cleavable protease cleaving PABOH and PABOH-DMED, respectively (de Groot et. al. J. Org. Chem. 2001 , 66 (26), 8815-8830; de Groot et. al. J. Med. Chem. 2000, 43 (16), 3093-3102; Burke, P. J et. al. Bioconjug. Chem. 2009, 20 (6), 1242-1250. The cathepsin B cleavable protease cleaves the PABOH and PABOH-DMED linker to release the effective drug as shown in FIG. 15.

[000150] In some embodiments pharmaceutical formulations comprising any of the compounds described herein are provided. The pharmaceutical formulations can be administered orally, intravenously, or through other common administration routes.

[000151] In some embodiments methods of treating cancer are provided including administering any of the compounds described herein to a mammal in need thereof. The method can include contacting any of the compounds described herein with a cathepsin B protease such that all or a portion of the cathepsin B protease cleavable portion of the compound is cleaved by the cathepsin B protease. The methods can also include contacting any of the compounds described herein with a cancer cell such that all or a portion of the linker is cleaved intracellularly.

[000152] Methods are also provided including contacting in vivo a cathepsin B protease with any of the compounds described herein such that all or a portion of the cathepsin B protease cleavable portion of the compound is cleaved by the cathepsin B protease.

[000153] Methods are also provided including contacting in a cell a cathepsin B protease with any of the compounds described herein such that all or portion of the cathepsin B protease cleavable portion of the compound is cleaved by the cathepsin B protease.

[000154] Methods are also provided including contacting in a cell a lysosome or peptidase with any of the compounds described herein such that all or a portion of the linker is cleaved.

[000155] Methods for making the linkers and for attaching the linkers to the pay loads described herein are also provided. As noted above, the linkers can be bound to the payload through an amino or hydroxyl group on the payload. In some cases an intermediate can be formed, such as an iso-cyanate group, on the payload followed by connecting the linker as shown in the example illustrated in FIGS. 4-5.

EXAMPLES

[000156] Synthesis of Cathepsin B Cleavable Linker. Various synthesis schemes are shown in FIGS. 1 A-1 B. A dipeptide maleimidocaproyl linker, which is recognized and cleaved by the cathepsin B protease, was selected for covalent attachment to our biologically active VDAs, as shown synthetically in FIGS. 1 A-I B. Different synthetic approaches were tried with the best results obtained from the scheme shown in FIG. 1 A. Much higher yields were obtained with the scheme shown in FIG. 1 A versus the scheme in FIG. I B. For example, the yield obtained in FIG. 1 A was above about 96%. In contrast, the average yields for the schemes shown in FIG. IB were typically around 25-30%. The yields for the second reaction step were also much higher in the scheme illustrated in FIG. 1A versus FIG. I B. For example, the yield for the second step in FIG. 1 A was about 80% and the yield for the second step in FIG. 1 B was typically around 25%-30%. The number of carbons in the carbon chain on the dipeptide maleimodcaproyl linker can be adjusted based on the desired properties of the linker.

[000157] FIG. 1 A shows a revised synthetic route for the preparation of the Val-Cit cleavable cathepsin B linker that proceeds in good yield and with high diastereoselectivity. This linear synthetic route proceeds sequentially by Fmoc protection of L-citrulline followed by reaction with para-amino benzyl alcohol (PABOH) followed by Fmoc deprotection, followed by dipeptide formation by reaction with Fmoc-Val-OSu, followed by Fmoc deprotection, followed by reaction with activated maleimido caproic acid (MC-OSu) to form the requisite linker. The overall reaction conditions presented in FIG. 1 A can be important for the improved yield and overall success of the revised synthetic route. For example, this route avoids epimerization that often plagues other synthetic routes towards this linker, such as the route shown in FIG. I B.

[000158] Synthesis of Fmoc-Cit: A solution of I-citrulline (1.1 equiv) and sodium bicarbonate (2.2 equiv) in water (0.2 M) was stirred at room temperature for 1.0 h. Then DME was added to the reaction mixture (DME: water = 1 :1 and overall concentration 0.1 M) followed by Fmoc chloride (1.0 equiv) and allowed to stir at room temperature for 24.0 h. Solvent DME was removed under reduced pressure. The aqueous solution was extracted thrice with EtOAc. Aqueous phase was then acidified with aqueous 2M HC1 (pH = 1 ) when white precipitate was formed. To the precipitate in water 10% iPrOH-EtOAc was added and was stirred well when all the white precipitate dissolved in organic solvent. Organic phase was collected and aqueous layer was further extracted twice with same 10% iPrOH-EtOAc solvent. Collected organic phase was dried over sodium sulfate. Solvent evaporation under reduced pressure generated clear viscous liquid. Sonication of the liquid with diethyl ether yielded white solid powder product Fmoc-Cit. White solid was further dried under vacuum. Yield: > 96%.

[000159] Synthesis of Fmoc-Cit-PABOH: To a solution of Fmoc-Cit (1 .0 equiv) and PABOH (3.0 equiv) in DMF (0.2 M), DIPEA (1.0 equiv) was added and was stirred for 30 min at room temperature. Then HATU (1.1 equiv) was added to the reaction mixture and allowed to stir at room temperature for 30.0 h in dark. Solvent DMF was removed under reduced pressure. To the residue water and 10% iPrOH- EtOAc were added and was stirred to get clear phases. Organic phase was separated and aqueous phase was further extracted twice with 10% iPrOH-EtOAc. Collected organic phase was dried over sodium sulfate. Solvent was partially evaporated under reduced pressure and then slurry of crude product was made in celite for solid loading in column. Product was purified by silica gel column flash

chromatography using 1 -12% MeOH-CH₂Cl₂. Compound Fmoc-Cit-PABOH was obtained as yellowish solid. Yield: 80%. [000160] Synthesis of Fmoc-Val-Cit-PABOH: A solution of Fmoc-Cit-PABOH (1.0 equiv) in DMF (0.2 M) was treated with piperidine (5.0 equiv) and allowed to stir at room temperature for 4 h. Solvent DMF and excess piperidine were removed under reduced pressure. Resulted residue was sonicated in ether and ether discarded. Then the solid was washed with CH₂C1₂. Solid was dried under vacuum and then dissolved in DMF (0.2 M). To the solution Fmoc-Val-OSu (1.1 equiv) was added and allowed to stir at room temperature for 16 h. The solvent DMF was removed under reduced pressure and crude was dissolved in methanol. Compound slurry was prepared in celite for solid loading in column. Product was purified by silica gel flash column chromatography using 1 -12% MeOH-CH₂Cl₂. Compound Fmoc-Val- Cit-PABOH was obtained as white solid. Yield: 85% (2 steps).

[000161] Synthesis of MC-Val-Cit-PABOH (5): A solution of Fmoc-Val-Cit-PABOH (1.0 equiv) in DMF (0.2 M) was treated with piperidine (5.0 equiv) and allowed to stir at room temperature for 4 h. Solvent DMF and excess piperidine were removed under reduced pressure. Resulted residue was sonicated in ether and ether discarded. Then the solid was washed with CH₂C1₂. Solid was dried under vacuum and then dissolved in DMF (0.2 M). To the solution MC-OSu (compound 4) (1.1 equiv) was added and allowed to stir at room temperature for 16 h. The solvent DMF was removed under reduced pressure and crude was dissolved in methanol. Compound slurry was prepared in celite for solid loading in column. Product was purified by silica gel flash column chromatography using 1 -12% MeOH-CH₂Cl₂. Compound MC-Val-Cit-PABOH (5) was obtained as white solid. Yield: 95% (2 steps).

[000162] FIG. 1 A also illustrates another synthetic scheme for making an activated succinimide ester 4. As shown in FIG. 1 A, to an ice cold suspension of carboxylic acid (1.0 equiv) and disuccinimidyl carbonate (1.05 equiv) in DMF (0.5M), triethylamine (1.0 equiv) was added with constant stirring when all the suspension dissolved to give clear solution with constant bubbling of gas. Gas bubbling stopped after 15 min. Reaction mixture was further stirred at rt for 2h.Then DMF was removed under reduced pressure and residue was dissolved in EtOAc. To the solution saturated sodium bicarbonate solution was added and stirred when gas bubbling was observed. After gas generation stopped, organic layer was separated. Aqueous layer was washed one more time with EtOAc. Collected organic layer was dried over sodium sulfate. Solvent evaporation under reduced pressure gave crude oily product. Compound was further was purified by silica gel flash chromatography using 0-30% EtOAc-CH₂Cl₂. Compound 4 was white solid with a 90% yield.

[000163] 1 H NMR of final compound, MC-Val-Cit-PABOH: NMR was taken in 600 MHz Bruker NMR instrument in DMSO solvent. l HNMR (600MHz, DMSO-d6) δ 9.90 (brs, 1 H), 8.06-8.05 (d, J = 7.59 Hz, 1 H), 7.81 -7.80 (d, J = 8.67 Hz, 1H), 7.55-7.53 (d, J = 8.44 Hz, 2H), 7.23-2.22 (d, J = 8.43 Hz, 2H), 7.00 (s, 2H), 5.99-5.97 (bit, J= 5.53 Hz, 1H), 5.40 (s, 2H), 5.10-5.08 (t, J = 5.72 Hz, 1 H), 4.42-4.41 (d, 7= 5.50 Hz, 2H), 4.39-4.34 (m, 1 H), 4.20-4.17 (dd, J = 15.44, 6.95 Hz, 1 H), 3.38-3.35 (t, J = 7.06 Hz, 2H), 3.04-2.91 (m, 2H), 2.20-2.09 (m, 2H), 1.99-1.93 (sex, J= 6.78 Hz, 1 H), 1.71 -1 .66 (m, 1H), 1.61 - 1.55 (m, 1 H), 1.53-1.15 (m, 8 H), 0.85-0.84 (d, J = 6.78 Hz, 3H), 0.82-0.81 (d, J = 6.78 Hz, 3H).

[000164] A dipeptide maleimidocaproyl linker, which is recognized and cleaved by the cathepsin B protease, was selected for covalent attachment to our biologically active VDAs, as shown synthetically in FIG. 1 B. Fmoc-Val-OSu was reacted with L-citrulline to afford Fmoc-protected dipeptide 1 in high yield (82%). By using N-ethoxycarbonyl-2-ethoxy-l ,2-dihydroquinoline (EEDQ) as a peptide coupling reagent, p-aminobenzyl alcohol (PABOH) was then coupled to Fmoc-protected dipeptide 1 in the dark for 1.5 days to obtain compound 2 with an 25%-30% yield. The Fmoc protecting group was removed by treatment with diethylamine (as a base) at ambient temperature for 16 h to afford compound 3 after trituration with CH₂C1₂ and filtration. Synthesis of the desired cathpsin B cleavable linker 5 was achieved by utilizing an activated succinimide ester 4, which is synthesized by reacting 6-maleimidocaproic acid with disuccinimide carbonate, to react with compound 3.

[000165] Synthesis of Cathepsin B-KGP156 Conjugate. KGP156, was selected as a payload to incorporate onto the cathepsin B cleavable linker 5 to form the linker-drug conjugate because of its accessible amino moiety. At first, a synthetic approach using an activated linker was employed. Initially, the para-nitrophenyl carbonate activating group could be installed on linker 5 by reacting linker 5 with para-nitrophenyl chloroformate (PNPOCOC1) (FIG. 2). This reaction was successful in generating the activated linker 6, but the yield (7%) was not promising. As a result, a modified reaction was then utilized while using bis(para-nitrophenyl) carbonate (Bis-PNP) instead of PNPOCOC1 to react with linker 5, and the activated linker 6 was afforded with an improved yield of 74%.

[000166] An additional synthetic approach for making cathepsin B- GP156 conjugate 7 is shown in FIG. 3. Conjugate 7 was successfully obtained from a nucleophilic substitution of activated linker 6 with GP156 with the addition of a catalytic amount of hydroxybenzotriazole (HOBt) in the presence of Hunig's base.

[000167] FIG. 4 illustrates yet another alternative route for the synthesis of cathepsin B- GP156 conjugate 7. A solution of KGP156 in CH₂C1₂ was first treated with triphosgene under a base-free condition to afford the corresponding isocyanate intermediate 10, which was obtained quantitatively and structurally confirmed by high resolution mass spectroscopy (HRMS) (FIG. 5). After solvent removal, the resulting solid was then subjected to a nucleophilic addition by linker 5 in DMF at 45°C to produce the desired conjugate 7.

[000168] Synthesis of Cathepsin B- GP18 Conjugates: Several different methodologies were attempted to synthesize the cathepsin B- GP18 conjugate with a carbonate conjugation. FIG. 6A illustrates a scheme using another spacer, Ν,Ν'-dimethylethylenediamine (DMED), that was installed between a cathepsin B linker and KGP18 for the purpose of generation of two carbamate linkages. To generate the bis-DMED-added linker 14, a reaction was employed reacting three equivalents of DMED with the activated cathepsin B linker 6 to form the bis-DMED added linker 14 as confirmed by HRMS (FIG. 6B). FIG. 6B shows HRMS of Compound 14.

[000169] FIG. 7A shows one synthetic approach to Cathepsin B- GP18 Conjugate 15. A small test reaction was performed by reacting the bis-DMED-added linker 14 with the GP18-activated compound 12 in the presence of Et₃N as shown in FIG. 7A. KGP18 was successfully added to the linker 14 through nucleophilic substitution, but there were two possible carbamate conjugates (15a or 15b) obtained after isolation with flash column chromatography and confirmation with HRMS (FIG. 7B). [000170] Additional methods for the synthesis of the cathepsin B-KGP18 conjugate 16 was explored as shown in FIGS. 8A-8B. Unlike the installation of DMED on the linker 6, DMED was first attached to KGP18 to form a KGP18-DMED hydrochloride salt 17 by reacting DMED with a KGP18-activated compound 12 followed by work-up under acidic conditions. The formation of the hydrochloride salt is useful because it is a more stable form of the KGP18-DMED complex. The hydrochloride salt functionalization deters the spontaneous cyclization of the DMED functional group which leads to the release of KGP18. The desired cathepsin B-KGP18 conjugate 16 was afforded by reacting GP18- DMED salt 17 with an activated linker 6 in DMF at ambient temperature.

[000171] FIG. 8B illustrates another synthesis scheme for producing cathepsin B-KGP18 conjugate 16. As shown in FIG. 8B, a solution of 6 (2.0 equiv) and 17 (1 .0 equiv) in DMF (0.1 M) was treated with

DIPEA (5.0 equiv) and stirred at rt for 16 h. Reaction was quenched with water and made acidic (pH ~ 3) with 2M HC1 and solvent was removed under reduced pressure. To the residue water and 10% nBuOH- EtOAc were added and stirred to get clear phases. Organic layer was separated. Aqueous layer was extracted twice with 10% nBuOH-EtO Ac. Collected organic layer was dried over sodium sulfate. Solvent was removed under reduced pressure to get crude product. Then the crude was dissolved in methanol and slurry of crude was prepared in celite to load in column. Product was purified by silica gel flash column chromatography using 0-10% MeOH-CH₂Cl₂ as solvent system. Compound 16 obtained as white solid with a yield of 28%.

[000172] Several synthetic approaches were employed for the synthesis of cathepsin B-KGP18 conjugate 18.

[000173] FIG. 9 illustrates a synthesis scheme for obtaining a MC-KGP156 Conjugate. FIG. 9 shows that to a solution of GP156 (1.0 equiv) and 6-Maleimidohexanoic (2.0 equiv) acid in DMF (0.1M), DIPEA (2.0 equiv) was added and stirred for 15 min at rt. Then HATU (2.0 equiv) was added to the reaction mixture and stirred at rt for 18h. Solvent DMF was removed under reduced pressure and the residue was dissolved in CH₂C1₂ and water and NaHC0₃ solution was added and stirred get clear phases. Organic layer was separated. Aqueous layer was extracted two more times with CH₂C1₂ and collected organic layer dried over sodium sulfate. Product was purified by silica gel flash column chromatography using 0-30% EtOAc-CH₂Cl₂ as solvent. Compound was isolated as slight greenish white solid: Yield: 80%. [000174] FIG. 10 illustrates a synthesis scheme for obtaining a MC-KGP 18 Conjugate. FIG. 10 shows that to a solution of KGP18 (1 .0 equiv) and 6-Maleimidohexanoic (2.0 equiv) acid in DMF (0.1 M), DIPEA (3.0 equiv) was added and stirred for 15 min at rt. Then HATU (2.0 equiv) was added to the reaction mixture and stirred at rt for 20h. Solvent DMF was removed under reduced pressure and the residue was dissolved in CH₂C1₂ and water and NaHC0₃ solution was added and stirred get clear phases. Organic layer was separated. Aqueous layer was extracted two more times with CH₂C1₂ and collected organic layer dried over sodium sulfate. Product was purified by silica gel flash column chromatography using 0-35% EtOAc-CH₂Cl₂ as solvent. Compound was slight yellowish solid. Yield: 26%.

[000175] FIG. 1 1 illustrates a synthesis scheme for obtaining MC-DMED-KGP 18 Conjugates. FIG. 1 1 shows that to a solution of 6-Maleimidohexanoic acid (1 .0 equiv) and oxalyl chloride (3.0 equiv) in

CH₂C1₂, DMF was added using a syringe. It was stirred for 2h. Excess oxalyl chloride was removed under vacuum. Now a solution of this crude acid chloride (3.0 equiv) and compound 17 (1 .0 equivalent) in DMF was treated with DIPEA (5.0 equiv) and stirred at rt for 20 h. Solvent DMF was removed reduced pressure. To the residue water and EtOAc were added and stiired to get clear phases. Organic layer was separated. Aqueous layer was extracted twice more with EtOAc. Collected organic layer was dried over sodium sulfate. Product was purified by silica gel flash column chromatography using 10-70% EtOAc- CH₂C1₂. Yield: 12% (2 steps). Product is slight yellowish solid.

[000176] FIG. 12 illustrates a synthesis scheme for obtaining MC-PABA-DMED-KGP18 conjugates. Procedure to synthesize MC-PABOH: To a solution of 6-Maleimidohexanoic acid (1 .0 equiv) and 4- aminobenzyl alcohol ( 1.5 equiv) in DMF (0.2M), DIPEA (2.0 equiv) was added and stirred for 15 min at rt. Then HATU ( 1 .5 equiv) was added to that and stirred at rt for 40 h in dark. Solvent DMF was removed under reduced pressure. Residue was dissolved in CH₂C1₂ and washed with saturated NaHC0₃ solution. Then organic layer was washed with 2M HC1. The collected organic layer was dried over anhydrous sodium sulfate. Product was purified by silica gel column chromatography flash using 5-10% EtOAc- CH₂C1₂. Yield: 96%. Compound MC-PABOH was white solid.

[000177] Procedure to synthesize MC-PABA-PNP: A solution of MC-PABOH (1 .0 equiv) in CH₂C1₂ was treated with DIPEA (2.0 equiv) and stirred at rt for 15 min. Then 4-nitrophenylcarbonate (3.0 equiv) was added to the reaction mixture and stirred at rt for 20h. Reaction was quenched with water and stiired to get clear phases. Organic layer was separated. Aqueous layer was washed twice with CH₂C1₂ and collected organic layer was dried over sodium sulfate. Product was purified by silica gel flash column chromatography using 0-25% EtOAc-CH₂Cl₂ as solvent. Compound MC-PABA-PNP is slight yellowish solid. Yield: 62%.

[000178] Procedure to synthesize MC-PAB A-DMED-KGP 18 : A solution of MC-PABA-PNP (2.0 equiv) and compound 1 7 (1 .0 equiv) in DMF (0.1 M) was treated with DIPEA (5.0 equiv) and stirred at rt for 20 h. Solvent DMF was removed under reduced pressure. To the residue water and EtOAc were added and stirred to get clear phases. Organic layer was separated. Aqueous layer was extracted twice with EtOAc and collected organic layer was dried over sodium sulfate. Product was purified by silica gel flash column chromatography using 5-40% EtOAc-CH₂Cl₂.Yield: 36%. Compound MC-PABA-DMED-

KGP18 is slight yellowish solid.

[000179] FIG. 13 illustrates a synthetic scheme for making a maleimidocaproyl KGP05 conjugate in accordance with some embodiments. KGP05 (0.58 mmol), 6-Maleimidocaproic acid (1 .17 mmol) and N- Ethoxycarbonyl-2-ethoxy-l , 2-dihydroquinoline (1 .17 mmol) were added to an oven-dried flask. To the reaction flask a 2:1 mixture of CH₂C1₂/CH₃0H (15 ml) was added and the reaction was stirred under nitrogen at room temperature for 20 hours. The solvent was removed by vacuum distillation and the resulting light brown solid was purified by flash column chromatography (0- 10% CH₃0H/CH₂C1₂) affording the desired maleimidocaproyl KGP05 conjugate (0.62 mmol, 42% yield) as an off white solid.

[000180] FIG. 14 illustrates a synthetic scheme to make Mc-Val-Cit-PABC-KGP05 in accordance with some embodiments. Synthesis of the isocyanate of KGP05 : GP05 was added to an oven dried flask and dissolved in CH₂C1₂ (5 ml). Triphosgene (0.29 mmol) and saturated NaHC0₃ were then added to the reaction flask. The reaction was allowed to stir at room temperature under nitrogen for 1 hour. The organic phase was separated and dried with Na₂S0₄. After filtration the solvent was removed by vacuum distillation. The resulting light brown solid was then purified by flash column chromatography (0-40% EtOAc/hexanes) affording the desired isocyanate (0.25 mmol, 84% yield).

[000181] Synthesis of Mc-Val-Cit-PABC- GP05 : KGP05 isocyanate (.04 mmol) and Mc-Val-Cit- PAB-OH (.04 mmol) were added to an oven dried flask and dissolved in DMF (.8 ml). The reaction was allowed to stir under nitrogen at room temperature for 3 days. The solvent was removed by vacuum distillation and the resulting oil was purified by flash column chromatography (0-20% CH₃0H/CH₂C1₂). The desired carbamate (.01 mmol, 28% yield) was obtained was a brown solid.

[000182] AcOH, CH₂C1₂, CH₃OH, 1 ,2-dimethoxyethane (DME), DMF, N-methyl-2-pyrrolidone (NMP), Pyridine, and THF were used in their anhydrous forms or as obtained from the chemical suppliers. Reactions were performed under N₂. Thin-layer chromatography (TLC) plates (precoated glass plates with silica gel 60 F254, 0.25 mm thickness) were used to monitor reactions. De-ionized (DI) water was used to quench and wash the reaction mixture as appropriate. Purification of intermediates and products was carried out with a Biotage Isolera flash purification system using silica gel (200-400 mesh, 60 A) columns or manually in glass columns. Intermediates and products synthesized during this study were characterized on the basis of their Ή NMR (500 or 600 MHz), ¹³C NMR (125 or 150

MHz)spectroscopic data using a Varian VNMRS 500 MHz or a Bruker DRX 600 MHz instrument.

Spectral data were recorded in CDC1₃, D₂0, DMSO-d6, or CD₃OD. All chemical shifts are expressed in ppm (δ), coupling constants (J) are presented in Hz, and peak patterns are reported as broad singlet (bs), singlet (s), doublet (d), triplet (t), quartet (q), pentet (p), double doublet (dd), triplet of doublets (td), doublet of triplets (dt) and multiplet (m).

[000183] Purity of the final compounds was further analyzed at 25 °C using an Agilent 1200 HPLC system with a diode-array detector (λ = 190-400 nm), a Zorbax XDB-C 1 8 HPLC column (4.6 mm A~ 1 50 mm, 5 μηι), and a Zorbax reliance cartridge guard-column; method A: solvent A, acetonitrile, solvent B, 0.1 % TFA in H₂0; or method B: solvent A, acetonitrile, solvent B, H₂0; gradient, 10% A/90% B to 100% A/0% B over 0 to 40 min; post-time 10 min; monitored at wavelengths of 210, 254, 230, 280, and 360 nm. Mass spectrometry was carried out under positive or negative electrospray ionization (ESI) using a Thermo Scientific LTQ Orbitrap Discovery instrument. Compound numbering for the combretastatin molecular scaffold followed this protocol: the trimethoxy A ring was numbered 1-6, and the B ring was numbered 1 '-6'. The ethylene bridging atoms were numbered as 1 a and 1 a' respectively for the carbons connected to the A ring and B ring, respectively.

[000184] Fmoc-Val-Cit 1. To a solution of l-citrulline (0.274 g, 1.56 mmol) and NaHC0₃ (0.131 g, 1 .56 mmol) in water (4 mL) was added a solution of Fmoc-Val-Osu (0.650 g, 1.49 mmol) in DME (4 mL). THF (2 mL) was added to aid solubility, and the reaction mixture was stirred for 16 h at ambient temperature. HC1 (2 M, 8 mL) was added, and the white solid product began to precipitate but remained in the organic layer. The mixture was extracted with isopropanol/EtOAc ( 1 : 9, 2 ^χ 20 mL), and the combined organic suspension was washed with water (2 ^χ 20 mL). The resultant suspension was concentrated under reduced pressure, and then treated with Et₂0 (20 mL). After sonication and trituration with Et₂0, the crude product was collected by filtration, and the crude product was purified by flash chromatography using a pre-packed 50 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B (1 CV), 0%A / 100%B→ 30%A / 70%B (10 CV), 30%A / 70%B (2 CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] to afford Fmoc-protected dipeptide 1 (0.610 g, 1.22 mmol, 82% yield) as a white solid.

[000185] Ή NMR (500 MHz, DMSO-d6) δ 8.18 (1 H, d, J = 7.3 Hz), 7.90 (2H, d, J= 7.5 Hz), 7.75 (2H, t, J= 8.0 Hz), 7.41 (3H, q, J = 8.1 Hz), 7.33 (2H, td, J= 6.6, 3.2 Hz), 5.94 (1H, t, J= 5.5 Hz), 5.38 (2H, bs), 4.33 - 4.26 (1 H, m), 4.26 - 4.19 (2H, m), 4.19 - 4.12 (2H, m), 3.93 (1H, dd, J= 9.0, 7.2 Hz), 2.95 (2H, q, J = 6.6 Hz), 2.04 - 1.92 (1H, m), 1.76 - 1.65 (1 H, m), 1.62 - 1.52 (1H, m), 1.46 - 1.34 (2H, m), 0.89 (3H, d, J= 6.8 Hz), 0.87 (3H, d, J= 6.8 Hz).

[000186] ¹ C NMR (125 MHz, DMSO-d6) δ 173.9, 171.8, 159.2, 156.5, 144.4, 144.2, 141.2, 128.1 , 127.5, 125.9, 120.6, 66.1 , 60.3, 52.4, 47.1 , 31.0, 28.8, 27.2, 19.7, 18.7.

[000187] Fmoc-Val-Cit-PABOH 2. To a solution of Fmoc-protected dipeptide 1 (0.30 g, 0.60 mmol) in CH₂Cl₂/MeOH (2: 1 , 9 mL) was added P-aminobenzyl alcohol (0.12 g, 0.98 mmol ) and EEDQ (0.24 g, 0.98 mmol), and the reaction mixture was stirred in the dark for 1.5 d. The solvents were evaporated, and the resultant was triturated with Et₂0 (25 mL). The resulting suspension was sonicated for 20 min and left to stand for 30 min. The crude product was collected by filtration and then purified by flash

chromatography using a pre-packed 50 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B (1 CV), 0%A / 100%B -→ 20%A / 80%B (10 CV), 20%A / 80%B (2 CV); flow rate: 40 mL/min; monitored at 254 and 280 nm] to afford Fmoc-protected compound 2 (0.31 g, 0.51 mmol, 85% yield) as a brown solid.

[000188] Ή NMR (500 MHz, DMSO-d6) 6 10.00 (1 H, s), 8.13 (1 H, d, J = 7.7 Hz), 7.92 (2H, d, J = 7.5 Hz), 7.81 - 7.71 (2H, m), 7.57 (2H, d, J= 8.4 Hz), 7.49 - 7.40 (3H, m), 7.35 (2H, t, J= 7.4 Hz), 7.26 (2H, d, J = 8.3 Hz), 5.99 (1 H, t, J = 6.5 Hz), 5.43 (2H, s), 5.12 (1 H, t, J= 5.6 Hz), 4.45 (2H, d, J= 5.4 Hz), 4.32 (1H, d,J= 10.1 Hz), 4.29 - 4.19 (3H, m), 4.00 - 3.88 (1H, m), 3.12 - 2.91 (2H, m), 2.09 - 1.94 (1H, m), 1.80-1.31 (4H, m), 0.90 (3H, d, .7=6.6 Hz), 0.88 (3H, d,J=6.7 Hz).

[000189] ^I3C NMR (125 MHz, DMSO-d6) δ 171.7, 170.9, 159.3, 156.6, 144.4, 144.2, 141.2, 138.0, 137.9, 128.1, 127.6, 127.4, 125.8, 120.6, 119.3, 66.2, 63.1, 60.6, 47.2,30.9,30.0, 27.3, 19.7, 18.8, 15.0.

[000190] Val-Cit-PABOH 3. To a solution of Fmoc-protected compound 2 (0.31 g, 0.51 mmol) in NMP (5 mL) was added diethylamine (1 mL), and the reaction mixture was stirred for 16 h at ambient temperature. The solvent was then evaporated under reduced pressure, and the resulting oil was triturated with CH₂C1₂, and the mixture was sonicated for 30 min. The solid was collected by filtration and washed with CH₂C1₂ (3 x 10 mL) to afford desired compound 3 (0.19 g, 0.49 mmol, 93% yield) as a brown solid.

[000191] Ή NMR (500 MHz, DMSO-d6) δ 10.03 (1H, s), 8.18 (1H, d, J= 6.2 Hz), 7.52 (2H, d, J= 8.4 Hz), 7.22 (2H, d,J = 8.4 Hz), 5.97 (1H, t, J = 5.8 Hz), 5.39 (2H, s), 5.08 (1H, s), 4.51 - 4.43 (3H, m), 3.13 - 2.95 (2H, m), 2.92 (1H, dd, J= 13.2, 6.3 Hz), 1.93 (2H, dd, J= 11.9, 6.5 Hz), 1.74 - 1.61 (1H, m), 1.61 - 1.49 (1H, m), 1.47-1.29 (2H,m),0.87 (3H, d, ,7=6.8 Hz), 0.78 (3H, d, .7=6.8 Hz).

[000192] ⁿC NMR (125 MHz, DMSO-d6) δ 170.9, 159.3, 137.9, 137.9, 127.4, 119.4, 63.0, 59.8, 53.0, 31.6,30.5,27.1, 19.8, 17.5.

[000193] 6-Maleimidohexanoic acid N-hydroxysuccinimide ester 4. Sodium bicarbonate (0.0512 g, 0.483 mmol ) was added to a solution of 6-maleimidocaproic acid (0.211 g, 1.000 mmol) in DI water (10 mL). After the reagents were dissolved in DI water, the DI water was evaporated by blowing with air. The resulting solid was dissolved in anhydrous DMF (3 mL) and the solution was cooled to 0 °C.

Disuccinimide carbonate (0.282 g, 1.10 mmol) was added into the solution and the reaction was stirred at 0 °C for 1 h. CH₂C1₂ (25 mL) was added into reaction which was then washed with water (3 x 10 mL). The resulting organic layer was dried over Na₂S0₄, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B (1 CV), 0%A / 100%B→ 2%A / 98%B (10 CV), 2%A / 98%B (2 CV); flow rate: 25 mL/min; monitored at 210 and 280 nm] to afford ester 4 (0.215 g, 0.697 mmol, 70% yield) as a light yellow liquid.

[000194] Ή NMR (500 MHz, CDC1₃) 66.69 (2H, s), 3.53 (2H, t, J= 7.1 Hz), 2.83 (4H, s), 2.60 (2H, t,

.7=7.4 Hz), 1.78 (2H,p, .7=7.5 Hz), 1.63 (2H,p, .7=7.4 Hz), 1.41 (2H,p,J=7.6 Hz).

[000195] ¹³CNMR(125 MHz, CDC1₃) δ 170.9, 169.2, 168.5, 134.2,37.5,30.8,28.1,25.9,25.7,24.1.

[000196] Mc-Val-Cit-PABOH 5. Compound 3 (0.19 g, 0.49 mmol) was added to a solution of ester 4 (0.18 g, 0.57 mmol) in NMP (7 mL), and the reaction was stirred for 16 h. The solvent was evaporated at reduced pressure, and then the residue was triturated with Et₂0 (30 mL). The crude product was collected by filtration and washed with Et₂0 (3 x 15 mL). The crude product was purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B (1 CV), 0%A / 100%B→ 30%A / 70%B (10 CV), 30%A / 70%B (2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] to afford linker 5 (0.26 g, 0.45 mmol, 92% yield) as a brown solid.

[000197] Ή NMR (500 MHz, DMSO-d6) δ 9.82 (1H, s), 7.98 (1H, d,J= 8.5 Hz), 7.73 (1H, d,J= 8.5 Hz), 7.46 (2H, d,J= 7.8 Hz), 7.14 (2H, d, 7= 8.0 Hz), 6.92 (2H, s), 5.89 (1H, t,J= 6.0 Hz), 5.33 (2H, s), 5.01 (1H, t, J= 5.7 Hz), 4.34 (2H, d, J= 4.5 Hz), 4.29 (1H, q,J= 7.4 Hz), 4.14 - 4.05 (1H, m), 3.29 (2H, t, .7=6.3 Hz), 2.99-2.82 (2H, m), 2.17- 1.96 (1H, m), 1.94- 1.80 (2H, m), 1.67-1.03 (8H, m), 0.76 (3H, d, .7=6.4 Hz), 0.73 (3H, d, .7=6.1 Hz).

[000198] ¹³C NMR (125 MHz, DMSO-d6) δ 173.2, 172.7, 171.5, 170.8, 159.3, 138.0, 137.8, 134.9, 127.3, 119.2, 63.0, 58.0, 53.5, 37.4, 35.4, 30.8, 29.8, 28.2, 26.2, 25.7, 25.4, 19.7, 18.6.

[000199] Mc-Val-Cit-PABC-PNP 6. To a solution of linker 5 (0.51 g, 0.89 mmol) in anhydrous DMF (20 mL) was added Bis-para-nitropheny] carbonate (0.14 g, 4.5 mmol) and DIEA (0.44 mL, 2.7 mmol), and the reaction was stirred for 18 h at ambient temperature. The solvent was removed and the resulting residue was purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B (1 CV), 0%A / 100%B→ 30%A /70%B (10 CV), 30%A / 70%B (2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] to afford activated linker 6 (0.47 g, 0.64 mmol, 74% yield) as a tan solid.

[000200] Ή NMR (500 MHz, CD₃OD) δ 8.24 (2H, d, J= 8.0 Hz), 7.59 (2H, d, / = 7.3 Hz), 7.54 (1H, d, J= 8.5 Hz), 7.50 - 7.41 (1H, m), 7.36 (2H, d, J= 7.1 Hz), 7.30 (2H, d, J= 8.2 Hz), 7.27 - 7.21 (1H, m), 6.70 (2H, s), 5.21 (2H, s), 4.54 (1H, m), 4.28 - 4.08 (1H, m), 3.45 (2H, t, J= 6.4 Hz), 3.14 (2H, t, J = 28.2 Hz),2.22(2H,q, J=8.1 Hz), 2.10 - 1.96 (1H, m), 1.87 (2H, m), 1.74 - 1.47 (8H, m), 1.35-1.17 (2H, m),0.94 (3H,d,J=8.3 Hz), 0.91 (3H,d,J=5.9 Hz).

[000201] l,3-Bis(3-methoxy-9-(3,4,5-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-yl)urea9. Pyridine (0.0900 mL, 1.12 mmol) was added to a solution of KGP156 (0.0355 g, 0.100 mmol) in CH₂ ¾ (1.5 mL) at 0 °C, and triphosgene (0.0356 g, 0.120 mmol) was then added to the reaction mixture. The reaction was stirred for 18 h while warming to ambient temperature. The solvent was evaporated, and the resulting residue was purified by flash chromatography using a pre-packed 10 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B (1 CV), 0%A / 100%B→ 4%A / 96%B (10 CV), 4%A / 96%B (2 CV); flow rate: 12 mL/min; monitored at 254 and 280 nm] to afford &W-KGP156 urea 9 (0.0230 g, 0.0312 mmol, 31 % yield) as a tan solid.

[000202] Ή NMR (500 MHz, CDC1₃) δ 6.98 (1H, d,J= 8.6 Hz), 6.78 (1H, d, J= 8.6 Hz), 6.51 (2H, s), 6.39 (1H, t, J= 7.3 Hz), 6.03 (1H, s), 3.87 (3H, s), 3.86 (3H, s), 3.81 (6H, s), 2.84 (2H, t, J= 7.1 Hz), 2.27 (2H, p,J=7.0 Hz), 1.99 (2H, q, J =7.1 Hz).

[000203] ^l3CNMR(125 MHz, CDC1₃) δ 157.5, 153.0, 149.9, 142.7, 138.4, 137.5, 136.1, 133.7, 129.4, 127.8, 123.7, 108.5, 105.4, 61.0, 56.3, 55.7, 34.3, 26.8, 25.8.

[000204] HRMS: Obsvd 737.3462 [M + H]⁺, Calcd for C₄3H49N₂0₉: 737.3433.

[000205] Mc-Val-Cit-PABC- GPl 56 Carbamate 7. To a flask containing a solution of KGP156 (0.100 g, 0.281 mmol) in CH₂C1₂ (5 mL) was added triphosgene (0.0417 g, 0.141 mmol), and the reaction was stirred for 18 h at ambient temperature. The solvent was evaporated by blowing with N₂ gas followed by rotavapor evaporation at 40 °C to obtain the isocyanate intermediate 10 as a tan solid. To the flask containing isocyanate 10 was added a solution of linker 5 (0.240 g, 0.420 mmol) in THF (2 mL), and the reaction was stirred for 16 h at 45 °C. The solvent was evaporated under reduced pressure, and the resulting residue was purified by flash chromatography using a pre-packed 10 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B ( 1 CV), 0%A / 100%B→ 20%A / 80%B ( 10 CV), 20%A / 80%B (2 CV); flow rate: 12 mL/min; monitored at 254 and 280 nm] to afford a crude product. The crude product was further purified by preparative TLC (MeOH/CH₂Cl₂, 1 :9) followed by re- crystallization with MeOH to obtain desired carbamate 7 (0.013 g, 0.0136 mmol, 5% yield) as a tan solid.

[000206] Ή NMR (600 MHz, CD₃OD) δ 7.63 (2H, d, J = 7.2 Hz), 7.42 (2H, d, J = 7.2 Hz), 6.93 (1 H, d, J = 8.5 Hz), 6.90 (1H, d, J = 8.8 Hz), 6.80 (2H, s), 6.55 (2H, s), 6.39 (1H, t, J= 7.3 Hz), 5.51 (2H, s), 5.17 (2H, s), 4.53 ( 1 H, t, J = 7.0 Hz), 4.18 (1H, d, 7 = 7.5 Hz), 3.84 (3H, s), 3.78 (3H, s), 3.77 (6H, s), 3.49 (2H, t, J = 7.1 Hz), 3.26 - 3.18 ( 1 H, m), 3.13 (1 H, dt, J = 13.3, 6.5 Hz), 2.66 (2H, t, J = 6.7 Hz), 2.29 (2H, t, J = 7.4 Hz), 2.17 - 2.10 (1 H, m), 2.09 (1 H, dt, 7 = 13.7, 6.8 Hz), 1.93 (2H, q, J = 12.3, 9.6 Hz), 1 .78 (1 H, ddt, ./ = 17.7, 13.3, 6.6 Hz), 1 .65 (3H, p, J = 7.4 Hz), 1 .59 (3H, p, J = 7.3 Hz), 1 .34 - 1 .29 (2H, m), 0.99 (6H, t, .7 = 7.4 Hz).

[000207] ¹³C NMR ( 150 MHz, CD₃OD) δ 176.3, 174.0, 172.6, 172.3, 162.3, 158.4, 156.2, 154.2, 144.2, 142.6, 140.0, 139.4, 138.4, 135.3, 135.3, 134.5, 134.2, 130.2, 129.6, 128.2, 121.1 , 109.8, 106.5, 67.4, 61 .2, 60.6, 56.5, 56.2, 55.0, 54.8, 38.4, 36.5, 35.0, 31.7, 30.4, 27.8, 27.4, 26.5, 26.4, 20.9, 19.8, 18.9.

[000208] HRMS: Obsvd 976.4421 [M + Na]⁺, Calcd for C₅₀H₆₃N₇NaO₁₂: 976.4427.

[000209] 3-Methoxy-9-(3 ,4,5-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-yl (4- nitrophenyl) carbonate 12. To a flask containing a solution of GP 18 (0.108 g, 0.303 mmol) in CH₂C1₂ (5 mL) was added pora-nitrophenyl chloroformate (0.363 g, 1 .80 mmol) and Et₃N (0.253 mL, 1 .80 mmol), and the reaction was stirred for 16 h at ambient temperature. Water (10 mL) was added to the reaction, and the resulting mixture was extracted by EtOAc (3 x 15 mL). The combined organic layer was washed with brine, dried over Na₂S0 , filtered, concentrated under reduced pressure and purified by flash chromatography using a pre-packed 10 g silica column [solvent A: MeOH; solvent B: CH₂C1₂; gradient: 0%A / 100%B ( 1 CV), 0%A / 100%B→ 4%A / 96%B (10 CV), 4%A / 96%B (2 CV); flow rate: 12 mL/min; monitored at 254 and 280 nm] to afford activated KGP18 derivative 12 (0.150 g, 0.288 mmol, 90% yield) as a yellow solid.

[000210] Ή NMR (500 MHz, CDC1₃) δ 8.39 - 8.25 (2H, m), 7.59 - 7.47 (2H, m), 6.99 (1 H, d, J = 8.4 Hz), 6.86 (1 H, d, J = 8.7 Hz), 6.50 (2H, s), 6.39 (1H, t, J = 7.3 Hz), 3.92 (3H, s), 3.87 (3H, s), 3.82 (6H, s), 2.72 (2H, t, J = 6.9 Hz), 2.1 7 (2H, p, J = 7.1 Hz), 1 .99 (2H, q, J = 7.2 Hz).

[000211] ¹³C NMR ( 125 MHz, CDC1₃) δ 155.8, 153.1 , 151 .0, 149.8, 145.7, 142.3, 137.9, 137.7, 137.4, 135.1 , 133.9, 128.3, 127.9, 125.5, 121.8, 109.6, 105.5, 61.1 , 56.4, 56.2, 34.1 , 25.5, 24.9.

[000212] HRMS: Obsvd 522.1756 [M + H]⁺, Calcd for C₂₈H₂₈N0₉: 522.1759. HPLC (Method B): 1 8.65min.

[000213] 3-Methoxy-9-(3 ,4,5-trimethoxyphenyl)-6,7-dihydro-5H-benzo[7]annulen-4-yl methyl(2- (methylamino)ethyl)carbamate hydrochloride 17. DMED (0.598 g, 6.79 mmol) was added to a solution of carbonate 12 (0.354 g, 0.679 mmol) in CH₂C1₂ (10 mL), and the reaction was stirred for 15 min. Water ( 10 mL) was added, and the mixture was extracted with CH₂C1₂ (3 x 10 mL). The combined organic layer was washed with brine, dried over Na₂S0₄, filtered, and the filtrate was added a HC1 solution (4 N in dioxane, 2 mL). The resulting solution was evaporated to dryness under reduced pressure, and the resulting crude solid was then washed with Et₂0 (3 < 15 mL) to afford desired hydrochloride salt 17

(0.282 g, 0.559 mmol, 82% yield) as a red solid.

[000214] Ή NMR (600 MHz, CD₃OD) δ 6.94 ( 1 H, d, J = 8.6 Hz), 6.87 (1 H, d, J = 8.7 Hz), 6.53 (2H, s), 6.39 (1 H, t, J = 6.9 Hz), 3.87 - 3.83 (3H, m), 3.76 (3H, s), 3.75 (6H, s), 3.73 (3H, s), 3.40 - 3.36 (2H, m), 3.12 - 3.08 (2H, m), 2.87 - 2.75 (3H, m), 2.67 - 2.49 (2H, m), 2.18 - 2.05 (2H, m), 1 .96 -1 .88 (2H, m)

[000215] HRMS: Obsvd 471.2491 [M - Cl]⁺, Calcd for C₂₆H₃₅N₂0₆: 471.2490.

[000216] Mc-Val-Cit-PABC-DMED- GP 18 Bis-Carbamate 16. Activated linker 6 (0.029 g, 0.039 mmol) was dissolved in anhydrous DMF, which was then added hydrochloride salt 17 (0.020 g, 0.039 mmol) and DIEA (0.026 g, 0.16 mmol). The reaction was stirred for 16 h and the solvent was evaporated under reduced pressure. The resulting thick oil was purified by prep reversed TLC (ACN/H₂0, 1 : 1 ) to afford drug-linker conjugate 16 (0.0032 g, 0.0030 mmol, 8% yield) as a white solid.

[000217] HRMS: Obsvd 1091.5029 [M + Na]⁺, Calcd for C₅5H₇₂N₈Na0₁₄: 1091.5060.

[000218] tert-Butyl (4-(hydroxymethyl)phenyl)carbamate 19. To a solution of ra-aminobenzyl alcohol (0.123 g, 0.0999 mmol) in AcOH (10% in water, 8 mL) was added a solution of di-feri-butyl dicarbonate in THF (1 M, 1.05 mL, 1 .05 mmol), and the reaction was stirred for 18 h at ambient temperature. Water (30 mL) was added, and the mixture was basified with a NaOH solution (2 M) to pH 14. The resulting mixture was extracted with Et₂0 (3 ^χ 30 mL). The combined organic layer was washed with water (2 x 1 5 mL), dried over Na₂S0₄, filtered, and concentrated under reduced pressure to afford boc-protected aminobenzyl alcohol 19 (0.166 g, 0.743 mmol, 74% yield) as a light yellow solid.

[000219] Ή NMR (600 MHz, CDC1₃) δ 7.34 (2H, d, J = 8.0 Hz), 7.28 (2H, d, J = 8.4 Hz), 4.62 (2H, s), 1 .52 (9H, s).

[000220] ¹³C NMR (150 MHz, CDC1₃) δ 152.9, 138.0, 135.6, 128.0, 1 18.7, 65.1 , 31.3, 28.5.

[000221] HRMS: Obsvd 246.1 102 [M + Na]⁺, Calcd for C₁₂H₁₇NNa0₃: 246.1 101.

[000222] 4-Nitrobenzyl methanesulfonate 23. To a flask containing a solution of 4-Nitrobenzyl alcohol (0.613 g, 4.00 mmol) in CH₂C1₂ (16 mL) at 0 °C were added methanesulfonyl chloride (0.325 mL, 4.20 mmol) and Et₃N (0.665 mL, 4.80 mmol). The reaction was stirred for 1 h at 0 °C, and water (20 mL) was added. The reaction mixture was extracted with CH₂C1₂ (3 ^χ 20 mL), and the combined organic layer was washed with brine, dried over Na₂S0₄, filtered, and concentrated under reduced pressure to afford desired mesylate 23 (0.829 g, 3.59 mmol, 90% yield) as a yellow solid.

[000223] Ή NMR (600 MHz, CDC1₃) 6 8.28 (2H, d, J= 8.8 Hz), 7.60 (2H, d, J = 8.8 Hz), 5.33 (2H, s), 3.07 (3H, s)

[000224] ^l3C NMR (150 MHz, CDC1₃) δ 148.3, 140.7, 129.0, 124.1 , 69.1 , 38.3.

[000225] 3-Methoxy-4-((4-nitrobenzyl)oxy)-9-(3,4,5-trimethoxyphenyl)-6,7-dihydro-5H- benzo[7]annulene 24. To a solution of KGP1 8 (0.250 g, 0.702 mmol) and K₂C0₃ (0280 g, 2.03 mmol) in anhydrous DMF (25 mL) was added mesylate 23 (0.260 g, 1 .12 mmol), and the reaction was stirred for 16 h at ambient temperature. The solvent was removed by evaporation under reduced pressure, and water (30 mL) was added to the residue, which was then extracted with EtOAc (3 ^χ 30 mL). The combined organic layer was washed with brine, dried over Na₂S0₄, filtered, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: EtOAc; solvent B : hexanes; gradient: 0%A / 100%B (1 CV), 12%A / 88%B→ 80%A / 40%B (10 CV), 80%A / 40%B (2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] to afford activated KGP18 derivative 24 (0.324 g, 0.660 mmol, 94% yield) as a white solid.

[000226] Ή NMR (600 MHz, CDC1₃) δ 8.27 (2H, d, J = 8.1 Hz), 7.71 (2H, d, J = 8.1 Hz), 6.83 (1 H, d, J = 8.5 Hz), 6.80 (1 H, d, J = 8.5 Hz), 6.48 (2H, s), 6.34 (1 H, t, J = 7.2 Hz), 5.15 (2H, s), 3.90 (3H, s), 3.87 (3H, s), 3.81 (6H, s), 2.74 (2H, t, J= 6.8 Hz), 2.06 (2H, p, J = 7.2 Hz), 1 .94 (2H, q, J = 7.0 Hz).

[000227] ⁿC NMR (150 MHz, CDCI₃) δ 1 3.0, 151.4, 147.6, 145.7, 144.4, 142.8, 138.3, 137.5, 136.0, 1 34.0, 128.1 , 127.3, 126.0, 123.8, 109.5, 105.3, 73.8, 61.1 , 56.3, 55.8, 34.6, 25.7, 24.5.

[000228] HRMS: Obsvd 514.1834 [M + Na]⁺, Calcd for C₂₈H₂₉N a0₇: 514.1836. HPLC (Method B): 19.55min.

[000229] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/".

[000230] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or

"approximately," even if the term does not expressly appear. The phrase "about" or "approximately" may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1 % of the stated value (or range of values), +/- 1 % of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +1- 1 0% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[000231] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims. [000232] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term "invention" merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.

Claims

What is claimed is:

A composition comprising a compound having the formula la:

or a pharmaceutically acceptable salt thereof,

wherein the phenyl ring "Z" is bonded to either carbon "a" or "b";

R, and R₃, are each, independently, selected from the group consisting of H, lower alkoxy, phosphate, hydroxyl, and a linker (Y), wherein at least one of R] and R₃ is the linker (Y);

( and R₅ are each, independently, selected from the group consisting of H, lower alkoxy, phosphate, and hydroxyl;

X is selected from the group consisting of a single bond and C(O); and

n is 1 , 2, 3 or 4.

2. The composition of claim 1 , wherein the linker (Y) comprises a cathepsin B protease cleavable compound.

3. The composition of claim 1 , wherein the linker (Y) comprises a non-cleavable linker adapted to be cleaved or degraded intracellularly or not cleaved.

4. The composition of any of claims 1 -3, wherein R₃ is H;

R₄ and R₅ are OCH₃;

X is a single bond;

n is 2.

5. The composition of claim 4, wherein the compound has the formula:

, wherein R, is the linker (Y).

6. The composition of any of claims 1 -3, wherein R₃ is H;

R, and R₅ are OCH₃;

X is a single bond;

n is 1 , 3, or 4.

7. The composition of claim 6 having the formula:

, wherein R, is the linker (Y).

8. A composition comprising a compound having the formula:

, wherein Y is a linker and R' is hydrogen or hydroxyl.

9. The composition of claim 8, wherein the linker (Y) comprises a cathepsin B protease cleavable compound.

10. The composition of claim 8, wherein the linker (Y) comprises a non-cleavable linker adapted to be cleaved or degraded intracellularly or not cleaved. 1 1 . A composition comprising a compound having the formula:

, wherein Y is a linker.

12. The composition of claim 1 1 , wherein the linker (Y) comprises a cathepsin B protease cleavable compound.

13. The composition of claim 1 1 , wherein the linker (Y) comprises a non-cleavable linker adapted to be cleaved or degraded intracellularly or not cleaved. 14. The composition of any one of claims 1 - 13, wherein Y comprises:

5. The composition of any one of claims 1-13, wherein Y comprises:

The composition of any one of claims 1-13, wherein Y comprises:

17. The composition of any one of claims 1-13, wherein Y comprises:

The composition of claim 5, having the formula:

The composition of claim 5, having the formula:

The composition of claim 5, having the formula:

The composition of claim 5, having the formula:

The composition of claim 5, having the formula:

7. The composition of claim 5, having the formula:

The composition of claim 5, having the formula:

The composition of claim 7, having the formula:

The composition of claim 7, having the formula:

31. The composition of claim 7, having the formula:

The composition of claim 7, having the formula:

The composition of claim 7, having the formula:

34. The composition of claim 7, having the formula:

The composition of claim 7, having the formula:

The composition of claim 7, having the formula:

The composition of claim 8, having the formula:

40. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 39.

41 . A method of treating cancer, comprising:

administering to a mammal in need thereof a compound according to any of claims 1 -40.

42. The method of claim 41 , further comprising: contacting the compound according to any of claims 1 -40 with a cathepsin B protease such that all or a portion of the cathepsin B protease cleavable portion of the compound is cleaved by the cathepsin B protease. 43. The method of claim 41 , further comprising: contacting the compound according to any of claims 1 -40 with a cancer cell such that all or a portion of the linker is cleaved intracellularly or such that the linker is not cleaved.

44. A method comprising contacting in vivo a cathepsin B protease with a compound according to any one of claims 1 to 40 such that all or a portion of the cathepsin B protease cleavable portion of the compound according to any one of claims 1 -40 is cleaved by the cathepsin B protease.

45. A method comprising contacting in a cell a cathepsin B protease with a compound according to any one of claims 1 to 40 such that all or a portion of the cathepsin B protease cleavable portion of the compound according to any one of claims 1 -40 is cleaved by the cathepsin B protease.

46. A method comprising contacting in a cell a lysosome or peptidase with a compound according to any one of claims 1 to 40 such that all or a portion of the linker of the compound according to any one of claims 1 -40 is cleaved. 47. A method for making a Val-Cit cleavable cathepsin B linker, comprising the following steps:

(a) Fmoc protection of L-citrulline followed by reaction with para-amino benzyl alcohol (PABOH) followed by Fmoc deprotection

(b) dipeptide formation by reacting the product of step (a) with Fmoc-Val-OSu followed by Fmoc deprotection;

(c) reacting the product of step (b) with activated maleimido caproic acid (MC-OSu) to form the Val-Cit cleavable cathepsin B linker.