Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06008—Dipeptides with the first amino acid being neutral
  • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
  • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
  • C07K5/06052—Val-amino acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
  • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
  • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide

Definitions

• the present application relates to small molecules for treating cancer that can include a cytotoxic payload and a linker.
• compositions including an antibody linker bound to a highly cytotoxic compound 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.
• the present invention relates to novel compounds including a vascular targeting or disrupting agent and a linker along with methods of use.
• 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.
• composition including a compound having the formula la:
• R 3 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 R 3 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 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.
• R 3 is H;
• R4 and R 5 are OCH 3 ;
• X can be a single bond;
• n can be 2.
• the compound can have the formula:
• R can be the linker (Y).
• R 3 is H; R 4 and R 5 can be OCH 3 ; X can be a single bond; n can be 1 , 3, or 4.
• the composition can have the formula:
• can be the linker (Y).
• 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.
• 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.
• Y ean include:
• Y can include:
• Y can include:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• composition can have the formula:
• 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.
• 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.
• PABOH para-amino benzyl alcohol
• MC-OSu activated maleimido caproic acid
• FIGS. 1 A-1 B illustrate examples of synthetic schemes in accordance with some embodiments
• FIG. 2 illustrates two synthetic schemes in accordance with some embodiments.
• FIG. 3 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 4 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 5 shows a High Resolution Mass Spectroscopy (HRMS) of an isocyanate intermediate compound in accordance with some embodiments.
• FIG. 6A illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 6B illustrates HRMS of a compound in accordance with some embodiments.
• FIG. 7A illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 7B illustrates HRMS of a compound in accordance with some embodiments.
• FIGS. 8A-8B illustrate synthetic schemes in accordance with some embodiments.
• FIG. 9 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 10 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 1 1 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 12 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 13 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 14 illustrates a synthetic scheme in accordance with some embodiments.
• FIG. 15 is a schematic illustration of examples of a cathepsin B protease cleaving a portion of a linker attached to an effective drug.
• ADCs Antibody-drug conjugates
• GI 50 sub- nanomolar to picomolar range against human cancer cell lines
• VDAs potent vascular disrupting agents
• 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.
• the payloads can incorporate amino or hydroxyl moieties that can be suitable for covalent attachment to the appropriate linker.
• the payloads attached to the cleavable or non- cleavable linker have a cytotoxicity in the sub-nanomolar range.
• the IC 50 value can be less than 1 x 10 "9 M.
• the payloads attached to the linker can have a high VDA activity alone or in combination with a high cytotoxicity.
• 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.
• 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.
• cyste 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.
• 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 ).
• 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-
• cathepsin B-like proteases include cathepsin B, T. cruzi-B, and L. mexicana-B.
• IC 50 refers to the concentration of compound that results in half-maximal inhibition of enzyme.
• 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 (CH 3 -), ethyl (CH 3 CH 2 -), n-propyl (CH 3 CH 2 CH 2 -), isopropyl ((CH 3 ) 2 CH-), n-butyl (CH 3 CH 2 CH 2 CH 2 -), isobutyl ((CH 3 ) 2 CHCH 2 -), sec-butyl
• 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.
• 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, substituted al
• Amino refers to the group -NH 2 .
• 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.
• 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.
• 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 (-CH 2 C ⁇ CH).
• 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.
• suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like.
• Halo or "halogen” refers to fluoro, chloro, bromo, and iodo and is preferably fluoro, bromo, or chloro.
• 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.
• 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.
• Heterocycle refers 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.
• the nitrogen and/or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or -S0 2 - moieties.
• 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
• Neitro refers to the group -N0 2 .
• Niroso refers to the group -NO.
• 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.
• a group that is substituted has 1 , 2, 3, or 4 substituents, 1 , 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.
• 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.
• pyrazoles imidazoles, benzimidazoles, triazoles, and tetrazoles.
• Patient refers to human and non-human animals, especially mammals.
• “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,
• “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.
• 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.
• solvent 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.
• impermissible substitution patterns e.g., methyl substituted with 5 fluoro groups.
• impermissible substitution patterns are easily recognized by a person having ordinary skill in the art.
• the compound including the payload and cathepsin B protease
• R] and R 3 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 R 3 is the linker (Y);
• R4 and R 5 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);
• n 1 , 2, 3 or 4.
• the linker (Y) can be any of the linkers described herein including cleavable linkers and non-cleavable linkers.
• R4 and R 5 are OCH 3 .
• 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.
• R 3 is H
• R 4 and R 5 are OCH 3
• X is a single bond
• n is
• the linker (Y) can be linked through a hydroxyl or amino group on R] .
• R 3 is H
• R 4 and R 5 are OCH 3
• X is a single bond
• n is
• the linker (Y) can be linked through a hydroxyl or amino group on R,.
• Ri and R 3 are H or OH.
• R, is OH or NH 2 and R 3 is H.
• R, and R 3 are OCH 3 .
• the linker can be linked through a hydroxyl or amino group on R, or R 3 .
• either of Ri or R 3 can be linked to the linker.
• 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 R 3 .
• the compound including the payload and linker includes a compound of the following general Formula lb:
• X is selected from the group consisting of a single bond, CH 2 , O, S, N(H), and C(O);
• X, , X 2 , X 3 , X4 and X 5 are each, independently, selected from the group consisting of C, C(H), N, N(H), O and S;
• Ri, R 2 , R 3 , R 4 , R 5 , R6 and R 7 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, R 2 , and R 3 is or is attached to the linker (Y), and ring "Z" is bonded to either carbon "a” or "b.”
• X is a single bond
• Ri, R 2 , R 2 , and R 3 is or is attached to the linker (Y), and ring "Z"
• X is a single bond
• R4, R 5 and 3 ⁇ 4 are each, independently, selected from the group consisting of H, OCH 3 , phosphate, amino, and OH.
• the linker (Y) can be any of the linkers described herein including cleavable linkers and non-cleavable linkers.
• either of R R 2 , or R 3 can be linked to the cathepsin B protease cleavable linker or non-cleavable linkers.
• any of the cathepsin B protease cleavable compounds described herein can be attached to compound lb through either R R 2 , or R 3 .
• 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.
• 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:
• 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.
• 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:
• 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:
• 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).
• CA4 combretastatin A-4
• 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 ⁇ 8006 is shown below:
• 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.
• 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.
• 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.
• the non-cleavable linker and payload may have activity after being only partially cleaved or not substantially cleaved after antibody targeting.
• any of the compounds described herein can be attached to an antibody that can be used to target the specific cancer cells.
• the payload can have the formula below, with Y as any of the linkers describe herein:
• the payload can have the formula below, with Y as any of the linkers
• 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):
• the pay load can have the formula below, with Y as any of the linkers described herein:
• 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.
• the linker is a cathepsin B protease cleavable linker with the formula of:
• the linker is a cathepsin B protease cleavable linker with the formula of:
• the linker is a cathepsin B protease cleavable linker with the formula of:
• the linker is a cathepsin B protease cleavable linker with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• the linker may be a non-cleavable linker that can all or partially cleave intracellularly with the formula of:
• 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.
• composition includes a payload and a cathepsin B protease cleavable linker with the following formula (also referred to as KGP477):
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a cathepsin B protease cleavable linker with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a cathepsin B protease cleavable linker with the following formula:
• composition includes a payload and a cathepsin B protease cleavable linker with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved ula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• composition includes a payload and a linker that may be cleaved intracellularly with the following formula:
• the composition includes a payload and a cathepsin B protease cleavable linker with the following formula (also referred to as KGP475):
• the composition includes a payload and a cathepsin B protease cleavable linker with the following formula (also referred to as GP474):
• 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 IC 50 values are reported along with standard deviations. For KGP474 an IC 50 of 0.203 ⁇ 0.0467 and 0.712 ⁇ 0.21 1 for DU 145 and NCI-H460, respectively, was observed.
• 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.
• compositions comprising any of the compounds described herein are provided.
• the pharmaceutical formulations can be administered orally, intravenously, or through other common administration routes.
• methods of treating cancer 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.
• 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.
• 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.
• 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.
• linkers can be bound to the payload through an amino or hydroxyl group on the payload.
• 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.
• 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.
• the yield obtained in FIG. 1 A was above about 96%.
• 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.
• 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.
• 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.
• PABOH para-amino benzyl alcohol
• MC-OSu activated maleimido caproic acid
• FIG. 1 A also illustrates another synthetic scheme for making an activated succinimide ester 4.
• carboxylic acid 1.0 equiv
• disuccinimidyl carbonate (1.05 equiv)
• DMF 0.5M
• triethylamine 1.0 equiv
• 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%).
• 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.
• 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.
• HOBt hydroxybenzotriazole
• FIG. 4 illustrates yet another alternative route for the synthesis of cathepsin B- GP156 conjugate 7.
• a solution of KGP156 in CH 2 C1 2 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.
• HRMS high resolution mass spectroscopy
• FIG. 6A illustrates a scheme using another spacer, ⁇ , ⁇ '-dimethylethylenediamine (DMED), that was installed between a cathepsin B linker and KGP18 for the purpose of generation of two carbamate linkages.
• DMED dimethylethylenediamine
• FIG. 6B shows HRMS of Compound 14.
• 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 Et 3 N 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).
• Additional methods for the synthesis of the cathepsin B-KGP18 conjugate 16 was explored as shown in FIGS. 8A-8B.
• 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.
• 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
• 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 CH 2 C1 2 and water and NaHC0 3 solution was added and stirred get clear phases. Organic layer was separated.
• 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.
• 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
• 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 CH 2 C1 2 and washed with saturated NaHC0 3 solution.
• KGP18 is slight yellowish solid.
• 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.
• a 2:1 mixture of CH 2 C1 2 /CH 3 0H (15 ml) was added and the reaction was stirred under nitrogen at room temperature for 20 hours.
• 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 CH 2 C1 2 (5 ml). Triphosgene (0.29 mmol) and saturated NaHC0 3 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 Na 2 S0 4 . 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).
• Mass spectrometry was carried out under positive or negative electrospray ionization (ESI) using a Thermo Scientific LTQ Orbitrap Discovery instrument.
• ESI electrospray ionization
• the trimethoxy A ring was numbered 1-6
• 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.
• Fmoc-Val-Cit 1. To a solution of l-citrulline (0.274 g, 1.56 mmol) and NaHC0 3 (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.
• n C NMR 125 MHz, DMSO-d6) ⁇ 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.
• 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. CH 2 C1 2 (25 mL) was added into reaction which was then washed with water (3 x 10 mL).
• 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 Et 2 0 (30 mL). The crude product was collected by filtration and washed with Et 2 0 (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: CH 2 C1 2 ; 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.
• 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.
• Mc-Val-Cit-PABC- GPl 56 Carbamate 7 To a flask containing a solution of KGP156 (0.100 g, 0.281 mmol) in CH 2 C1 2 (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 N 2 gas followed by rotavapor evaporation at 40 °C to obtain the isocyanate intermediate 10 as a tan solid.
• 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/H 2 0, 1 : 1 ) to afford drug-linker conjugate 16 (0.0032 g, 0.0030 mmol, 8% yield) as a white solid.
• 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 Et 2 0 (3 ⁇ 30 mL).
• reaction mixture was extracted with CH 2 C1 2 (3 ⁇ 20 mL), and the combined organic layer was washed with brine, dried over Na 2 S0 4 , filtered, and concentrated under reduced pressure to afford desired mesylate 23 (0.829 g, 3.59 mmol, 90% yield) as a yellow solid.
• n C NMR 150 MHz, CDCI 3 ) ⁇ 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.
• 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.

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