Classifications

• • 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
  • A61K47/54—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 organic compound
  • A61K47/554—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 organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile 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
  • A61K47/54—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 organic compound
  • A61K47/545—Heterocyclic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
• • 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
  • A61K47/54—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 organic compound
  • A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug
  • A61K47/551—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic 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
  • A61K47/62—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 a protein, peptide or polyamino acid
  • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
  • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
  • C07J43/003—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton not condensed
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
  • C07J71/0005—Oxygen-containing hetero ring
  • C07J71/0026—Oxygen-containing hetero ring cyclic ketals
  • C07J71/0031—Oxygen-containing hetero ring cyclic ketals at positions 16, 17

Definitions

• Macrophages are a diverse group of white blood cells known for eliminating pathogens through phagocytosis.
• macrophages were classified by the organ in which they were found: Kupffer cells in the liver, Langerhans cells in the skin, microglia in the brain and spinal cord, and osteoclasts in the bone.
• M2 macrophages are alternatively activated by exposure to certain cytokines such as IL-4, IL-10, or IL-13. M2 macrophages will produce either polyamines to induce proliferation or proline to induce collagen production. These macrophages are associated with wound healing and tissue repair.
• the disclosure relates to the design, synthesis, and testing of a series of compounds. Accordingly, the disclosure provides compounds comprising folate or related compounds as a ligand linked to a steroid via a linker.
• the linker can be any suitable linker, such as a hydrophilic linker.
• the linker can comprise one or more amino acids, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, or a combination of any of the foregoing.
• the linker can comprise an oligomer of peptidoglycans, glycans, anions, or a combination of any of the foregoing.
• the compounds polarize macrophages from pro-inflammatory (Ml) to anti-inflammatory (M2).
• the ligand is a folate analog or an antifolate.
• the steroid can be any suitable steroid, such as dexamethasone, betamethasone or betamethasone 17-valerate.
• the steroid can be releasable, such as via reduction, oxidation, or hydrolysis, or non-releasable.
• the steroid can release via a self-immolative moiety.
• G 1 is a folate radical, an antifolate radical, or a folate analog radical
• L is a linker
• L is a linker
• G 1 is an antifolate radical or a folate analog radical comprising an amino acid selected from the group consisting of aspartic acid, lysine, tyrosine, cysteine, threonine, serine, histidine, and arginine.
• G 1 is a radical of an antifolate of any one of formulas in Table 4 as described herein.
• the steroid polarizes macrophages from pro-inflammatory (Ml) to anti-inflammatory (M2).
• G 2 is a radical of a steroid selected from the group consisting of betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone- 17-valerate, budesonide, flumethazone, fluticasone proprionate, fluorocortisone, fludrocortisone, paramethasone, eplerenone, and an ester of any of the foregoing.
• G 2 is a radical of dexamethasone.
• G 2 is a radical of prednisone.
• G 2 is a radical of prednisolone. In some embodiments, G 2 is a radical of methylprednisolone. In some embodiments, G 2 is a radical of budesonide. In some embodiments, G 2 is a radical of triamcinolone. In some embodiments, G 2 is a radical of betamethasone. In some embodiments, the linker is releasable. In some embodiments, the linker is non-releasable. In some embodiments, the linker comprises one or more of an amino acid, an alkyl chain, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, or a combination of any of the foregoing.
• PEG polyethylene glycol
• the linker increases the water solubility of the compound.
• the linker comprises an oligomer of peptidoglycans, glycans, anions, or a combination of any of the foregoing.
• the linker comprises at least one 2,3-diaminopropionic acid group, at least one glutamic acid group, and at least one cysteine group.
• the linker comprises a repeating unit of the formula: wherein q is an integer from 1 to 10.
• the linker comprises the formula: wherein q is an integer from 1 to 10.
• the linker comprises the formula: wherein q is an integer from 1 to 10.
• the linker comprises the formula: wherein X can be O, NH, NR, or S, and q is an integer from 1 to 10, wherein R is Ci- 6 alkyl.
• the linker comprises the formula:
• the linker is a bivalent linker. In some embodiments, the linker is polyvalent and has multiple attachment points for one or more additional chemical groups. In some embodiments, the additional chemical groups comprise one or more additional G 1 groups. In some embodiments, the additional chemical groups comprise one or more binding ligands that are not G 1 groups. In some embodiments, the linker comprises a PEG oligomer with 2-16 PEG units. In some embodiments, the linker comprises a PEG oligomer with 12 PEG units. In some embodiments, the linker comprises an albumin ligand. In some embodiments, the albumin ligand comprises
• the linker comprises a dimethylcysteine group.
• the dimethylcysteine group is linked to a succinimide to form:
• the linker comprises a phosphate or pyrophosphate group.
• the linker comprises a cathepsin B cleavable group.
• the cathepsin B cleavable group is valine-citrulline.
• the linker comprises a carbamate moiety.
• the linker comprises a b-glucuronide.
• the linker comprises an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate or enzyme -cleavable amino acid sequence.
• the linker comprises a self-immolative moiety. In some embodiments, the linker comprises a self-immolative disulfide and or sterically protected disulfide bond. In some embodiments, the linker comprises a self-immolative cathepsin- cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative furin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative b-glucuronidase -cleavable moiety. In some embodiments, the linker comprises a self-immolative phosphatase-cleavable moiety.
• the linker comprises a self-immolative sulfatase- cleavable moiety.
• the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof.
• the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof.
• the compound has the formula:
• the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof. In some embodiments, the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof. In some embodiments, the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof. In some embodiments, the compound has the formula:
• the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof. In some embodiments, the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof. In some embodiments, the compound has the formula: or a pharmaceutically acceptable salt, polymorph, prodrug, solvate or clathrate thereof. In some embodiments, the compound is selected from the compounds in the specification.
• compositions comprising: (a) a compound disclosed herein; and (b) a pharmaceutically acceptable excipient.
• the pharmaceutically acceptable excipient is part of a nanoparticle, a liposomal formulation or an exosomal formulation.
• a method of shifting macrophages from Ml to M2 in a subject in need thereof comprising: administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• methods for treating an inflammatory disease or disorder (“disease” and “disorder” can be used interchangeably) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• G 2 is a radical of dexamethasone.
• G 2 is a radical of prednisone.
• G 2 is a radical of prednisolone. In some embodiments, G 2 is a radical of methylprednisolone. In some embodiments, G 2 is a radical of budesonide. In some embodiments, G 2 is a radical of triamcinolone. In some embodiments, G 2 is a radical of betamethasone. In some embodiments, the inflammatory disorder is Crohn’s disease, lupus, inflammatory bowel disease (IBS), Addison’s disease, Grave’s disease,
• Sjogren’s syndrome celiac disease, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, reactive arthritis, psoriatic arthritis, pernicious anemia, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, or fibrotic disease, graft vs.
• GVHD host disease
• fatty liver disease asthma, osteoporosis, sarcoidosis, ischemia-reperfusion injury, prosthesis osteolysis, glomerulonephritis, scleroderma, psoriasis, with autoimmune myocarditis, spinal cord injury, central nervous system, viral infection, influenza, coronavirus infection, cytokine storm syndrome, bone damage, inflammatory brain disease, atherosclerosis, cancer, or a tumor.
• the disorder is treated with reduced adverse effects relative to when the steroid is administered without linkage to the folate ligand.
• G 2 is a radical of dexamethasone.
• G 2 is a radical of prednisone.
• G 2 is a radical of prednisolone.
• G 2 is a radical of methylprednisolone.
• G 2 is a radical of budesonide.
• G 2 is a radical of triamcinolone.
• G 2 is a radical of betamethasone.
• a subject in need thereof is a method for treating inflammation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• the inflammation is treated with substantially reduced side effects relative to when the steroid is administered without linkage to the folate ligand.
• the inflammation is associated with an autoimmune disease.
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, fludrocortisone, beclomethasone, fluticasone, mometasone, ciclesonide, cortisone, cortivazol, hydrocortisone, 21-hydroxypregnenolone, meprednisone, dexamethasone, triamcinolone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of dexamethasone.
• G 2 is a radical of prednisone.
• G 2 is a radical of prednisolone. In some embodiments, G 2 is a radical of methylprednisolone. In some embodiments, G 2 is a radical of budesonide. In some embodiments, G 2 is a radical of triamcinolone. In some embodiments, G 2 is a radical of betamethasone.
• the inflammation is associated with Crohn’s disease, lupus, inflammatory bowel disease (IBS), Addison’s disease, Grave’s disease, Sjogren’s syndrome, celiac disease, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, reactive arthritis, psoriatic arthritis, pernicious anemia, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, or fibrotic disease, graft vs.
• IBS inflammatory bowel disease
• Addison’s disease Grave’s disease
• Sjogren’s syndrome celiac disease
• Hashimoto’s thyroiditis myasthenia gravis
• autoimmune vasculitis reactive arthritis
• psoriatic arthritis pernicious anemia
• ulcerative colitis ulcerative colitis
• rheumatoid arthritis type 1 diabetes
• multiple sclerosis or fibrotic disease
• GVHD host disease
• fatty liver disease asthma, osteoporosis, sarcoidosis, ischemia- reperfusion injury, prosthesis osteolysis, glomerulonephritis, scleroderma, psoriasis, with autoimmune myocarditis, spinal cord injury, central nervous system, viral infection, influenza, coronavirus infection, cytokine storm syndrome, bone damage, inflammatory brain disease, atherosclerosis, cancer, or a tumor.
• the inflammation is associated with inflammatory bowel disease, and wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, hydrocortisone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with Addison’s disease, and G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, hydrocortisone, fludrocortisone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with Grave’s disease
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with Sjogren’s syndrome
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with celiac disease
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with Hashimoto’s thyroiditis, and wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with myasthenia gravis
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with autoimmune vasculitis
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with reactive arthritis
• G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with psoriatic arthritis
• G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with pernicious anemia
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with ulcerative colitis
• G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, triamcinolone, beclomethasone, betamethasone, dexamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with multiple sclerosis, and G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with asthma, and G 2 is a radical of a steroid selected from the group consisting of triamcinolone, fluticasone, budesonide, mometasone, beclomethasone, ciclesonide, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with osteoporosis
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with sarcoidosis
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with a fibrotic disease
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, and prednisone.
• the inflammation is associated with graft vs. host disease (GVHD)
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with fatty liver disease
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with ischemia-reperfusion injury
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with prosthesis osteolysis, and G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with scleroderma, and G 2 is a radical of a steroid selected from the group consisting of dexamethasone, budesonide, betamethasone, triamcinolone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with psoriasis
• G 2 is a radical of a steroid selected from the group consisting of budesonide, betamethasone, triamcinolone, prednisone, prednisolone, methylprednisolone, hydrocortisone, dexamethasone, hydrocortisone- 17- valerate, diflorasone, meprednisone, halobetacol, tixocortol, amcinonide, desonide, fluocinolone acetonide, fluocinonide, halcinonide, beclomethasone, and halometasone.
• the inflammation is associated with a viral infection
• G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with influenza
• G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with SARS-CoV-2 (COVID-19), and G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, and prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with cytokine storm syndrome, and G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with damage to bone
• G 2 is a radical of a steroid selected from the group consisting of cortisone, cortivazol, hydrocortisone, 21-hydroxypregnenoIone, meprednisone, dexamethasone, triamcinolone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with inflammatory brain disease
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with atherosclerosis
• G 2 is a radical of a steroid selected from the group consisting of fluticasone, budesonide, beclomethasone, ciclesonide, dexamethasone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with a tu or, and wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, clobetasol, dexamethasone, betamethasone, budesonide, meprednisone, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, clobetasol, dexamethasone, betamethasone, budesonide, meprednisone, prednisone, prednisolone, and methylprednisolone.
• the inflammation is associated with cancer
• G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, clobetasol, dexamethasone, betamethasone, budesonide, meprednisone, prednisone, prednisolone, and methylprednisolone.
• a disease or disorder that involves polarizing macrophages from Ml to M2 in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• FIG. 1 exemplifies the chemical structure for compound 101 of the disclosure.
• FIG. 2 exemplifies a reaction scheme for the activation of dexamethasone for linkage to a ligand comprising folate and a moiety that becomes a linker when reacted with the activated dexamethasone.
• FIG. 3 exemplifies the chemical structure of a ligand comprising folate and a moiety that becomes a linker when reacted with the activated dexamethasone shown in FIG. 2.
• FIG. 4 exemplifies a reaction scheme for the formation of compound 101, by reaction of the activated dexamethasone with the compound of FIG. 3.
• FIG. 5 exemplifies structures and names of steroids described in the disclosure.
• FIG. 7 is a plot of CD206 (a marker for M2 macrophages) expression vs. percent of maximum (Max) showing flow cytometry results of compound 101 in the absence and presence of the competitor folate -glucosamine compared to untreated cells and cells treated with free dexamethasone.
• FIG. 8 is a plot of CD86 (a marker for Ml macrophages) expression vs. percent of maximum (Max) showing flow cytometry results of compound 101 in the absence and presence of the competitor folate -glucosamine compared to untreated cells and cells treated with free dexamethasone.
• FIG. 9 is a set of plots of flow cytometery results for F4/80 rodent colon cells in a peritonitis model, and includes results for cells treated with compound 106, untreated, and vehicle.
• FIG. 10 is a bar graph of flow cytometery results for F4/80 rodent colon cells in a peritonitis model, and includes results for cells treated with compound 106, untreated, and vehicle.
• Ly6G and CD8 rodent colon cells in a peritonitis model, and includes results for cells treated with compound 107, and untreated.
• FIG. 13 is a set of plots of flow cytometery results for F4/80, CD4,
• FIG. 14 is a bar graph of flow cytometery results for F4/80, CD4, Ly6G, and CD8 rodent colon cells in a peritonitis model, and includes results for cells treated with compound 108, and untreated.
• FIG. 17 is a set of LC-MS traces for compound 106.
• FIG. 20 is a set of LC-MS traces for compound 108.
• FIG. 21 is a set of LC-MS traces for compound 124.
• FIG. 22 is a set of LC-MS traces for compound 126.
• FIG. 23 is a set of LC-MS traces for compound 127.
• Folate receptor beta is expressed on the surface of activated macrophages that are present at sites of inflam ation. These macrophages have been shown to be crucial in maintaining levels of pro-inflammatory signals in a variety of diseases.
• Steroids work by decreasing inflammation and reducing the activity of the immune system inflammation is a process in which the body’s white blood cells and chemicals can protect against infection and foreign substances such as bacteria and viruses. In certain diseases, however, the body’s defense system (immune system) doesn’t function properly. This might cause inflammation to work against the body’s tissues and cause damage. Signs of inflammation include redness, warmth, swelling, and pain.
• alkoxycycloalkylenecarbonyl would be understood to be an alkoxy as defined herein bonded to a cycloalkylene as defined herein, and the cycloalkylene is in turn bonded to a carbonyl group, which is not defined herein but is generally understood to organic chemists, with an open valence on the carbonyl.
• radical refers to a fragment of a molecule, wherein that fragment has an open valence for bond formation.
• a monovalent radical has one open valence such that it can form one bond with another chemical group.
• a radical of a molecule e.g., a radical of a steroid
• a radical is created by removal of one hydrogen atom from that molecule to create a monovalent radical with one open valence at the location where the hydrogen atom was removed.
• a radical can be divalent, divalent, etc., wherein two, three or more hydrogen atoms have been removed to create a radical which can bond to two, three, or more chemical groups.
• a radical open valence may be created by removal of other than a hydrogen atom (e.g., a halogen atom), or by removal of two or more atoms (e.g., a hydroxyl group), as long as the atoms removed are a small fraction (20% or less of the atom count) of the total atoms in the molecule forming the radical.
• a radical is formed from a folate, antifolate, or folate analog by removal of a hydroxyl group.
• substituted or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto another group (e.g., on an aryl or an alkyl group).
• substituents include, but are not limited to, a halogen (e.g., F, Cl, Br, and I), OR, OC(0)N(R) 2 , CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R) 2 , SR, SOR, SO2R, S0 2 N(R) 2 , SO3R, -(CH 2 )O- 2 P(0)(OR) 2 , C(0)R, C(0)C(0)R, C(0)CH 2 C(0)R, C(S)R, C(0)0R, 0C(0)R, C(0)N(R) 2 , 0C(0)N(R) 2 , C(S)N(R) 2 , (CH2)O- 2 N(R)C(0)R, (CH 2 )O- 2 N(R)C(0)OR, (CH 2 )O- 2 N(R)
• An “individual,” “subject” or “patient,” as used herein, is a mammal, preferably a human, but can also be an animal.
• Alkyl generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as having from one to fifteen carbon atoms ( e.g ., C 1 -C 15 alkyl). Disclosures provided herein of an “alkyl” are intended to include independent recitations of a saturated “alkyl,” unless otherwise stated. In certain embodiments, an alkyl comprises one to thirteen carbon atoms (e.g., C 1 -C 13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (e.g., C 1 -C 8 alkyl).
• an alkyl comprises one to five carbon atoms (e.g., C 1 -C 5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (e.g., C 1 -C4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (e.g., C 1 -C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (e.g., C 1 -C 2 alkyl). In other embodiments, an alkyl comprises one carbon atom (e.g.,
• the alkyl group is selected from methyl, ethyl, 1 -propyl (n-propyl), 1- methylethyl (Ao-propyl), 1 -butyl ( «-butyl), 1-methylpropyl (.sec -butyl), 2- methylpropyl (/50-butyl), 1,1-dimethylethyl (ferf-butyl), 1-pentyl ( «-pentyl).
• the alkyl is attached to the rest of the molecule by a single bond.
• Alkylene or “alkylene chain” generally refers to a straight or branched divalent alkyl group linking the rest of the molecule to a radical group, such as having from one to twelve carbon atoms, for example, methylene, ethylene, propylene, /-propylene, «-butylene, and the like.
• Aryl refers to a radical derived from an aromatic monocyclic or multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom.
• the aromatic monocyclic or multicyclic hydrocarbon ring system contains only hydrogen and carbon from five to eighteen carbon atoms, where at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) p-electron system in accordance with the Hiickel theory.
• the ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene.
• alkyl or “aryl-alkyl” refers to a radical of the formula -R c -aryl where R c is an alkylene chain as defined above, for example, methylene, ethylene, and the like.
• R c is an alkylene chain as defined above, for example, methylene, ethylene, and the like.
• the alkylene chain part of the aralkyl radical is optionally substituted as described above for an alkylene chain.
• Halo or halogen refers to bromo, chloro, fluoro or iodo substituents.
• Haloalkyl refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, 2,2,2-trifluoroethyl, l-fluoromethyl-2-fluoroethyl, and the like.
• heteroalkyl refers to an alkyl group as defined above in which one or more skeletal carbon atoms of the alkyl are substituted with a heteroatom (with the appropriate number of substituents or valencies - for example, -CH2- may be replaced with -NH- or -0-).
• each substituted carbon atom is independently substituted with a heteroatom, such as wherein the carbon is substituted with a nitrogen, oxygen, selenium, or other suitable heteroatom.
• each substituted carbon atom is independently substituted for an oxygen, nitrogen (e.g.
• a heteroalkyl is attached to the rest of the molecule at a carbon atom of the heteroalkyl. In some embodiments, a heteroalkyl is attached to the rest of the molecule at a heteroatom of the heteroalkyl. In some embodiments, a heteroalkyl is a C 1 -C 18 heteroalkyl. In some embodiments, a heteroalkyl is a C 1 -C 12 heteroalkyl.
• a heteroalkyl is a C 1 -C 6 heteroalkyl. In some embodiments, a heteroalkyl is a Ci- C4 heteroalkyl. In some embodiments, heteroalkyl includes alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, and heterocycloalkyl, heterocycloalkylalkyl, as defined herein.
• Heteroalkylene refers to a divalent heteroalkyl group defined above which links one part of the molecule to another part of the molecule.
• Heterocyclyl refers to a stable 3- to 18-membered non-aromatic ring radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which optionally includes aromatic, fused, and/or bridged ring systems. The heteroatoms in the heterocyclyl radical are optionally oxidized. The heterocyclyl radical is partially or fully saturated.
• heterocyclyl is intended to include independent recitations of heterocyclyl comprising aromatic and non-aromatic ring structures, unless otherwise stated.
• the heterocyclyl is attached to the rest of the molecule through any atom of the ring(s).
• heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5.6.7.8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl,
• Heteroaryl refers to a radical derived from a 3- to 18-membered aromatic ring radical that comprises two to seventeen carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
• the heteroaryl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one of the rings in the ring system is fully unsaturated, i.e., it contains a cyclic, delocalized (4n+2) p-electron system in accordance with the Hiickel theory.
• Heteroaryl includes fused or bridged ring systems.
• the heteroatom(s) in the heteroaryl radical is optionally oxidized.
• heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
• heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzol /?] [1,4] dioxepinyl, benzo[b] [ 1 ,4]oxazinyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl (benzothiophenyl), benzothieno [3 ,2 -d] pyrimi
• heterocycloalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein.
• Representative heterocycloalkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl methyl, and indol- 2-yl propyl.
• heterocycloalkylalkyl refers to a heterocycloalkyl group attached to an alkyl group, as defined herein.
• heteroarylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
• amine refers to primary, secondary, and tertiary amines having, e.g., the formula N(group) 3 + wherein each group can independently be H or non-H, such as alkyl, aryl, and the like.
• Amines include but are not limited to R-Nth, for example, alkylamines, arylamines, alkylarylamines; R2NH wherein R is defined herein, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines, and the like.
• amine also includes ammonium ions as used herein.
• amino group refers to a substituent of the form -NH2, -NHR, -NR2, -NR3 + , wherein each R is defined herein, and protonated forms of each, except for -NR3 + , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine.
• An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.
• alkylamino includes a monoalkylamino, dialkylamino, and trialkylamino group.
• alkylamino is -NH-alkyl and -N(alkyl)2.
• the compounds disclosed herein in some embodiments, contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that are defined, in ter s of absolute stereochemistry, as ( R )- or (5)-. Unless stated otherwise, it is intended that all stereoisomeric forms of the compounds disclosed herein are contemplated by this disclosure. When the compounds described herein contain alkene double bonds, and unless specified otherwise, it is intended that this disclosure includes both E and Z geometric isomers (e.g., cis or trans.) Likewise, all possible isomers, as well as their racemic and optically pure forms, and all tautomeric forms are also intended to be included.
• geometric isomer refers to E or Z geometric isomers (e.g., cis or trans ) of an alkene double bond.
• positional isomer refers to structural isomers around a central ring, such as ortho-, meta-, and para- isomers around a benzene ring.
• linker generally refers to a portion of a compound that forms a chemical bond with a G 1 (e.g., a binding ligand) and/or G 2 (e.g., a therapeutic agent).
• G 1 e.g., a binding ligand
• G 2 e.g., a therapeutic agent
• a “linker” can connect two or more functional parts of a molecule to form a compound provided herein.
• the linker may comprise atoms selected from C, N, O, S, Si, and P; C, N, O, S, and P; or C, N, O, and S.
• the linker may connect different functional capabilities of the compound, such as the folate ligand and the therapeutic agent.
• the linker may comprise a several linker groups, such as, for example, in the range from about 2 to about 100 atoms in the contiguous backbone.
• the linker is a releasable linker.
• the linker is a non-releasable linker.
• a “pteroyl” radical, moiety, or group as used herein has the following structure: wherein the asterisk denotes the point of attachment of the carbonyl carbon to another chemical group, such as the linker L.
• a “pteroyl-amino acid” radical, moiety, or group as used herein has the following structure: wherein the asterisk denotes the point of attachment of the carbonyl carbon to another chemical group, such as the linker L, and wherein H 2 N-Ax-COOH is an amino acid.
• Reduced adverse events in a subject refers to a reduction of the prevalence and/or severity of adverse events occurring in a treated patient, wherein that reduction is significant with at least a 95% confidence level.
• “Adverse events” as used herein include serious events causing death, hospitalization and/or disability (temporary or permanent), as well as less serious events causing discomfort (such as skin rash, sleep problems, hair loss, headache) which is noticeable by the subject.
• salts and “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.
• pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids.
• Pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
• such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.
• inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric
• organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic
• salts can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
• such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
• Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 21st ed., Lippincott Williams & Wilkins, 2006, e.g., Chapter 38, the disclosure of which is hereby incorporated by reference.
• solvate means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non- covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
• prodrug means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo ) to provide an active compound, particularly a compound disclosed herein.
• prodrugs include, but are not limited to, derivatives and metabolites of a compound disclosed herein that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
• Specific prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid.
• the carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule.
• Prodrugs can typically be prepared using well-known methods, such as those described by Burger’s Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).
• polymorph generally refers to crystalline materials that have the same chemical composition but different molecular packing.
• crystalline salt includes crystalline structures with the same chemical materials, but incorporating acid or base addition salts within the molecular packing of the crystalline structure.
• clathrate generally refers to a compound in which molecules of one component (e.g., solvent) are physically trapped within tire crystal structure of another.
• non-releasable linker or “non-cleavable linker” are used interchangeably. As used herein, they refer to a linker that cannot be cleaved under extracellular physiological conditions (e.g., a pH-labile, acid-labile, oxidatively-labile, or enzyme -labile bond). However, such a linker may include bonds that may be cleaved after entry into a cell.
• Releasable linker refers to a linker that includes at least one bond that can be cleaved under extracellular physiological conditions (e.g., a pH-labile, acid-labile, oxidatively-labile, or enzyme -labile bond).
• Releasable groups also include photochemically-cleavable groups. Examples of photochemically-cleavable groups include 2-(2-nitrophenyl)-ethan- 2-ol groups and linkers containing o-nitrobenzyl, desyl, trans-o-cinnamoyl, m- nitrophenyl or benzylsulfonyl groups (see, for example, Dorman and Prestwich, Trends Biotech. 18:64-77 (2000); and Greene and Wuts, Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, New York (1991)).
• the cleavable bond or bonds may be present in the interior of a cleavable linker and/or at one or both ends of a cleavable linker.
• physiological conditions resulting in bond cleavage include standard chemical hydrolysis reactions that occur, for example, at physiological pH, or as a result of compartmentalization into a cellular organelle such as an endosome having a lower pH than cytosolic pH.
• the bivalent linkers described herein can undergo cleavage under other physiological or metabolic conditions, such as by the action of a glutathione mediated mechanism.
• the lability of the cleavable bond may be adjusted by including functional groups or fragments within the bivalent linker L that are able to assist or facilitate such bond cleavage, also termed anchimeric assistance.
• the lability of the cleavable bond can also be adjusted by, for example, substitutional changes at or near the cleavable bond, such as including alpha branching adjacent to a cleavable disulfide bond, increasing the hydrophobicity of substituents on silicon in a moiety having a silicon-oxygen bond that may be hydrolyzed, homologating alkoxy groups that form part of a ketal or acetal that may be hydrolyzed, and the like.
• additional functional groups or fragments may be included within the bivalent linker L that are able to assist or facilitate additional fragmentation of the compounds after bond breaking of the releasable linker, when present.
• the terms “subject,” “patient,” and “individual” are used interchangeably. None of the terms are intended to require the continuous supervision of a medical professional.
• the subject can be any mammal, for example a human.
• treating encompasses therapeutic treatment (e.g., a subject with signs and symptoms of a disease state being treated) and/or prophylactic treatment.
• Prophylactic treatment encompass prevention and inhibition or delay of progression of a disease state.
• terapéuticaally effective amount refers to that amount of one or more compounds of the various embodiments described herein (e.g. a compound of the formula (I)) that elicits a biological or medicinal response in a tissue system, animal or human, that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the signs or symptoms of the disease or disorder being treated.
• an “antifolate” is a compound that binds to a folate receptor and antagonizes the biological actions of folic acid or one of its naturally occurring forms such as dihydrofolate, 5-methyltetrahydrofolate, or methylene tetrahydrofolates.
• this disclosure relates to the design, synthesis, and testing of a series of compounds. According, provided are compounds of the formula
• G 1 is a folate radical, an antifolate radical, or a folate analog radical
• L is a linker
• G 2 is a radical of a steroid.
• the folate radical is a group of the formula: wherein the asterisk (*) denotes the point of attachment of the carbonyl carbon to the linker L.
• G 1 can be a radical of an antifolate (e.g., a folate antagonist or a folic acid antagonist) or a folate analog radical.
• G 1 is a radical of an antifolate of any one of formulas in Table 4 as described herein
• G 1 is a folate analog radical.
• the folate analog is a pteroyl moiety or a pteroyl-amino acid moiety or a folate analog of any one of formulas V-X as described herein.
• G 1 is a pteroyl-amino acid radical.
• G 1 is an antifolate radical or a folate analog radical comprising an amino acid selected from the group consisting of aspartic acid, lysine, tyrosine, cysteine, threonine, serine, histidine, and arginine.
• G 1 is a radical of a folate analog of any one of formulas V-X as described herein.
• G 1 is a radical of an antifolate of any one of formulas in Table 4 as described herein.
• G 1 is a folate radical of the formula: wherein the asterisk denotes the point of attachment of the carbonyl carbon to the linker L; or a pteroyl-amino acid radical;
• G 2 is a radical of a corticosteroid.
• G 1 is a radical of a folate analog which is a pyrido[2,3-d]pyrimidine analog ligand, a functional fragment or analog thereof with an affinity (for example, and without limitation, a high specificity) for the folate receptor.
• a folate analog which is a pyrido[2,3-d]pyrimidine analog ligand, a functional fragment or analog thereof with an affinity (for example, and without limitation, a high specificity) for the folate receptor.
• such folate analogs may have a relative affinity for binding folate receptor, such as folate receptor beta (FR ⁇ ) of about 0.01 or greater as compared to folic acid at a temperature about 20 °C/25 °C/30 °C/physiological temperature.
• FR ⁇ folate receptor beta
• G 1 targeting moiety (or radical thereof) of the present disclosure may comprise any ligand (or radical thereof) useful to target folate receptors and is not limited to the structures specified herein.
• the ligand (or radical thereof) may bind to the folate receptor.
• a group such as C(O)Z
• a substituent which substitutes for any hydrogen bonded to a ring atom of that ring. This includes substitution of hydrogens that are explicitly shown or described (e.g., hydrogens as part of X 1 - X 9 groups in formulas V-XI), as well as hydrogens that are not explicitly shown but would be understood to be present.
• G 1 has a structure of formula V or a radical thereof: wherein
• X 1 , X 2 , X 3 , X 4 , X 5 , X 6, X 7 , X 8 , and X 9 are each independently N, NH, CH, CH 2 , O, or S; Y is C, CH, CH 2 , N, NH, O, or S;
• Z is glutamic acid or valine
• Ri and R 2 are each independently NH 2 , OH, SH, C3 ⁇ 4, or H;
• R 3 is H or an alkyl; m and n are each independently 0, 1, or between 0 and 1; and is representative of either a single or double bond C-C.
• formula V has a structure of VI or a radical thereof: wherein
• Xi, X 2 , X 3 , X 5 , Xe, X 7 , Xs, and X 9 are each independently N, NH, CH, CH 2 , O, or S;
• Y is C, CH, CH 2 , N, NH, O, or S;
• Z is glutamic acid or valine
• R 1 and R 2 are each independently NH 2 , OH, SH, CH 3 , or H;
• R 3 is H or an alkyl; m and n are each independently 0, 1, or between 0 and 1; and is representative of either a single or double bond C-C.
• formula V has a structure of formula VII or a radical thereof: wherein
• Xi, X 2 , X 3 , X 4 , X 5 , Xe, X 7 , Xs, and X 9 are each independently N, NH, CH, CH 2 , O, or S;
• Y is C, CH, CH 2 , N, NH, O, or S;
• Z is glutamic acid or valine;
• R 1 and R2 are each independently NH2, OH, SH, CH3, or H;
• R 3 is H or an alkyl; m and n are each independently 0, 1, or between 0 and 1; and is representative of either a single or double bond C-C.
• formula V has the structure of formula VIII or a radical thereof: wherein
• Xi, X2, X3, X5, Xe, X7, Xs, and X9 are each independently N, NH, CH, CH2, O, or S;
• Z is glutamic acid or valine
• R1 and R2 are each independently NH2, OH, SH, CH3, or H;
• R3 is H or an alkyl; m is 0, 1 , or between 0 and 1 ; and
• ⁇ is representative of either a single or double bond C-C.
• formula V has the structure of formula IX or ra radical thereof: wherein
• Xi, X2, X3, X5, Xe, X7, Xs, and X9 are each independently N, NH, CH, CH2, O, or S;
• Y is C, CH, CH 2 , N, NH, O, or S;
• Z is glutamic acid or valine;
• Ri and R 2 are each independently NH 2 , OH, SH, CH3, or H;
• R3 is H or an alkyl; m is 0, 1 , or between 0 and 1 ; and is representative of either a single or double bond C-C.
• formula V has the structure of formula X or a radical thereof: wherein
• Y is C, CH, CH 2 , N, NH, O, or S;
• Z is glutamic acid, valine, or a substrate
• Ri and R 2 are each independently NH 2 , OH, SH, C3 ⁇ 4, or H;
• R3 is H or an alkyl; m is 0, 1 , or between 0 and 1 ; and is representative of either a single or double bond C-C.
• formula V has the structure of formula XI or a radical thereof: wherein
• Xi, X 2 , X3, X4, X5, Xe, X7, Xs, and X9 are each independently N, NH, CH, CH 2 , O, or S;
• Y is C, CH, CH 2 , N, NH, O, or S;
• Z is glutamic acid, valine, or a substrate;
• Ri and R2 are each independently NH2, OH, SH, CH3, or H;
• R3 is hours or an alkyl; m is 0, 1 , or between 0 and 1 ; and is representative of either a single or double bond C-C.
• G 1 moiety (e.g., or radicals thereof) of the present disclosure are provided in the following
• Table 1 provides non-limiting examples of additional embodiments of formula VIII.
• Table 2 provides non-limiting examples of additional embodiments of formula IX.
• the G 1 moiety (e.g., a radical thereof) is a nonclassical antifolate such as, for example, pyrido[2,3- d] pyrimidine or similar analogs (or radicals thereof) having the formulas (e.g., radicals of the formulas) set forth in Table 4 below (or an analog or functional fragment thereof).
• a substituent of 2’,3’-(C 4 H 4 ) indicates an aryl ring fused at the 2’ and 3’ positions of the substituted aryl ring to form a bicyclic aryl system.
• the steroid present in the compounds described herein can be any suitable steroid that polarizes macrophages from pro-inflammatory (Ml) to anti inflammatory (M2).
• G 2 is a radical of a corticosteroid.
• corticosteroids examples include betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone- 17-valerate, fluorocortisone, fludrocortisone, paramethasone, eplerenone, amcinonide, alclometasone, beclomethasone, ciclesonide, clobetasol, clocortolone, desonide, deflazacort, desoximetasone, diflorasone, fluocinolone, fluocinonide, fluprednidene, flunisolide, fluticasone, fluclorolone, fludroxycortide, fluorometholone, flucorolone, halcinonide, halometasone, 21-hydroxypregnenolone, halobetacol
• G 2 is a radical of a steroid selected from the group consisting of betamethasone, cortisone, cortivazol, difluprednate, hydrocortisone, prednisolone, methylprednisolone, prednisone, dexamethasone, hydrocortisone- 17- valerate, budesonide, flumethazone, fluticasone proprionate, fluorocortisone, fludrocortisone, paramethasone, eplerenone, and an ester of any of the foregoing.
• the steroid is betamethasone.
• the steroid is cortisone.
• the steroid is fluorocortisone. In some embodiments, the steroid is fludrocortisone. In some embodiments, the steroid is paramethasone. In some embodiments, the steroid is eplerenone. In some embodiments, the steroid is amcinonide. In some embodiments, the steroid is alclometasone. In some embodiments, the steroid is beclomethasone. In some embodiments, the steroid is ciclesonide. In some embodiments, the steroid is clobetasol. In some embodiments, the steroid is clocortolone. In some embodiments, the steroid is desonide.
• the steroid is deflazacort. In some embodiments, the steroid is desoximetasone. In some embodiments, the steroid is diflorasone. In some embodiments, the steroid is fluocinolone. In some embodiments, the steroid is fluocinonide. In some embodiments, the steroid is fluprednidene. In some embodiments, the steroid is flunisolide. In some embodiments, the steroid is fluticasone. In some embodiments, the steroid is fluclorolone. In some embodiments, the steroid is fludroxycortide. In some embodiments, the steroid is fluorometholone.
• the linker present in the compounds described herein can be any suitable linker.
• the linker can be a hydrophilic linker, such as a linker that comprises one or more of an amino acid (which can be the same or different), an alkyl chain, a polyethylene glycol (PEG) monomer, a PEG oligomer, a PEG polymer, or a combination of an any of the foregoing.
• the linker can comprise an oligomer of peptidoglycans, glycans, or anions.
• the linker comprises a chemical group, that group includes one or more of its atoms in the backbone of the linker.
• said chemical group is not be required to include atoms in the backbone of L when the group is for binding purposes (such as an albumin binding group), is a glucuronide, or is a “W” group as described herein.
• atoms in the backbone of L when the group is for binding purposes (such as an albumin binding group), is a glucuronide, or is a “W” group as described herein.
• a linker that comprises one or more PEG units all carbon and oxygen atoms of the PEG units are part of the backbone unless otherwise specified.
• a cleavable bond for a releasable linker is part of the backbone.
• the “backbone” of the linker L is the shortest chain of contiguous atoms forming a covalently bonded connection between G 1 and G 2 .
• a polyvalent linker has a branched backbone, with each branch serving as a section of backbone linker until reaching a terminus.
• L groups described herein can have any suitable length and chemical composition.
• L can have a chain length of at least about 7 atoms in length.
• L is at least about 10 atoms in length.
• L is at least about 14 atoms in length.
• L is between about 7 and about 31 (such as, about 7 to 31, 7 to about 31 or 7 to 31) between about 7 and about 24 (such as, about 7 to 24, 7 to about 24 or 7 to 24), or between about 7 and about 20 (such as, about 7 to 20, 7 to about 20 or 7 to 20) atoms in length.
• L is between about 14 and about 31 (such as, about 14 to 31, 14 to about 31 or 14 to 31), between about 14 and about 24 (such as, about 14 to 24, 14 to about 24 or 14 to 24), or between about 14 and about 20 (such as, about 14 to 20, 14 to about 20 or 14 to 20) atoms in length.
• L has a chain length of at least 7 atoms, at least 14 atoms, at least 20 atoms, at least 25 atoms, at least 30 atoms, at least 40 atoms; or from 1 to 15 atoms, 1 to 5 atoms, 5 to 10 atoms, 5 to 20 atoms, 10 to 40 atoms or 25 to 100 atoms.
• L linker group having a chain length of 1 to 5 atoms is a group of the formula: wherein R zi is H, alkyl, arylalkyl, or -alkyl-S-alkyl or the side-chain of any naturally- or non-naturally occurring amino acid, and the like; and the numbers represent the atoms that are counted as being part of the chain, which in this case is 3 atoms.
• R zi examples include H (i.e., side chain of glycine), alkyl (e.g., side chain of alanine, valine, isoleucine, and leucine), -alkyl-S-alkyl (e.g., side chain of methionine), arylalkyl (e.g., side chain of phenylalanine, tyrosine, and tryptophan), and the like.
• the atom to which R zi is attached is chiral and can have any suitable relative configuration, such as a D- or L- configuration.
• the atoms used in forming L can be combined in all chemically relevant ways, such as chains of carbon atoms forming alkylene groups, chains of carbon and oxygen atoms forming polyoxyalkylene groups, chains of carbon and nitrogen atoms forming poly amines, and others.
• the bonds connecting atoms in the chain may be either saturated or unsaturated, such that for example, alkanes, alkenes, alkynes, cycloalkanes, arylenes, imides, and the like may be divalent radicals that are included in L.
• the atoms forming the linker may also be cyclized upon each other to form saturated or unsaturated divalent cyclic radicals in the linker, such as radicals of the formulae: wherein each X 5 is independently Cth, N (when there is a bond attached to X 5 ), NH or O and each X 6 is independently N, C (when there is a bond attached to X 6 ) or CH.
• the foregoing groups can be of the formulae: the like.
• the chain forming the linker may be substituted or unsubstituted.
• L has suitable substituents that can affect the hydrophobicity or hydrophilicity of L.
• L can have a hydrophobic side chain group, such as an alkyl, cycloalkyl, aryl, arylalkyl, or like group, each of which is optionally substituted.
• L can contain hydrophobic amino acid side chains, such as one or more amino acid side chains from phenylalanine (Phe) and tyrosine (Tyr), including substituted variants thereof, and analogs and derivatives of such side chains.
• Variants, analogs, and derivatives of these side chains include, for example, groups such as: which are respectively a variant of tyrosine, an amine analog of tyrosine, and a methoxy derivative of tyrosine. Other variants, analogs, and derivatives are contemplated.
• L comprises portions that are neutral under physiological conditions.
• L comprises portions that can be protonated or deprotonated to carry one or more positive or one or more negative charges, respectively.
• L comprises neutral portions and portions that may be protonated to carry one or more positive charges.
• neutral portions include poly hydroxyl groups, such as sugars, carbohydrates, saccharides, inositols, and the like, and/or polyether groups, such as polyoxyalkylene groups including polyoxyethylene, polyoxypropylene, and the like.
• portions that can be protonated to carry one or more positive charges include amino groups, such as polyaminoalkylenes including ethylene diamines, propylene diamines, butylene diamines and the like, and/or heterocycles including pyrrolidines, piperidines, piperazines, and other amino groups, each of which can be optionally substituted.
• portions that can be deprotonated to carry one or more negative charges include carboxylic acids, such as aspartic acid, glutamic acid, and longer chain carboxylic acid groups, and sulfuric acid esters, such as alkyl esters of sulfuric acid.
• Illustrative polyoxyalkylene groups include those of a specific length range from about 4 to about 20 polyoxyalkylene (e.g., polyethylene glycol) groups, such as about 4 to 20, 4 to about 20 or 4 to 20 polyoxyalkylene groups.
• Illustrative alkyl sulfuric acid esters may also be introduced with click chemistry directly into the backbone.
• Illustrative L groups comprising polyamines include L groups comprising EDTA and DTPA radicals:
• each R31 is independently H, alkyl, arylalkyl, heterocyclylalkyl, ureido, aminoalkyl, alkylthio or amidoalkyl, such as in the side chains of naturally-occurring amino acids like alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serine, threonine, asparagine, methionine, lysine, arginine, and histidine.
• Non-naturally occurring amino acids are also contemplated herein.
• the releasable linker comprises about 1 to about 30 atoms (e.g., about 1 to 30, 1 to about 30, and 1 to 30 atoms), or about 2 to about 20 atoms (e.g., about 2 to 20, 2 to about 20, and 2 to 20 atoms).
• Lower molecular weight linkers e.g., those having an approximate molecular weight of about 30 g/mol to about 1,000 g/mol, such as from about 30 g/mol to about 300 g/mol, about 100 g/mol to about 500 g/mol or about 150 g/mol to about 600 g/mol
• Precursors to such linkers may be selected to have either nucleophilic or electrophilic functional groups, or both, optionally in a protected form with a readily cleavable protecting group to facilitate their use in synthesis of the intermediate species.
• the linker is a bivalent linker (e.g., connecting a single G 1 to a single G 2 ). In some embodiments, the linker is a multivalent linker (e.g., connecting two or more G 1 to a single G 2 ). In some embodiments, the linker is polyvalent and has multiple attachment points for one or more additional chemical groups (e.g., the additional chemical groups comprise one or more additional G 1 groups; or the additional chemical groups comprise one or more binding ligands that are not G 1 groups). In some embodiments, the linker is a releasable linker. In some embodiments, the linker is a non-releasable linker.
• L is (L 1 ) 0 -Y-(L 2 ) P , wherein: each L 1 is a first linker; each L 2 is a second linker;
• Y is a template that connects multiple arms of the compound; o is an integer from 1-5; and p is an integer from 1-5.
• L 1 and L 2 are the same. In some embodiments, L 1 and L 2 are different. In some embodiments, each L 1 is connected to an G 1 group (and the Y group). In certain embodiments, each L 2 is connected to a G 2 group (and the Y group). In certain embodiments, o and m are the same, such as 1-6, 1-3, or 1. In some embodiments, p is 1. In some embodiments, o is 1. In some embodiments, p and o are each 1.
• each L 1 and L 2 independently comprise a oligoethylene glycol (chain), a polyethylene glycol (chain), an alkyl (chain), an oligopeptide (chain), or a polypeptide (chain). In some embodiments, each L 1 and L 2 independently comprise an oligoethylene glycol (chain) or a polyethylene glycol (chain).
• each L 1 and L 2 independently comprise a triazole or an amide.
• each L 1 and L 2 independently comprise an oligopeptide (chain) or a polypeptide (chain). In some embodiments, each L 1 and L 2 independently comprise a peptidoglycan (chain).
• p is an inter from 1-5. In some embodiments, p is an integer from 1-3. In some embodiments, p is 1.
• G 1 is a folate radical, an antifolate radical, or a folate analog radical
• S is a spacer (e.g., having a length for the arms of the multivalent targeting ligand (e.g., drug) to reach multiple adjacent folate receptors on a target cell);
• Y is a template that connects multiple arms of the compound;
• L is a (e.g., bi-functionalized) linker connecting G 1 to G 2 (e.g., through a first covalent bond connecting L to G 1 and a second covalent bond linking L to G 2 ); and
• S is a peptidoglycan.
• the spacer is a rigid linker.
• S is a rigid linker, such as, for example, an oligoproline or an oligopiperidine
• S is a length of at least 15 angstroms (A). In some embodiments, S is a length of at most 200 angstroms (A). In some embodiments, S is a length from 15-200 angstroms (A).
• Y is a template that connects multiple arms of the compound.
• Y has a repeating structure.
• Y comprises a releasable bond.
• L comprises a disulfide bond.
• Y comprises at least one citric acid group (or a radical thereof).
• Y comprises one or more triazole.
• Y comprises one or more amine.
• Y comprises one or more amide.
• Y has an aromatic core (e.g., an aryl core or a heteroaryl core).
• Y has an alkyl(ene) core.
• Y has an amine core.
• Y is N(L 1 ) 3 (e.g., wherein L 1 is described elsewhere herein).
• Y is phenyl substituted with three L 1 (e.g., wherein L 1 is described elsewhere herein).
• Y is CriJ p (e.g., wherein L 1 is described elsewhere herein).
• Y is attached to a single L 1 . In some embodiments, Y is attached to a single L 2 . In some embodiments, Y is attached to a single L 1 and a single L 2 . In some embodiments, Y is independently connected to each L 1 and L 2 by an amide bond. In some embodiments, Y is attached to L.
• Y is a template (e.g., a multivalent template) that connects multiple arms of the compound.
• Y has a repeating structure.
• Y comprises at least one citric acid group (or a radical thereof).
• the template has the following structure:
• Y is a template (e.g., a multivalent template) that connects multiple arms of the compound and comprises a template (e.g., a repeating unit) of the following structure:
• Y is a template that connects multiple arms of the compound that has a citric acid-based template.
• Y is a template (e.g., a multivalent template) that connects multiple arms of the compound and has a (e.g., citric acid-based) template of the following structure:
• Y is a template (e.g., a multivalent template) that connects multiple arms of the compound and has a (e.g., citric acid-based) template of the following structure:
• L comprises at least one linker group, each linker group selected from the group consisting of polyethylene glycol (PEG), alkyl, sugar, and peptide.
• the linker is a polyethylene glycol- (PEG-) (e.g., pegylated-), alkyl-, sugar-, and peptide-based dual linker.
• the linker comprises a PEG oligomer with 2-16 PEG units. In some embodiments, the linker comprises a PEG oligomer with 12 PEG units.
• L is a non-releasable linker (e.g., bivalently (e.g., covalently) attached to G 2 and G 1 ). In some embodiments, L is a releasable linker (e.g., bivalently (e.g., covalently) attached to G 2 and G 1 ).
• L can comprise one or more releasable linkers that cleave under the conditions described herein by a chemical mechanism involving beta elimination.
• releasable linkers include beta-thio, beta-hydroxy, and beta-amino substituted carboxylic acids and derivatives thereof, such as esters, amides, carbonates, carbamates, and ureas.
• linkers also include 2- and 4- thioarylesters, carbamates, and carbonates.
• Assisted cleavage of releasable portions of L can include mechanisms involving benzylium intermediates, benzyne intermediates, lactone cyclization, oxonium intermediates, beta-elimination, and the like.
• the initial cleavage of the releasable linker may be facilitated by an anchimerically assisted mechanism.
• the hydroxyalkanoic acid which may cyclize, facilitates cleavage of the methylene bridge, by for example an oxonium ion, and facilitates bond cleavage or subsequent fragmentation after bond cleavage of the releasable linker.
• acid catalyzed oxonium ion-assisted cleavage of the methylene bridge may begin a cascade of fragmentation of this illustrative bivalent linker, or fragment thereof.
• acid-catalyzed hydrolysis of the carbamate may facilitate the beta elimination of the hydroxyalkanoic acid, which may cyclize, and facilitate cleavage of methylene bridge, by for example an oxonium ion. It is appreciated that other chemical mechanisms of bond cleavage under the metabolic, physiological, or cellular conditions described herein may initiate such a cascade of fragmentation. It is appreciated that other chemical mechanisms of bond cleavage under the metabolic, physiological, or cellular conditions described herein can initiate such a cascade of fragmentation.
• Illustrative mechanisms for cleavage of the bivalent linkers described herein include the following 1,4 and 1,6 fragmentation mechanisms for carbonates and carbamates:
• Nuc is an exogenous or endogenous nucleophile, glutathione, or bioreducing agent, and the like, and R a and X a are connected through other portions of the bivalent linker.
• the location of R a and X a can be switched such that, e.g., the resulting products are X a -S-Nuc and HO-R a H2N-R a .
• the bond cleavage can also occur by acid catalyzed elimination of the carbamate moiety, which may be anchimerically assisted by the stabilization provided by either the aryl group of the beta sulfur or disulfide illustrated in the above examples.
• the releasable linker is the carbamate moiety.
• the fragmentation can be initiated by a nucleophilic attack on the disulfide group, causing cleavage to form a thiolate.
• the thiolate can intermolecularly displace a carbonic acid or carbamic acid moiety and form the corresponding thiacyclopropane.
• the resulting phenyl thiolate can further fragment to release a carbonic acid or carbamic acid moiety by forming a resonance- stabilized intermediate.
• the releasable nature of the illustrative bivalent linkers described herein may be realized by whatever mechanism may be relevant to the chemical, metabolic, physiological, or biological conditions present. [00152] As described above, therefore, releasable linkers can comprise a disulfide group.
• releasable linkers comprised in L include divalent radicals comprising alkyleneaziridin-l-yl, alkylenecarbonylaziridin-1- yl, carbonylalkylaziridin-l-yl, alkylenesulfoxylaziridin-l-yl, sulfoxylalkylaziridin-l-yl, sulfonylalkylaziridin-l-yl, or alkylenesulfonylaziridin-l-yl groups, wherein each of the releasable linkers is optionally substituted.
• releasable linkers comprised in L include divalent radicals comprising methylene, 1-alkoxyalkylene, 1- alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, 1- alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl) carbonyl, carbonyl(biscarboxyaryl)carbonyl, haloalkylenecarbonyl, alkylene(dialkylsilyl) , alkylene(alkylarylsilyl) , alkylene(diarylsilyl), (dialkylsilyl)aryl, (alky larylsilyl) aryl, (diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy, oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideniminyl, iminocyclo
• releasable linkers comprised in L include an oxygen atom and methylene, 1-alkoxyalkylene, 1- alkoxycycloalkylene, 1- alkoxyalkylenecarbonyl or 1-alkoxycycloalkylenecarbonyl groups, wherein each of the releasable linkers is optionally substituted.
• the releasable linker includes an oxygen atom and a methylene group, wherein the methylene group is substituted with an optionally substituted aryl, and the releasable linker is bonded to the oxygen to form an acetal or ketal.
• the releasable linker includes an oxygen atom and a sulfonylalkyl group, and the releasable linker is bonded to the oxygen to form an alkylsulfonate.
• releasable linkers comprised in L include a nitrogen and iminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, and carbonylcycloalkylideniminyl groups, wherein each of the releasable linkers is optionally substituted and the releasable linker is bonded to the nitrogen to form an hydrazone.
• the hydrazone is acylated with a carboxylic acid derivative, an orthoformate derivative, or a carbamoyl derivative to form various acylhydrazone releasable linkers.
• releasable linkers comprised in L include an oxygen atom and alkylene(dialkylsilyl), alkylene(alkylarylsilyl), alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl or (diarylsilyl)aryl groups, wherein each of the releasable linkers is optionally substituted and the releasable linker is bonded to the oxygen to form a silanol.
• releasable linkers comprised in L include two independent nitrogens and carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, or carbonyl(biscarboxyaryl)carbonyl.
• the releasable linker is bonded to the heteroatom nitrogen to form an amide, and also bonded to X a or R a via an amide bond.
• releasable linkers comprised in L include an oxygen atom, a nitrogen, and a carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl, or carbonyl(biscarboxyaryl)carbonyl.
• the releasable linker forms an amide, and in some embodiments is bonded to X a or R a via an amide bond.
• L comprises an optionally substituted 1- alkylenesuccinimid-3-yl group and a releasable portion comprising methylene
• L comprises carbonyl, thionocarbonyl, alkylene, cycloalky lene, alkylenecycloalkyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, l-alkylenesuccinimid-3-yl, l-(carbonylalkyl)succinimid- 3-yl, alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl, alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, 1 -(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl or l-(carbonyltetrahydrofuranyl)succinimid-3-yl, each of which is optionally substituted.
• L further comprises an additional nitrogen such that L comprises alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl or l-(carbonylalkyl)succinimid-3-yl groups, each of which is optionally substituted, bonded to the nitrogen to form an amide.
• L further comprises a sulfur atom and alkylene or cycloalkylene groups, each of which is optionally substituted with carboxy, and is bonded to the sulfur to form a thiol.
• L comprises a sulfur atom and l-alkylenesuccinimid-3-yl and l-(carbonylalkyl)succinimid-3-yl groups bonded to the sulfur to form a succinimid-3-ylthiol.
• L comprises a nitrogen and a releasable portion comprising alkyleneaziridin-l-yl, carbonylalkylaziridin-l-yl, sulfoxylalkylaziridin-l-yl, or sulfonylalkylaziridin-l-yl, each of which is optionally substituted.
• L comprises carbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl, or l-(carbonyIaIkyI)succinimid-3-yl, each of which is optionally substituted, and bonded to the releasable portion to form an aziridine amide.
• L examples include alkylene-amino-alkylenecarbonyl, alkylene- thio-(carbonyIaikyIsuccinimid-3-yl), and the like, as further illustrated by the following formulae: wherein x’ and y’ are each independently 1, 2, 3, 4, or 5.
• L can have any suitable assortment of atoms in the chain, including C (e.g., -CH2-, C(O)), N (e.g., NH, NR b , wherein R b is, e.g., H, alkyl, alkylaryl, and the like), O (e.g., -0-), P (e.g., -0-P(0)(0H)0-), and S (e.g., -S-).
• C e.g., -CH2-, C(O)
• N e.g., NH, NR b , wherein R b is, e.g., H, alkyl, alkylaryl, and the like
• O e.g., -0-
• P e.g., -0-P(0)(0H)0-
• S e.g., -S-
• the atoms used in forming L may be combined in all chemically relevant ways, such as chains of carbon atoms forming alkyl groups, chains of carbon and oxygen atoms forming polyoxyalkyl groups, chains of carbon and nitrogen atoms forming polyamines, and others, including rings, such as those that form aryl and heterocyclyl groups (e.g., triazoles, oxazoles, and the like).
• the bonds connecting atoms in the chain in L may be either saturated or unsaturated, such that for example, alkanes, aikenes, alkynes, cycloalkanes, arylenes, imides, and the like may be divalent radicals that are included in L.
• the chain-forming F can be substituted or unsubstituted.
• F groups include the groups 1 -alkylsuccinimid- 3-yl, carbonyl, thionocarbonyl, alkyl, cycloalkyl, aikylcycloalkyl, aikylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, l-aIkyIsuccinimid-3-yl, 1- (carbonylalkyl)succinimid-3-yl, aikylsulfoxyl, sulfonylaikyl, alkylsulfoxylalkyl, alkylsulfonylalkyl, carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl, l-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and 1- (carbonyltetrahydrofuranyl)succinimid-3-yl,
• L groups are alkylcarbonyl, cycloalkylcarbonyl, carbonylalkylcarbonyl, l-(carbonyIaIkyI)succinimid-3-yl, and succinimid-3- ylthiol, wherein each group can be substituted or unsubstituted.
• L is formed via click chemistry/click chemistry- derived.
• click chemistry and “click chemistry-derived” generally refer to a class of small molecule reactions commonly used in conjugation, allowing the joining of substrates of choice with specific molecules. Click chemistry is not a single specific reaction, but describes a way of generating products that follow examples in nature, which also generates substances by joining small modular units. In many applications, click reactions join a biomolecule and a reporter molecule. Click chemistry is not limited to biological conditions: the concept of a “click” reaction has been used in pharmacological and various biomimetic applications. However, they have been made notably useful in the detection, localization and qualification of biomolecules.
• Click reactions can occur in one pot, typically are not disturbed by water, can generate minimal byproducts, and are “spring-loaded” — characterized by a high thermodynamic driving force that drives it quickly and irreversibly to high yield of a single reaction product, with high reaction specificity (in some cases, with both regio- and stereo-specificity). These qualities make click reactions suitable to the problem of isolating and targeting molecules in complex biological environments. In such environments, products accordingly need to be physiologically stable and any byproducts need to be non-toxic (for in vivo systems).
• Click chemistry examples include examples where L can be derived from copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), inverse electron demand Diels- Alder reaction (IEDDA), and Staudinger ligation (SL).
• CuAAC copper-catalyzed azide-alkyne cycloaddition
• SPAAC strain-promoted azide-alkyne cycloaddition
• IEDDA inverse electron demand Diels- Alder reaction
• SL Staudinger ligation
• each R b is independently H, alkyl, arylalkyl, -alkyl-S-alkyl or arylalkyl or the side -chain of any naturally- or non-naturally occurring amino acid and the like.
• the wavy line connected to X a and R a represents a linkage between X a and R a and the groups to which they are attached. It should be appreciated that in Schemes 1-5, the triazole, oxazole, and the -NH-S02-NH- group would be considered to be part of L.
• L is a linker selected from the group consisting of pegylated-, alkyl-, sugar-, and peptide- based dual linker; L is either a non- releasable linker or a releasable linker bivalently covalently attached to the folate ligand (or, in other embodiments, folate analogue or antifolate) and the steroid.
• L is: wherein x” is an integer from 0 to 10, and y” is an integer from 3 to 100.
• L is: wherein R37 is H or C 1 -C6 alkyl; 10 3 ⁇ 4, R35 b , R36 a , and R36 b each is independently H or C 1 -C6 alkyl.
• L comprises an amino acid.
• L comprises an amino acid selected from the group consisting of Lys, Asn, Thr, Ser, He, Met, Pro, His, Gin, Arg, Gly, Asp, Glu, Ala, Val, Phe, Leu, Tyr, Cys, and Trp.
• L comprises at least two amino acids independently selected from the group consisting of Glu and Cys.
• L comprises Glu-Glu, wherein the glutamic acids are covalently bonded to each other through the carboxylic acid side chains.
• L comprises one or more hydrophilic spacer linkers comprising a plurality of hydroxyl functional groups.
• L comprises at least one 2,3- diaminopropionic acid group, at least one glutamic acid group (e.g., unnatural amino acid D- Glutamic acid), and at least one cysteine group.
• glutamic acid group e.g., unnatural amino acid D- Glutamic acid
• cysteine group e.g., one or more cysteine groups.
• a linker is one having the non-natural amino acid, such as a linker having the repeating unit of the formula: wherein q is an integer from 1 to 10 (e.g., 1 to 3 and 2 to 5).
• L comprises the general formula:
• L comprises the formula: wherein the disulfide group is a part of a self-immolative group that can be generically described as a group of the formula -CH 2 -S-S-CH 2 -.
• the compounds described herein include linkages that cause the steroids described herein to be released by any suitable mechanism, including a release mechanism involving reduction, oxidation, or hydrolysis.
• a reduction mechanism includes reduction of a disulfide group into two separate sulfyhydryl groups.
• a group of the formula -CH 2 -S-S-CH 2 - would be reduced to two separate groups of the formula -CH 2 -SH, such that if the linker were of the formula:
• the steroid is attached to the linker via a self-immolative moiety (e.g., a disulfide group).
• a self-immolative moiety e.g., a disulfide group
• An example of a self-immolative disulfide also includes a sterically protected disulfide bond.
• the steroid can be attached to the linker via any other suitable self-immolative bond, including via a self-immolative cathepsin cleavable amino acid sequence; via a self-immolative furin cleavable amino acid sequence; via a self-immolative b-glucuronidase cleavable moiety; via a self- immolative phosphatase cleavable moiety; or via a self-immolative sulfatase cleavable moiety.
• Multiple self-immolative linkages are also contemplated herein.
• the linker comprises a self-immolative moiety. In some embodiments, the linker comprises a self-immolative disulfide and or sterically protected disulfide bond. In some embodiments, the linker comprises a self-immolative cathepsin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative furin-cleavable amino acid sequence. In some embodiments, the linker comprises a self-immolative b- glucuronidase -cleavable moiety. In some embodiments, the linker comprises a self-i mmol alive phosphatase-cleavable moiety. In some embodiments, the linker comprises a self-immolative sulfatase-cleavable moiety.
• the linker comprises a phosphate or pyrophosphate group. In some embodiments, the linker comprises a cathepsin B cleavable group. In some embodiments, the cathepsin B cleavable group is Valine-Citrulline. In some embodiments, the linker comprises a carbamate moiety. In some embodiments, the linker comprises a b-glucuronide.
• the compounds described herein include linkages where the steroid is attached to the linker via an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate or enzyme-cleavable amino acid sequence.
• the linker comprises an ester, phosphate, oxime, acetal, pyrophosphate, polyphosphate, disulfide, sulfate, hydrazide, imine, carbonate, carbamate or enzymelcleavable amino acid sequence
• L comprises one or more spacer linkers.
• Spacer linkers can be hydrophilic spacer linkers comprising a plurality of hydroxyl functional groups.
• a spacer “L” can comprise any stable arrangement of atoms.
• a spacer comprises one or more L’ .
• Each L’ is independently selected from the group consisting an amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., polyethylene glycol), cycloakyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof.
• a spacer comprises any one or more of the following units: an amide, ester, urea, carbonate, carbamate, disulfide, amino acid, amine, ether, alkyl, alkene, alkyne, heteroalkyl (e.g., PEG), cycloakyl, aryl, heterocycloalkyl, heteroaryl, carbohydrate, glycan, peptidoglycan, polypeptide, or any combination thereof.
• a spacer L or L’ comprises a solubility enhancer or PK/PD modulator W.
• a spacer comprises a glycosylated amino acid.
• a spacer comprises one or more monosaccharide, disaccharide, polysaccharide, glycan, or peptidoglycan.
• a spacer comprises a releasable moiety (e.g., a disulfide bond, an ester, or other moieties that can be cleaved in vivo).
• a spacer comprises one or more units such as ethylene (e.g., polyethylene), ethylene glycol (e.g., PEG), ethanolamine, ethylenediamine, and the like (e.g., propylene glycol, propanolamine, propylenediamine).
• a spacer comprises an oligoethylene, PEG, alkyl chain, oligopeptide, polypeptide, rigid functionality, peptidoglycan, oligoproline, oligopiperidine, or any combination thereof.
• a spacer comprises an oligoethylene glycol or a PEG.
• a spacer can comprise an oligoethylene glycol.
• a spacer comprises a PEG.
• a spacer comprises an oligopeptide or polypeptide.
• a spacer comprises an oligopeptide.
• a spacer comprises a polypeptide.
• a spacer comprises a peptidoglycan.
• a spacer does not comprise a glycan. In some embodiments, a spacer does not comprise a sugar. In some embodiments, a rigid functionality is an oligoproline or oligopiperidine. In some embodiments, a rigid functionality is an oligoproline. In some embodiments, a rigid functionality is an oligopiperidine. In some embodiments, a rigid functionality is an oligophenyl. In some embodiments, a rigid functionality is an oligoalkyne.
• an oligoproline or oligopiperidine has about two up to and including about fifty, about two to about forty, about two to about thirty, about two to about twenty, about two to about fifteen, about two to about ten, or about two to about six repeating units (e.g., prolines or piperidines).
• the compound comprises W groups to improve properties of the compound.
• one or more G 1 are replaced with W, provided that one or more G 1 are not W.
• one or more G 1 are replaced with W, provided that one or more G 1 are folate receptor ligands.
• the linker L comprises one or more W groups.
• W c comprises a solubility enhancer or PK/PD modulator.
• W comprises polyethylene glycol (PEG), sugar, peptide, or peptidoglycan. In some embodiments, W comprises a PEG, sugar, peptide, or peptidoglycan for achieving better solubility and PK/PD properties. In some embodiments, W comprises one or more monosaccharide, disaccharide, peptide, peptidoglycan, and/or serum albumin. In some embodiments, W comprises one or more PEG, peptide, peptidoglycan, or serum albumin. In some embodiments, W does not comprise a sugar. In some embodiments, W does not comprise a monosaccharide, disaccharide, or polysaccharide.
• PEG polyethylene glycol
• W does not comprise a glycan.
• W comprises a glycosylated amino acid.
• W comprises a glycosylate cysteine.
• W comprises a free carboxylic acid.
• W comprises a PEG.
• W comprises one or more monosaccharide, disaccharide, oligosaccharide, polysaccharide, peptide, peptidoglycan, serum albumin, solubility enhancer, PK/PD modulator, or a combination thereof.
• W modulates a pharmacological, pharmacokinetic, pharmacodynamic, or physicochemical property.
• W facilitates internalization.
• W improves aqueous solubility.
• W increases plasma protein binding.
• W modulates (e.g., reduces) the compound’s excretion, elimination, metabolism, stability (e.g., enzymatic stability, plasma stability), distribution, toxicity, or a combination thereof.
• a monosaccharide such as found in W exists in an equilibrium between its linear and cyclic form.
• a monosaccharide is linear.
• a monosaccharide is cyclic.
• a monosaccharide exists as a D isomer.
• a monosaccharide exists as an L isomer.
• W comprises one or more monosaccharides selected from the following: ribose, galactose, mannose, glucosefructose, N- acetylglucosamine, /V-acctylmuramic acid or derivatives thereof (e.g., cyclic or linear forms, methylated derivatives, acetylated derivatives, phosphorylated derivatives, am mated derivatives, oxidized or reduced derivatives, D or L isomers, isotopes, stereoisomers, regioisomers, tautomers, or combinations thereof).
• monosaccharides selected from the following: ribose, galactose, mannose, glucosefructose, N- acetylglucosamine, /V-acctylmuramic acid or derivatives thereof (e.g., cyclic or linear forms, methylated derivatives, acetylated derivatives, phosphorylated derivatives, am mated derivatives,
• W comprises a glycosylated amino acid such as:
• W is a pharmacokinetic modulator.
• the pharmacokinetic modulator is a peptide or protein that can modulate (e.g., enhance) protein binding.
• the pharmacokinetic modulator enhances plasma protein binding.
• the pharmacokinetic modulator reduces the rate of elimination, excretion, or metabolism.
• the pharmacokinetic modulator increases the duration of action of the compound.
• the linker comprises a dimethylcysteine group.
• the dimethylcysteine group is linked to a succinimide to form:
• Compounds of formula (I) include compounds of the formulae:
• the steroid is dexamethasone, flumethasone, betamethasone, betamethasone-17-valerate, and budesonide, respectively.
• the steroids are shown attached to L via the hydroxyl group a to the carbonyl of the D-ring, the connection of the steroids described and contemplated herein can be from any suitable portion of the steroid.
• the folate targeting ligand and the steroid portions of the compounds of the formula (I) contain chiral centers. All diastereomers of the compounds described herein are contemplated, as well as racemates.
• Compound 113 (Folate- AlbuminBinder-PEG2-Hydrolink-DimethylCysteine-Maleimide- CathepsinBCleavable-(CH2CH2)-DiCarbamate-Dexamethasone): Compound 114 (Folate- AlbuminBinder-PEG2-Hydrolink-DimethylCysteine-Maleimide- CathepsinBCleavable-Phosphate-Dexamethasone):
• Compound 115 (Folate- AlbuminBinder-PEG2-Hydrolink-DimethylCysteine-Maleimide- BetaGlucuronidaseCleavable-Dicarbamate-Dexamethasone): Compound 116 (Folate-AlbuminBinder-PEG12(CH 2 )-DimethylCysteine-Maleimide-
• BetaGlucuronidaseCleavable-Dicarbamate-Dexamethasone
• Compound 121 (Pteroyl-Serine -PyroPhosphate -Dexamethasone): Compound 122 (Pteroyl-Threonine-Phosphate-Dexamethasone):
• compositions comprising one or more compounds as described herein (e.g., a compound of the formula (I)) and one or more pharmaceutically acceptable excipients.
• Excipients are substances added to a pharmaceutical formulation which are not active ingredients.
• the class of excipients includes diluents (e.g., fillers used to, among other things, increase weight and improve content uniformity in tablets, including starches, hydrolyzed starches, partially pregelatinized starches; other examples of diluents include anhydrous lactose, lactose monohydrate, and sugar alcohols such as sorbitol, xylitol and mannitol).
• compositions may be specifically formulated for administration via one or more of a number of routes including, but not limited to, buccal, cutaneous, epicutaneous, epidural, infusion, inhalation, intraarterial, intracardial, intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, pulmonary, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, transdermal, and transmucosal.
• administration can be by means of capsule, drops, foams, gel, gum, injection, liquid, patch, pill, porous pouch, powder, tablet, or other suitable means of administration.
• compositions comprising any compound described herein and at least one pharmaceutically acceptable excipient that is part of a nanoparticle, a liposomal or an exosomal formulation.
• compositions generally do not provide any pharmacological activity to the formulation, though they provide chemical and/or biological stability, and release characteristics. Examples of suitable formulations can be found, for example, in Remington, The Science And Practice of Pharmacy, 20th Edition, (Gennaro, A. R., Chief Editor), Philadelphia College of Pharmacy and Science, 2000, which is incorporated by reference in its entirety.
• any suitable pharmaceutically acceptable excipient may be used in the compositions disclosed herein. Suitable excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible.
• the pharmaceutical composition is suitable for parenteral administration. Alternatively, the pharmaceutical composition is suitable for intravenous, intraperitoneal, intramuscular, sublingual, or oral administration.
• the pharmaceutical composition is a sterile aqueous solution or dispersion or a sterile powder for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions disclosed herein is contemplated. Supplementary active compounds can also be incorporated into the compositions.
• compositions may be sterile and stable under the conditions of manufacture and storage.
• the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• isotonic agents can be included in the pharmaceutical compositions, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.
• the compounds described herein can be formulated in a time-release formulation, for example in a composition that includes a slow-release polymer.
• the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled- release formulation, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, and polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are known to those skilled in the art.
• compositions may be orally administered as a capsule (hard or soft), tablet (film-coated, enteric- coated or uncoated), powder or granules (coated or uncoated) or liquid (solution or suspension).
• the formulations may be conveniently prepared by any of the methods well-known in the art.
• the pharmaceutical compositions may include one or more suitable production aids or excipients including fillers, binders, disintegrants, lubricants, diluents, flow agents, buffering agents, moistening agents, preservatives, colorants, sweeteners, flavors, and pharmaceutically compatible carriers.
• suitable production aids or excipients including fillers, binders, disintegrants, lubricants, diluents, flow agents, buffering agents, moistening agents, preservatives, colorants, sweeteners, flavors, and pharmaceutically compatible carriers.
• the compounds can be administered by a variety of dosage forms as known in the art. Any biologically- acceptable dosage form known to persons of ordinary skill in the art, and combinations thereof, are contemplated. Examples of such dosage forms include, without limitation, chewable tablets, quick dissolve tablets, effervescent tablets, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, tablets, multi-layer tablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatin capsules, caplets, lozenges, chewable lozenges, beads, powders, gum, granules, particles, microparticles, dispersible granules, cachets, douches, suppositories, creams, topicals, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, ingestibles, injectables (including subcutaneous, intramuscular, intravenous, and intradermal), infusions, and
• Other compounds which can be included by admixture, are, for example, medically inert ingredients (e.g., solid and liquid diluent), such as lactose, dextrosesaccharose, cellulose, starch or calcium phosphate for tablets or capsules, olive oil or ethyl oleate for soft capsules and water or vegetable oil for suspensions or emulsions; lubricating agents such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; gelling agents such as colloidal clays; thickening agents such as gum tragacanth or sodium alginate, binding agents such as starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuff; sweeteners; wetting agents such as lecit
• Liquid dispersions for oral administration can be syrups, emulsions, solutions, or suspensions.
• the syrups can contain as a carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol.
• the suspensions and the emulsions can contain a carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
• the amount of active compound in a composition may vary according to factors such as the disease state, age, gender, weight, patient history, risk factors, predisposition to disease, administration route, and pre-existing treatment regime (e.g., possible interactions with other medications). Dosage regimens may be adjusted to provide the optimum response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the situation.
• a therapeutically effective amount is provided, and the amount is sufficient to provide a therapeutic effect to a subject having inflammation, such as inflammation associated with a disease or a disorder, or a prophylactic effect to a subject at risk for developing inflammation associated with a disease or disorder.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms for use with the methods disclosed herein are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
• the compounds of the various embodiments described herein may be administered in an effective amount.
• the dosage can be administered once, twice, or thrice a day, although more frequent dosing intervals are possible.
• the dosage may be administered every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, and/or every 7 days (once a week).
• the dosage may be administered daily for up to and including 30 days, preferably between 7-10 days.
• the dosage may be administered twice a day for 10 days. If the patient requires treatment for a chronic disease or condition, the dosage may be administered for as long as signs and/or symptoms persist.
• the patient may require “maintenance treatment” where the patient is receiving dosages every day for months, years, or for life.
• the compositions disclosed herein may be to effect prophylaxis of recurring symptoms.
• the dosage may be administered once or twice a day to prevent the onset of symptoms in patients at risk, especially for asymptomatic patients.
• compositions described herein may be administered in any of the following routes: buccal, epicutaneous, epidural, infusion, inhalation, intraarterial, intracardial, intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, pulmonary, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, transdermal, and transmucosal.
• routes of administration are buccal and oral.
• the administration can be local, where the composition is administered directly, close to, in the locality, near, at, about, or in the vicinity of, the site(s) of disease, e.g., inflammation, or systemic, wherein the composition is given to the patient and passes through the body widely, thereby reaching the site(s) of disease.
• Local administration can be administration to the cell, tissue, organ, and/or organ system, which encompasses and/or is affected by the disease, and/or where the disease signs and/or symptoms are active or are likely to occur.
• Administration can be topical with a local effect; composition is applied directly where its action is desired.
• Administration can be enteral wherein the desired effect is systemic (nonlocal), composition is given via the digestive tract.
• Administration can be parenteral, where the desired effect is systemic, composition is given by other routes than the digestive tract.
• compositions comprising a therapeutically effective amount of one or more compounds of the various embodiments described herein (e.g. a compound of the formula (I)) are also contemplated.
• the compositions are useful in a method for treating inflammation in a subject, the method comprising administering a therapeutically effective amount of a compound described herein to the subject.
• the therapeutically effective amount can provide a prophylactic or therapeutic effect.
• the inflammation can be treated with reduced, or elimination of, adverse effects relative to when the steroid is administered without the linkage to the folate targeting ligand.
• Adverse effects of steroids such as those that are administered systemically include sodium (salt) and fluid retention, weight gain, edema, high blood pressure, muscle weakness, slow wound healing, and the like.
• the inflammation is associated with Crohn’s disease, lupus, inflammatory bowel disease (IBS), Addison’s disease, Grave’s disease, Sjogren’s syndrome, celiac disease, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, reactive arthritis, psoriatic arthritis, pernicious anemia, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, or fibrotic disease, graft vs.
• IBS inflammatory bowel disease
• Addison’s disease Grave’s disease
• Sjogren’s syndrome celiac disease
• Hashimoto’s thyroiditis myasthenia gravis
• autoimmune vasculitis reactive arthritis
• psoriatic arthritis pernicious anemia
• ulcerative colitis ulcerative colitis
• rheumatoid arthritis type 1 diabetes
• multiple sclerosis or fibrotic disease
• GVHD host disease
• fatty liver disease asthma, osteoporosis, sarcoidosis, ischemia-reperfusion injury, prosthesis osteolysis, glomerulonephritis, scleroderma, psoriasis, autoimmune myocarditis, spinal cord injury, central nervous system, viral infection, influenza, coronavirus infection, cytokine storm syndrome, bone damage, inflammatory brain disease, atherosclerosis, cancer, or a tumor.
• Ml and M2 macrophage profiles can be used to determine dosage, monitor efficacy of treatment (including therapeutic and prophylactic), and adjust dosage treatment plans.
• CD86 can be assessed to determine Ml macrophage levels
• CD206 can be assessed to determine M2 macrophage levels.
• TRMs tissue -resident macrophages
• the inflammatory disorder is Crohn’s disease, lupus, inflammatory bowel disease (IBS), Addison’s disease, Grave’s disease, Sjogren’s syndrome, celiac disease, Hashimoto’s thyroiditis, myasthenia gravis, autoimmune vasculitis, reactive arthritis, psoriatic arthritis, pernicious anemia, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, multiple sclerosis, or fibrotic disease, graft vs.
• IBS inflammatory bowel disease
• Addison’s disease Grave’s disease
• Sjogren’s syndrome celiac disease
• Hashimoto’s thyroiditis myasthenia gravis
• autoimmune vasculitis reactive arthritis
• psoriatic arthritis pernicious anemia
• ulcerative colitis ulcerative colitis
• rheumatoid arthritis type 1 diabetes, multiple sclerosis, or fibrotic disease, graft vs.
• GVHD host disease
• fatty liver disease asthma, osteoporosis, sarcoidosis, ischemia-reperfusion injury, prosthesis osteolysis, glomerulonephritis, scleroderma, psoriasis, with autoimmune myocarditis, spinal cord injury, central nervous system, viral infection, influenza, coronavirus infection, cytokine storm syndrome, bone damage, inflammatory brain disease, atherosclerosis, cancer, or a tumor.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of budesonide, fludrocortisone, beclomethasone, fluticasone, mometasone, ciclesonide, cortisone, cortivazol, hydrocortisone, 21-hydroxypregnenolone, meprednisone, dexamethasone, triamcinolone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of a steroid selected from the group consisting of budesonide, fludrocortisone, beclomethasone, fluticasone, mometasone, ciclesonide, cortisone, cortivazol, hydrocortisone, 21-hydroxypregnenolone, meprednisone, dexamethasone, triamcinolone, betamethasone
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, hydrocortisone, budesonide, betamethasone, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, hydrocortisone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, hydrocortisone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• methods for treating inflammation associated with celiac disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• methods for treating inflammation associated with autoimmune vasculitis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• Disclosed herein, in certain embodiments are methods for treating inflammation associated with psoriatic arthritis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, triamcinolone, beclomethasone, betamethasone, dexamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, triamcinolone, beclomethasone, betamethasone, dexamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• methods for treating inflammation associated with type 1 diabetes in a subject in need thereof comprising administering
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of triamcinolone, fluticasone, budesonide, mometasone, beclomethasone, ciclesonide, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, budesonide, betamethasone, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a fibrotic disease e.g., a fibrotic disease that results from inflammation
• methods for treating inflammation associated with a fibrotic disease comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, or a pharmaceutical composition disclosed herein.
• a fibrotic disease e.g., a fibrotic disease that results from inflammation
• a pharmaceutical composition comprising one or more compounds disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, and prednisone.
• GVHD graft vs. host disease
• graft vs. host disease comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• Disclosed herein, in certain embodiments are methods for treating inflammation associated with prosthesis osteolysis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, or a pharmaceutical composition disclosed herein.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein, wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, budesonide, betamethasone, triamcinolone, prednisone, prednisolone, and methylprednisolone.
• G 2 is a radical of a steroid selected from the group consisting of budesonide, betamethasone, triamcinolone, prednisone, prednisolone, methylprednisolone, hydrocortisone, dexamethasone, hydrocortisone-17-valerate, diflorasone, meprednisone, halobetacol, tixocortol, amcinonide, desonide, fluocinolone acetonide, fluocinonide, halcinonide, beclomethasone, and halometasone.
• G 2 is a radical of a steroid selected from the group consisting of budesonide, betamethasone, triamcinolone, prednisone, prednisolone, methylprednisolone, hydrocortisone, dexamethasone, hydrocortisone-17-valerate, diflorasone, mepre
• Disclosed herein, in certain embodiments are methods for treating inflammation associated with a viral infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, or a pharmaceutical composition disclosed herein.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, and prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of hydrocortisone, dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, cortivazol, hydrocortisone, 21-hydroxypregnenolone, meprednisone, dexamethasone, triamcinolone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• Disclosed herein, in certain embodiments are methods for treating inflammation associated with inflammatory brain disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein or a pharmaceutical composition disclosed herein.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of dexamethasone, betamethasone, budesonide, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of fluticasone, budesonide, beclomethasone, ciclesonide, dexamethasone, betamethasone, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, clobetasol, dexamethasone, betamethasone, budesonide, meprednisone, prednisone, prednisolone, and methylprednisolone.
• a compound disclosed herein or a pharmaceutical composition disclosed herein wherein G 2 is a radical of a steroid selected from the group consisting of cortisone, hydrocortisone, clobetasol, dexamethasone, betamethasone, budesonide, meprednisone, prednisone, prednisolone, and methylprednisolone.
• the therapeutically effective amount is that which may treat or alleviate the disease, signs or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment.
• the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment.
• the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the condition being treated and the severity of the condition; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well known to the researcher, veterinarian, medical doctor or other clinician. It is also appreciated that the therapeutically effective amount can be selected with reference to any toxicity, or other undesirable adverse effect, that might occur during administration of one or more of the compounds described herein.
• This example describes the synthesis of a compound comprising folate as a ligand, a linker, and dexamethasone as a steroid.
• FIG. 1 shows the chemical structure of a “drug compound,” otherwise known as compound 101.
• the drug compound from left to right is composed of folate as the ligand.
• the folate ligand is covalently linked to a hydrophilic linker composed of alternating unnatural amino acid D-glutamic acid, diaminopropionic acid (Dap), and the natural amino acid cysteine.
• the cysteine is then connected via a disulfide self-immolative linker to dexamethasone.
• the structure shown is also named compound 101, or Folate-Hydrolink-Cysteine-Carbonate-Dexamethasone.
• the compound shown in FIG. 1 can be synthesized via the scheme shown in FIG.
• the compound in FIG. 3 was synthesized via solid phase peptide synthesis (SPPS) using standard SPPS protocols along with N,N,N’,N’- tetramethyl-0-(lH-benzotriazol-l- yl)uronium hexafluorophosphate (HBTU) and the coupling reagent.
• SPPS solid phase peptide synthesis
• HBTU hexafluorophosphate
• the resin was then washed with DCM/DMF/DCM, dried and loaded onto an automated solid phase peptide synthesis machine.
• the remainder of the molecule was synthesized via solid phase peptide synthesis (SPPS) using standard SPPS protocols along with N,N,N',N'-Tetramethyl-0-(lH-benzotriazol-l-yl)uronium hexafluorophosphate (HBTU) / 1- [Bis(dimethylamino)methylene]- 1H- 1 ,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) as the coupling reagents.
• SPPS solid phase peptide synthesis
• N 10 -trifluoroacetylated (N 10 -TFA) pteroic acid was coupled last using previously mentioned standard SPPS protocol, followed by N 10 -TFA deprotection using 50% ammonia hydroxide in DMF.
• the product was cleaved from the resin using TFA/TIPS/TCEP-HCl and precipitated in diethyl ether, centrifuged and washed three times with diethyl ether and verified by LC-MS.
• Scheme 7 Synthetic Scheme of Steroid-Carbamate-Pyridyl Disulfide Compounds [00309] As shown in Scheme 7. Steroid (1 eq, Dexamethasone, Betamethasone, Flumethasone or Budesonide) and p-nitrophenyl chloroformate (leq) were loaded into a capped vial containing a magnetic stir bar. Next, addition of either chloroform or dichloromethane created a final steroid concentration of 0.1M. Lastly, ⁇ 25eq of pyridine was added to the stirring solution and the reaction was monitored at room temperature by LC-MS.
• Betamethasone and 5.4mg p-nitrophenyl chloroformate were loaded into a capped vial containing a magnetic stir bar.
• addition of 200uL dichloromethane followed by 40uL of pyridine was added to the stirring solution and the reaction was monitored at room temperature by LC-MS.
• 5.7mg of 2-(pyridin-2-yldisulfaneyl)ethan-l -amine acid chloride salt was mixed in a separate container with 500uL dichloromethane and 13.3uL of diisopropylethylamine (DIPEA) and continued to be monitored by LC-MS.
• DIPEA diisopropylethylamine
• the reaction was monitored by LC-MS and upon completion the crude reaction mixture was precipitated with diethyl ether, dried, dissolved in 300uL DMF and 300uL of 20mM ammonium acetate buffer (pH 6) containing 20mg Fol-mPEG2-Hydrolink-DimethylCys was added quickly and monitored to completion by LC-MS.
• the reaction as then purified by RPHPLC utilizing 20mM ammonium acetate buffer/acetonitrile and verified by LC-MS.
• LC-MS of compound 106 is shown in LIG.
• Fmoc -Phosphate 50mg was dissolved in 0.4mL of DMF, followed by the addition of 0.02mL Trimethylamine and 56.7mg of carbonyl diimidizole (CDI) for approximately 30 minutes. Then 63.3mg of dexamethasone-21 phosphate was added, followed by 150mg of anhydrous zinc (II) chloride. The reaction was monitored by LC-MS and upon completion, acetonitrile and 20mM ammonium acetate buffer pH 7 were added and was lyophilized. This resulting residue was then dissolved in DMSO and purified by RPHPLC. (27.06mg yield) Synthesis of Fmoc-PEGl 2 -Pyro-Dex
• lOOmg of folic acid was dissolved in lOmL of DMSO followed by the addition of 31.3mg NHS and 46.7mg N,N'-Dicyclohexylcarbodiimide. The mixture was allowed to stir overnight at room temperature. The product was then precipitated in ethyl acetate, filtered by vacuum filtration and washed three times with diethyl ether. The resulting solid was used without further purification. (103.4mg yield)
• FIG. 6 is an example of compound 101 in flow cytometry of macrophage gated on F4/80 to show CD 206 expression (a marker for M2, anti-inflammatory macrophages). Briefly, 1.5 mL of 3% thioglycolate were injected into the intraperitoneal cavity of 12-week-old ND4 mice. Three days later the macrophages were isolated by intraperitoneal lavage and plated in a 12-well dish and incubated at 37°C/5% C02. After 24 hours, the media was replaced with media containing 20 nanomolar the compound in FIG. 1 or 20 nanomolar of the compound in FIG.
• FIG. 7 is another example of Compound 101 in flow cytometry of macrophage gated on F4/80 to show CD 206 expression with controls.
• FIG. 8 is an example of Compound 101 in flow cytometry of macrophage to show CD 86 expression with controls.
• CD 86 a marker for Ml, pro-inflammatory macrophages
• Controls included unstained cells as the negative control as well as the use of 1 drop of compensation beads per dye-antibody ( 1 pF) for a positive control incubated for 1 hour on ice and washed twice with lmF 2% FBS in PBS. All samples were centrifuged at 400xg for lOmin between washes, transfers and after incubation to remove supernatant.
• FIG. 9 shows F4/80 cytometry results for one set of samples in the top row and a second set of samples in the bottom row.
• On the left of each row is a graph with results for cells treated with compound 106, in the middle of each row is a graph with results for untreated cells, and on the right side of each row is a graph with results for vehicle treated cells.
• FIG. 10 is a bar graph summarizing the results of FIG. 9. As shown by FIG. 9 and FIG. 10, the percentage of F4/80 macrophages is lower in the treated group vs the untreated or vehicle. This shows that there is less inflammation, since macrophages are one of the main mediator of inflammation and there are less of them present in the peritoneal cavity after treatment with compound 106.
• FIG. 11 shows cytometry results for cells which are untreated or treated with compound
• FIG. 12 is a bar graph summarizing the results of FIG. 11.
• the percentage of PE-F4/80 or (mouse macrophage), FITC-CD4 (T helper cell), eFluor780-CD8 (cytotoxic T cell), and PerCP/Cy5.5-Ly6G (neutrophil) is lower in the treated group vs the untreated or vehicle.
• the immune cells highlighted above represent a major portion of cells involved in inflammation and there are fewer of them present in the peritoneal cavity after compound 107 treatment.
• FIG. 13 shows cytometry results for cells which are untreated or treated with compound
• FIG. 14 is a bar graph summarizing the results of FIG. 13.
• PE-F4/80 or (mouse macrophage) FITC-CD4 (T helper cell)
• eFluor780-CD8 cytotoxic T cell
• PerCP/Cy5.5-Ly6G neutral
• the immune cells highlighted above represent a major portion of cells involved in inflammation and there are fewer of them present in the peritoneal cavity after Compound 108 treatment.
• mice 8-12 week old C57BL/6 mice were maintained on a folate free diet for at least three week dextran sodium sulfate (DSS) was added to their drinking water at a final concentration of 2.5% for 6 days.
• DSS dextran sodium sulfate
• the mice On days 3 and 4 the mice were give a single i.v. injection consisting of lOnmols of Compound 107 in PBS containing 2% DMSO.
• the mice were euthanized by C02 affixation followed by cervical dislocation. Then the colons were excised and prepared for histology as described previously (J Vis Exp. 2012; (60): 3678.). Briefly, the colons were cut out and the fecal contents were gently removed and washed in PBS.
• the colons were fixed in 10% buffered formalin for 24hrs followed by transfer to 70% ethanol. These samples were submitted to the Purdue Veterinary Pathology lab where they were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The results are shown in FIG. 15, with an image of the untreated colon on the left and an image of the treated colon on the right. Note that the treated group has more crypt architecture, fewer infiltrating immune cells and has a higher number of goblet cells, indicating a healthier colon.

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