WO2016081281A1 - Bisphosphonates lipophiles et procédés d'utilisation - Google Patents

Bisphosphonates lipophiles et procédés d'utilisation Download PDF

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WO2016081281A1
WO2016081281A1 PCT/US2015/060451 US2015060451W WO2016081281A1 WO 2016081281 A1 WO2016081281 A1 WO 2016081281A1 US 2015060451 W US2015060451 W US 2015060451W WO 2016081281 A1 WO2016081281 A1 WO 2016081281A1
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hydrochloride
compound
cancer
hydrogen
aliphatic
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PCT/US2015/060451
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Inder M. Verma
Yonghui Zhang
Eric Oldfield
Yifeng Xia
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Salk Institute For Biological Studies
The Board Of Trustees Of The University Of Illinois
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    • 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
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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    • 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
    • A61K47/02—Inorganic compounds
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
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    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • A61K9/2018—Sugars, or sugar alcohols, e.g. lactose, mannitol; Derivatives thereof, e.g. polysorbates
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    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; Cyclodextrin
    • A61K9/2054—Cellulose; Cellulose derivatives, e.g. hydroxypropyl methylcellulose
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    • A61P35/00—Antineoplastic agents
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    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/3843—Polyphosphonic acids containing no further substituents than -PO3H2 groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3839—Polyphosphonic acids
    • C07F9/3873—Polyphosphonic acids containing nitrogen substituent, e.g. N.....H or N-hydrocarbon group which can be substituted by halogen or nitro(so), N.....O, N.....S, N.....C(=X)- (X =O, S), N.....N, N...C(=X)...N (X =O, S)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/576—Six-membered rings
    • C07F9/58—Pyridine rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having three nitrogen atoms as the only ring hetero atoms
    • C07F9/6518—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6536—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having nitrogen and sulfur atoms with or without oxygen atoms, as the only ring hetero atoms
    • C07F9/6539—Five-membered rings

Definitions

  • This application relates to lipophilic bisphosphonates and methods of their use, for example in combination with an autophagy inducer, to treat cancer.
  • Bisphosphonates are a class of drugs as the treatment of choice for various diseases of excessive bone resorption like osteoporosis, Paget' s disease and the skeletal complications of malignancy, etc. Bisphosphonate drugs share some common properties but there are chemical, biochemical, and pharmacological differences among each bisphosphonate drugs.
  • First generation bisphosphonates such as etidronate and clodronate act by forming "toxic-ATP" analogs that inhibit the mitochondrial adenine nucleotide translocase (ANT) in osteoclasts.
  • the second and third generation drugs so-called nitrogen-containing bisphosphonates (e.g., pamidronate, alendronate, risedronate, ibandronate and zoledronate), inhibit the enzyme farnesyldiphosphate synthase (FPPS), blocking isoprenoid bio synthesis.
  • FPPS farnesyldiphosphate synthase
  • the disclosure provides, inter alia, novel bisphosphonate compounds and methods of making and using the compounds.
  • the disclosed compounds and methods can be used in connection with research and therapeutic applications, e.g., for treatment of cancers.
  • certain structural features significantly enhance the activity of the compounds.
  • the presence of particular alkyl substituents on a ring component in an organic bisphosphonate compound can contribute to desirable functional activity. Further variations are also provided.
  • the disclosure broadly provides bisphosphonate compounds and related methods of making and using such compounds. Specific embodiments concern organic bisphosphonate compounds and/or pharmaceutically acceptable salts or esters thereof. In addition, the disclosure provides other variations of bisphosphonate compounds. Particular species of functionally and/or therapeutically active bisphosphonates are also disclosed.
  • compositions that include one or more bisphosphonates are also disclosed.
  • the bisphosphonates are high potency bisphosphonates in one or more functional contexts.
  • the disclosure provides an imidazole that has a molecular weight of at least 150 and less than 1,000, such as from 200 to 900, or from 300 to 800, and in certain embodiments, from 350 to 400, and that is capable of activating a ⁇ T cell.
  • X is hydrogen, hydroxyl, SH, halogen, alkoxy, or aliphatic, particularly alkyl; each M is independently hydrogen, -(CH2) P -0-CO-R, -(CH2) P -CO-R, a cation or aliphatic, such as alkyl; each q is independently 0 or 1; R 11 and R 12 are independently H or aliphatic, particularly alkyl; p is 1 to 6; r is from 1 to 6; and R is hydrogen, aryl or aliphatic, particularly alkyl.
  • the compound is not (2-(lH-imidazol-l-yl)ethane-l,l-diyl)bis(phosphonic acid), (l-hydroxy-2-(lH- imidazol-4-yl)ethane-l,l-diyl)bis(phosphonic acid), sodium ( l-hydroxy-2-(l -methyl- 1H- imidazol-3-ium-3-yl)ethane- 1 , 1 -diyl)bis(hydrogen phosphonate), ( 1 -hydroxy-2-( 1H- 1 ,2,3- triazol-l-yl)ethane-l,l-diyl)bis(phosphonic acid), sodium (1-hydroxy-l-
  • M is a pharmaceutically acceptable cation.
  • M is Li + , Na + , K + , Ca 2+ , Mg 2+ , NH 4 + , N(R') 4 + , where each R' independently is hydrogen or aliphatic, particularly alkyl or substituted alkyl, such as trialkyl ammonium compounds where the alkyl groups are the same or different.
  • R' independently is hydrogen or aliphatic, particularly alkyl or substituted alkyl, such as trialkyl ammonium compounds where the alkyl groups are the same or different.
  • Certain particular examples include trimethyl ammonium, triethyl ammonium, and triethanol ammonium cations.
  • the compound has a formula
  • Ar is a heteroaryl.
  • exemplary heteroaryl compounds include imidazole, pyrazole, pyrole, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, 1,2,3- triazole, 1,2,4-triazole, tetrazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,4- oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5-thiadiazole or 1,3,4-thiadiazole, and in particular embodiments, Ar is a 5-membered nitrogen-containing ring, such as imidazole.
  • the compound may have a formula selected from
  • R 4 , R 6 and R 7 are each independently H, aliphatic, such as alkyl or alkenyl; alkoxy; alkenoxy; haloalkyl; aryl; alkylaryl; arylalkyl; nitro; or halogen; and R 5 and R 8 are each independently a lone pair of electrons; H; aliphatic, such as alkyl or alkenyl; haloalkyl; aryl; alkylaryl; or arylalkyl.
  • the disclosure provides compounds of the formula XA1:
  • X is hydrogen, hydroxyl, or halogen; each M independently is hydrogen, -(CH2) P -0-CO-R, -(CH2) P -CO-R, or aliphatic, such as alkyl; each q is independently 0 or 1; p is 1 to 6; and R is hydrogen, aryl, or aliphatic, such as alkyl; -OM can also be a salt of form -0 " A + , where A + is a cation; and Z is hydrogen, aryl, aliphatic, such as alkyl.
  • Z may be an alkyl group having a structure X-(CH 2 ) n - where n is an integer from 0 to 20, more typically 5-15, such as 8-12, and in particular examples, n is 8, and X is hydrogen or halogen.
  • R 5 , R 8 , or both R 5 and R 8 have a formula
  • R 9 is aliphatic, such as alkyl or alkenyl; alkoxy; haloalkyl; N0 2 ; OH; aryl; alkenyloxy; SO2R 10 ; or N(R 10 ) 2 .
  • R 10 is alkyl, and p is from 0 to 5.
  • composition comprising at least one disclosed compound, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier also is disclosed.
  • the compostion may comprise one compound disclosed herein, or may comprise two or more disclosed compounds.
  • the composition may further comprise one or more additional therapeutic compounds.
  • Embodiments of a method for synthesizing a compound or a pharmaceutical formulation thereof also are disclosed.
  • a composition provided herein is isolated and/or purified.
  • compositions disclosed herein can be used as a medicament.
  • a disclosed composition is used to prepare or manufacture a medicament, such as treatment of one or more conditions as disclosed herein (such as cancer).
  • Embodiments of a method for treating a medical condition comprising administering to a subject in need thereof a therapeutically effective amount of a compound or composition also are disclosed.
  • the disclosure provides a method of selectively inhibiting an FPPS enzyme and a PvGGPPS enzyme comprising contacting the enzyme or a cell containing the enzyme with a disclosed compound, wherein the compound is capable of selectively inhibiting said FPPS enzyme and PvGGPPS enzyme.
  • the disclosure provides a composition including a pharmaceutical formulation containing a compound of any formula herein.
  • the disclosure provides a medicament which includes a
  • the disclosure provides a method for making a medicament for treatment of a condition described herein.
  • a method of reducing or inhibiting growth of a cancer cell also is disclosed. Certain embodiments of the method include contacting the cancer cell with an effective amount of one or more compounds provided herein, and/or a pharmaceutical formulation thereof, for example in combination with an autophagy inducer (such as rapamycin or RAD001). Also disclosed are embodiments of a method for treating a cancer in vivo, for example by administering to a subject in need thereof a therapeutically effective amount of one or more compounds provided herein or a pharmaceutical formulation thereof, for example in combination with an autophagy inducer (such as rapamycin or RAD001).
  • an autophagy inducer such as rapamycin or RAD001
  • Exemplary cancers that can be treated with the disclosed embodiments include, but are not limited to, bladder cancer, lung cancer, breast cancer, melanoma cancer, colon cancer, rectal cancer, non-Hodgkin lymphoma cancer, endometrial cancer, pancreatic cancer, renal cancer, prostate cancer, leukemia cancer, and/or thyroid cancer.
  • the cancer is one expressing a KRAS mutation.
  • Embodiments of a method of stimulating a T cell are disclosed. In some examples such a method includes contacting the T cell with one or more compounds provided herein, and/or a pharmaceutical formulation thereof, for example in combination with an autophagy inducer (such as rapamycin).
  • the T cell is a gamma delta ( ⁇ ) T cell.
  • Embodiments of a method of immunotherapeutic treatment are disclosed.
  • a method of immunotherapeutic treatment includes administering to a subject in need thereof a therapeutically effective amount of one or more compounds provided herein, and/or a pharmaceutical formulation thereof, for example in combination with an autophagy inducer (such as rapamycin or RADOOl).
  • an autophagy inducer such as rapamycin or RADOOl
  • FIG. 1A is a graph illustrating KRAS tumor cell xenografts control groups.
  • FIG. IB is a graph illustrating KRAS tumor cell xenografts treated with BPH-1222 and rapamycin.
  • FIG. 1C is a graph illustrating KRAS tumor cell xenografts treated with rapamycin alone.
  • FIG. ID is a graph illustrating KRAS tumor cell xenografts treated with BPH-1222 alone.
  • FIG. 2A is a graph of tumor weight versus compound illustrating the tumor volume of mice treated with rapamycin, BPH-1222 and their combination.
  • FIG. 2B is a photograph showing tumors from the mice from FIG. 2A. Top row: mice treated with saline (left), rapamycin +9 (center) or 9 only (right). Bottom row: mice treated with rapamycin only. Combination therapy provides a large decrease in tumor volume.
  • FIG. 2C is a graph illustrating the body weight of mice treated with rapamycin, BPH- 1222 and their combination.
  • FIG. 3 is a schematic illustration of pathways involved in zoledronate analog activity in ⁇ T cells and in malaria parasites.
  • Green human cell
  • cyan malaria parasite.
  • HMG-CoA hydroxymethylglutaryl coenzyme A
  • IPP isopentenyldiphosphate
  • DMAPP dimethylse
  • FPP farnseyldiphosphate
  • GAP glyceraldehyl-3-phosphate
  • HMBPP 4-hydroxyl-3-methyl-but-2-enyl diphosphate
  • GGPP geranylgeranyldiphosphate
  • TNF-a tumor necrosis factor a.
  • FIG. 4A provides the structures of certain exemplary disclosed compounds.
  • FIG. 4B is a graph of IC50 versus sidechain length illustrating the chain length dependence of enzyme and cell growth inhibition/activation and effects of the 1-OH group on HsFPPS inhibition.
  • FIG. 4C is a graph of IC50 versus sidechain length illustrating the chain length dependence of enzyme and cell growth inhibition/activation and effects of the 1-OH group on ⁇ T cell activation/ TNF-a release.
  • FIG. 4D is a graph of IC50 versus sidechain length illustrating the chain length dependence of enzyme and cell growth inhibition/activation and effects of the 1-OH group on PvGGPPS inhibition.
  • FIG. 4E is a graph of IC50 versus sidechain length illustrating the chain length dependence of enzyme and cell growth inhibition/activation and effects of the 1-OH group on intra-erythrocytic P. falciparum cell growth inhibition.
  • FIG. 5A is a schematic illustration of the X-ray structure of HsFPPS/bisphosphonate 5 complex (cyan, PDB ID code 4GA3) superimposed on PvGGPPS structure (purple, PDB ID code 3RBM), with the Ca rmsd over 331 residues in 1.44 A.
  • FIG. 5B is a schematic illustration of the electron density map of 5 bound to HsFPPS.
  • FIG. 5C is a schematic illustration showing the comparison between the x-ray structures of 5 bound to HsFPPS, GPP (yellow) and FPP (green) bound to avian FPPS (PDB ID codes,
  • the bisphosphonate 5 binds to the allylic (GPP) site. Chain elongation in FPP is blocked by F98, F99, corresponding to decreased HsFPPS inhibition by bisphosphonate inhibitors with /V-alkyl chains longer than about CIO.
  • FIG. 5D is a schematic illustration showing the structure of HsPPPS/5 overlaid on 29 (BPH-703) bound to PvGGPPS (PDB IDcode 3RBM).
  • PvGGPPS PvGGPPS
  • FIG. 6 is a schematic illustration of lipophilic analogs of the bone resorption drug zoledronate killing malaria parasites in a direct manner by inhibiting parasite geranylgeranyldiphosphate synthase, in addition to activating ⁇ T cells to kill parasites in an indirect (TNF-a/granulysin mediated) manner.
  • FIG. 7A provides the chemical structure of zoledronate.
  • FIG. 7C provides the chemical structure of BPH-1222.
  • FIG. 7D is a schematic illustration showing the structure of BPH-1222 binding to FPPS was determined by single crystal X-ray crystallography. Zoledronate binding is shown superimposed with BPH-1222 (zoledronate in yellow and BPH-1222 in cyan).
  • FIG. 8 A is a graph of IC50 versus side-chain length illustrating the activity of zoledronate and its hydroxy analogs (see FIG. 10 for desoxy analogs) as determined in cell lines derived from KRAS-shp53 mouse lung cancer model (6# and L2) and control mouse embryonic fibroblasts (MEF) using MTT assay.
  • FIG. 8B is a graph of Ki versus side-chain length illustrating the Ki of compounds in FIG. 8 A measured in vitro against human FPPS or GGPPS.
  • FIG. 8C provides photographs of mouse lung cancer cells (6#) treated with a single drug (FTI-277, 15 ⁇ ; GGTI-298, 15 ⁇ ; BPH-1222, 10 ⁇ ; FOH, 10 ⁇ ; GGOH, 10 ⁇ ;
  • Ascorbic acid 50 ⁇ or drug combinations as indicated for 48 hours. Images were taken under phase-contrast microscope. Scale bars, 100 ⁇ .
  • FIG. 8D is a graph illustrating the cell survival of mouse lung cancer cells (L2) and embryonic fibroblasts (MEF, matched genetic background) treated with different concentrations of BPH-1222 for 3 days, measured using Cell Proliferation Reagent WST-1 from Roche.
  • FIG. 8E is a graph illustrating the cell survival of mouse embryonic fibroblasts transformed by KRAS-shp53 and MYCLl-shp53-shRBl treated with different concentrations of BPH-1222 for 3 days, measured using Cell Proliferation Reagent WST-1 from Roche.
  • FIG. 9A is a photograh of mouse lung cancer cells illustrating the FPPS/GGPPS activity and cell growth inhibited by bisphosphonates.
  • FIG. 9B is a graph of IC50 versus side-chain length illustrating the IC50 of desoxy zoledronate and its analogs determined in cell lines derived from KRAS-shp53 mouse lung cancer model (6# and L2) and control mouse embryonic fibroblasts (MEF) using MTT assay.
  • FIG. 9C is a graph of Ki versus side-chain length illustrating the Ki of the compounds in FIG. 9B measured in vitro against human FPPS or GGPPS.
  • FIG. 9D is a photograph of immunoblots illustrating mouse lung cancer cells (M3L2) that were treated with FTI-277 (15 ⁇ ), GGTI-298 (15 ⁇ ) or a combination thereof for 48 hours and whole cell lysates, analyzed by immunoblotting.
  • FIG. 9E is a graph illustrating the cell survival of the mouse lung cancer cells (M3L2) treated with different concentrations of FTI, GGTI or BPH-1222 for 3 days. Cell survival was measured using Cell Proliferation Reagent WST-1 from Roche.
  • FIG. 9F provides photographs illustrating mouse lung cancer cells (6#) that were treated with bisphosphonates (BPH-1222, 10 ⁇ ; BPH-714, 10 ⁇ ; zoledronate, 80 ⁇ ) and simvastatin (0.5 ⁇ ) alone, or in combination with other compounds (chloroquine, 30 ⁇ ; rapamycin, 0.1 ⁇ ; FOH, 10 ⁇ ; GGOH, 10 ⁇ ) for 48 hours. Images were taken under phase-contrast microscope. Scale bars, 100 ⁇ .
  • FIG. 10A is a photograph of immunoblots of U20S cells expressing Flag-KRAS G12D treated with bisphosphonates (BPH-1222, 10 ⁇ ; BPH-714, 10 ⁇ ; zoledronate, 20 ⁇ ) or chloroquine (30 ⁇ ) for 48 hours.
  • BPH-1222, 10 ⁇ ; BPH-714, 10 ⁇ ; zoledronate, 20 ⁇ bisphosphonates
  • chloroquine (30 ⁇ )
  • RAPIA RAPIA that require prenylation were examined by immunoblotting.
  • p pellet contains correctly prenylated proteins which bind avidly to membrane;
  • s supernatant contains unmodified proteins in cytoplasm.
  • * HRAS signal left on the membrane. **, autophagy activation determined by the presence of LC3-II (bottom band, PE-conjugated form).
  • FIG. 10B is a photograph of immunoblots of the cells from FIG. 10A treated with FTI- 277 (15 ⁇ ), GGTI-298 (15 ⁇ ) or BPH-1222 (5, 10, 15 ⁇ ) for 48 hours. Cell lysates were separated with 15-cm SDS-PAGE and blotted with indicated antibodies. *, KRAS mobility shift was observed.
  • FIG. IOC is a photograph of immunoblots of mouse lung cancer cells (M3L2) treated with BPH-1222 for 48 hours and analyzed for KRAS, AKT, Caspase-3 activation and LC3 conversion.
  • KRAS-GTP was pulled down from whole cell lysate with RAF-1 RBD beads and immunoblotted with total KRAS antibody.
  • FIG. 10D is a photograph of immunoblots of human cancer cells harboring KRAS mutations (Panc-1 and MiaPaCa2) treated with BPH-1222 (10 ⁇ ) for 48 hours and analyzed in the same way as in FIG. IOC.
  • FIG. 10E is a photograph of immunoblots of mouse lung cancer cells (6#) treated with BPH-1222 (10 ⁇ ) for 1, 2 or 3 days illustrating induced ER stress (CHOP, BiP), autophagy (PE-conjugated LC3II) and apoptosis (Caspase-3) in mouse lung cancer cell (6#) in a time-dependent manner.
  • induced ER stress CHOP, BiP
  • autophagy PE-conjugated LC3II
  • Caspase-3 apoptosis
  • FIG. 11A is a schematic diagram illustrating the Mouse KRAS G12D sequence for mass spectrum analysis.
  • FIG. 1 IB is a photograph of immunoblots of mouse lung cancer cells (6#) treated with simvastatin (0.5 ⁇ ), FTI-277 (10 ⁇ ), GGTI-298 (10 ⁇ ), BPH-1222 (10 ⁇ ), or
  • FIG. 11C is a photograph of immunoblots of mouse lung cancer cells (6#) treated with
  • FIG. 1 ID is a photograph of immunoblots of the same cells from FIG. 11C treated with drug combinations in the absence or presence of U0126 (10 ⁇ ) for 48 hours, and lysed for immunoblotting to check MAPK pathway activity.
  • FIG. 12 is a table of KRAS molecular weights (M.W.) measured by Mass spectrometry.
  • FIG. 13B is a photograph of mouse lung cancer cell (L2, infected with 5XKB-1UCI reporter) syngeneic grafts and treated with BPH-1222 (2 mg/kg) plus either chloroquine (60 mg/kg) or rapamycin (2.5 mg/kg) for 3 weeks. Scale bars, 10 mm.
  • FIG. 13C is a graph illustrating the NF- ⁇ activity in tumor grafts examined by in vivo luciferase imaging system (IVIS) immediately after the 3-week treatment.
  • IVIS in vivo luciferase imaging system
  • FIG. 13E provides photographs of L2 tumor samples treated with different combinations, sectioned and immuno- stained with Ki-67 and Cleaved Caspase-3 antibodies. Caspase-3 positive cells were marked with arrowheads. Scale bars, 100 ⁇ .
  • FIG. 13F is a graph illustrating the percentage of Ki-67 positive cells from the samples from FIG. 13E.
  • FIG. 13G is a graph illustrating the percentage of Caspase-3 positive cells from the samples from FIG. 13E.
  • FIG. 13H is a photograph of mouse lung cancer cells (L2) syngeneic grafts treated with
  • BPH-1222 (2 mg/kg), rapamycin (2.5 mg/kg) or combination for 3 weeks.
  • Scale bar 10 mm.
  • FIG. 131 is a photograph of immunoblots of mouse lung cancer cells (6#) treated with BPH-1222 (10 ⁇ ), rapamycin (0.1 ⁇ ) or combination for 48 hours and examined by immunoblotting .
  • FIG. 14A provides photographs of mouse lung cancer cell line (6#) and human lung cancer cell lines (A549 and A427) treated with BPH-1222 (10 ⁇ ), chloroquine (30 ⁇ ) or combination for 48 hours. Images were taken under phase-contrast microscope. Scale bars, 100 ⁇ .
  • FIG. 14B is a photograph of immunoblots of mouse lung cancer cells (6#) treated with BPH-1222 alone or together with rapamycin (0.1 ⁇ ) or chloroquine (30 ⁇ ) for 48 hours.
  • FIG. 14D is a photograph of the tumors from FVB mice carrying M3L2 cell syngeneic grafts treated with zoledronate (2 mg/kg), BPH-1222 (2 mg/kg), rapamycin (2.5 mg/kg) or combinations for 3 weeks. Each tumor was dissected and weighed. Scale bar, 10 mm.
  • FIG. 14E is a graph of tumor weight versus treatment illustrating the tumor weights.
  • FIGS. 15A-15H illustrate that combination therapy inhibits tumor growth in orthotopic graft model and KRAS-shp53 lentiviral model.
  • FIG. 15A provides photogarphs of the lungs from mouse orthotopic grafts that were induced by tail vein injection of M3L2 cells. Mice were left untreated or given treatment 16 days after the inoculation. Lungs were collected when mice reached IACUC clinical end-point.
  • FIG. 15D provides photographs of Luciferase imaging results of one mouse from Low- Ctrl group and one mouse from Low-Treat group, to show the shrinkage of tumor after the combination therapy.
  • Mouse lung adenocarcinomas were induced by intra-tracheally infection of KRAS-shp53 lentiviral vectors.
  • mice were left untreated or given treatment when luciferase signals from tumors were detectable (Low-Ctrl and Low-Treat groups, luciferase signal: 10 3 - 10 5 ; High-Ctrl and High-Treat groups, luciferase signal: > 10 5 ). See FIG. 16 for results from all mice.
  • FIG. 15E is a graph illustrating the fold changes of luciferase signal after 2- week treatment. Negative value means shrinkage in tumor size.
  • FIG. 15F is a graph of survival percentage versus days illustrating the Kaplan-Meier curves of mice from all groups.
  • FIG. 15G provides photographs of tumors from Low-Ctrl and Low-Treat groups that were sectioned and immuno- stained with different antibodies. Caspase-3 positive cells were marked with arrowheads. Scale bars, 100 ⁇ (insets in 4E-BPl-p staining: scale bars, 20 ⁇ ).
  • FIG. 15H is a graph quantifying the results shown in FIG. 15G.
  • FIGS. 16A-16C provides the results from the combination therapy in the KRAS-shp53 lentiviral model. All mice carrying KRAS-shp53 tumors described in FIGS. 16A-16C were examined for tumor load by in vivo luciferase imaging system (IVIS) every 10 days during the treatment. Legend names are all derived from mouse IDs and don't carry any real meanings.
  • IVIS in vivo luciferase imaging system
  • FIG. 17 is a graph of weight versus treatment illustrating the effect of treatment with BPH-1222 and either rapamycin (rapal222) or everolimus (eve 1222) in a syngeneic
  • FIG. 18 is a graph of weight versus treatment illustrating the effect of treatment with BPH-1222 and either rapamycin (rapal222) or everolimus (evel222) in an orthotopic model.
  • rapamycin rapal222
  • evel222 everolimus
  • references in the specification to "one embodiment,” “an embodiment,” etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of a person of ordinary skill in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.
  • ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values.
  • a recited range e.g., weight percents or carbon groups
  • Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, as used in an explicit negative limitation.
  • Administration To provide or give a subject an agent, such as one or more compounds provided herein, by any effective route.
  • routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, and intravenous), transdermal, intranasal, and inhalation routes.
  • Autophagy A cellular recycling pathway in which cytoplasm and organelles are engulfed within double-membrane vesicles, autophagosomes, and fused with lyosomes for degradation. Autophagy plays a role in cell survival and death and has been implicated in development, aging, neurodegeneration, and cancer.
  • autophagy inducers that can be used in the compositions and methods provided herein include but are not limited to: (1) inhibitors of mTOR activation, such as metformin, perifosine, rapamycin, everolimus, resveratrol, and tamoxifen; (2) activators of autophagosme formation, such as MG-132, SAHA (suberoylanilide hydroxamine), trichostatin A, valproic acid, 5-aza-cytidine, and Z-VAD-FMK.
  • inhibitors of mTOR activation such as metformin, perifosine, rapamycin, everolimus, resveratrol, and tamoxifen
  • activators of autophagosme formation such as MG-132, SAHA (suberoylanilide hydroxamine), trichostatin A, valproic acid, 5-aza-cytidine, and Z-VAD-FMK.
  • MG-132 inhibitors of mTOR activation
  • Autophagy inducers are administered at therapeutically effective amounts, such as at least 0.01 mg/kg, at least 0.1 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 5 mg/kg, or at least 10 mg/kg.
  • Cancer A malignant tumor characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
  • Metalastatic disease refers to cancer cells that have left the original tumor site and migrate to other parts of the body for example via the bloodstream or lymph system.
  • a cell targeted for removal by the disclosed methods is a cancer cell.
  • Contacting can occur in vitro or ex vivo, for example, with a sample, or in vivo by administering to a subject.
  • Increase or Decrease A statistically significant positive or negative change, respectively, in quantity from a control value.
  • An increase is a positive change.
  • an increase may be at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500% as compared to the control value.
  • a decrease is a negative change.
  • a decrease may be at least 20%, at least 25%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% decrease as compared to a control value.
  • Inhibit or reduce The slowing, halting, or reversing of something, such as the growth or progression of a disease (such as a cancer) condition, or group of cells.
  • inhibition or reduction can be a decrease of at least 20%, at least 40% at least, 60%, at least 80%, at least 90%, at least 95%, or at least 99%, for example, compared to a particular activity that occurs in the absence of a treatment or contact.
  • KRAS OMIM 190070. Includes KRAS nucleic acid molecules and proteins.
  • the protein product of the normal KRAS gene performs is involved in normal tissue signaling, and the mutation of a KRAS gene is a step in the development of many cancers.
  • This proto- oncogene is implicated in many cancers, such as lung adenocarcinoma, follicular and undifferentiated thyroid cancer, acute myeloid leukemia, mucinous adenoma, ductal carcinoma of the pancreas and colorectal carcinoma.
  • KRAS mutations in KRAS in some examples are the result of a single amino acid substitution, and can be used to predict which subjects are not likely to response to particular therapies, such as those that inhibit EGFR, such as erlotinib, gefitinib, panitumumab and cetuximab.
  • KRAS mutations frequently found in neoplasms include those at exon 2 (codons 12 and 13) and exon 3 (codon 61). Mutations in KRAS codons 12 and 13 have been associated with lack of response to EGFR- targeted therapies in both CRC and NSCLC patients
  • KRAS is upregulated/amplified in a cancer (such as cancers of the ovary, GI tract, uterus, and lung).
  • KRAS is located at 12pl2.1, spans approximately 38 kb, and encodes a 188- amino acid residue with a molecular weight of 21.6 kDa.
  • KRAS sequences are publically available, for example from the GenBank® sequence database (e.g., Accession Nos.
  • NP_004976.2, NP_203524.1 and NP_067259.4 provide exemplary KRAS protein sequences, while Accession Nos. NM_004985.4 and NM_021284.6 provide exemplary KRAS nucleic acid sequences).
  • One of ordinary skill in the art can identify additional KRAS nucleic acid and protein sequences, including KRAS variants.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • compositions and formulations suitable for pharmaceutical delivery of a bisphosphonate compound provided herein are known to persons of ordinary skill in the art. Additionally, Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of a bisphosphonate compound provided herein.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Pharmaceutically acceptable salts comprise pharaiaceutically-acceptable anions and/or cations.
  • Pharaiaceutically-acceptable cations include among others, alkali metal cations ⁇ e.g., Li + , Na + , K + ), alkaline earth metal cations (e.g., Ca 2+ , Mg 2+ ), non-toxic heavy metal cations and ammonium (NH 4 + ) and substituted ammonium
  • Pharaiaceutically-acceptable anions include among other halides (e.g., CI " , Br " ), sulfate, acetates (e.g., acetate, trifluoro acetate), ascorbates, aspartates, benzoates, citrates, and lactate.
  • the subject is a non-human mammalian subject, such as a monkey or other primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, horse, or cow.
  • the subject is a human subject.
  • the subject has a cancer, such as a cancer with a KRAS mutation (such as one at codon 12, 13 or 61).
  • Effective amount or Therapeutically effective amount The amount of agent, such as one or more bisphosphonate compounds provided herein (for example alone or with other agents, such as an autophagy inducer), that is an amount sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease.
  • an "effective amount" is sufficient to reduce or eliminate a symptom of a disease, such as cancer (such as a cancer expressing a KRAS mutation).
  • the therapeutically effective amount can be dependent on the subject being treated (e.g., the species or size of the subject), the severity of the disease in the recipient subject.
  • an effective amount of a bisphosphonate compound provided herein can be determined by various methods, including generating an empirical dose-response curve, predicting potency and efficacy using modeling, and other methods used in the art.
  • a therapeutically effective amount of bisphosphonate compound provided herein is at least 0.01 mg/kg, at least 0.1 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 5 mg/kg, or at least 10 mg/kg.
  • Specific assays for determining the therapeutically effective amount of bisphosphonate compounds provided herein are provided herein. For example, effects on cancer cells, such as cancer cell growth, can be measured in the recipient subject.
  • an "effective amount" of a bisphosphonate compound provided herein is an amount sufficient to reduce symptoms of a cancer, such as one that expresses a KRAS mutation, for example, by at least 10%, at least 20%, at least 50%, at least 70%, or at least 90% (as compared to no administration of the bisphosphonate compound provided herein).
  • an "effective amount" of a bisphosphonate compound provided (alone or in combination with effective amounts of other therapeutic agents, such as an autophagy inducer) herein can be an amount sufficient to reduce growth rate, size, volume, and/or metastasis of a cancer, such as one that expresses a KRAS mutation, for example, by at least 10%, at least 20%, at least 50%, at least 70%, or at least 90% (as compared to no administration of the
  • an "effective amount" of a bisphosphonate compound provided herein is an amount sufficient to increase stimulation of ⁇ T cells in a subject, for example by at least 10%, at least 20%, at least 50%, at least 70%, or at least 90% (as compared to no administration of a bisphosphonate compound provided herein).
  • Treating, Treatment, and Therapy Any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject' s physical or mental well-being, or prolonging survival.
  • the treatment may be assessed by objective or subjective parameters; including the results of a physical examination, blood and other clinical tests, and the like.
  • Tumor, neoplasia, malignancy or cancer A neoplasm is an abnormal growth of tissue or cells which results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the "tumor burden" which can be measured as the number, volume, or weight of the tumor. A tumor that does not metastasize is referred to as "benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as "malignant.”
  • a “non-cancerous tissue” is a tissue from the same organ wherein the malignant neoplasm formed, but does not have the characteristic pathology of the neoplasm. Generally, noncancerous tissue appears histologically normal.
  • a "normal tissue” is tissue from an organ, wherein the organ is not affected by cancer or another disease or disorder of that organ.
  • cancer-free subject has not been diagnosed with a cancer of that organ and does not have detectable cancer.
  • Exemplary tumors such as cancers, that can be treated with the disclosed
  • bisphosphonate compounds include solid tumors, such as breast carcinomas (e.g. lobular and duct carcinomas), sarcomas, carcinomas of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, for instance, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell adenocarcinoma, endometrial carcinomas (including, e.g., aden
  • the tumor is an adenocarcinoma.
  • the disclosed compounds can also be used to treat liquid tumors, such as a lymphatic, white blood cell, or other type of leukemia.
  • the tumor treated is a tumor of the blood, such as a leukemia (for example acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia , and adult T-cell leukemia), lymphomas (such as Hodgkin's lymphoma and non-Hodgkin's lymphoma), and myelomas).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogen
  • a phrase that is used to describe any environment that permits a desired activity is treatment of a cancer.
  • Aliphatic A substantially hydrocarbon-based compound, or a radical thereof (e.g.,
  • an aliphatic group contains from one to twenty- five carbon atoms; for example, from one to fifteen, from one to ten, from one to six, or from one to four carbon atoms.
  • the term "lower aliphatic” refers to an aliphatic group containing from one to ten carbon atoms.
  • An aliphatic chain may be substituted or
  • an aliphatic group can either be unsubstituted or substituted.
  • substituents include, but are not limited to, aliphatic, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, acyl, aldehyde, amide, amino, aminoalkyl, aryl, arylalkyl, carboxyl, cyano, cycloalkyl, dialkylamino, halo, haloaliphatic, hetero aliphatic, heteroaryl, heterocycloaliphatic, hydroxyl, oxo, sulfonamide, sulfhydryl, thioalkoxy, or other functionality.
  • Alkyl groups include straight-chain, branched and cyclic alkyl groups. Alkyl groups include those having from 1 to 25 carbon atoms. Alkyl groups include small alkyl groups having 1 to 3 carbon atoms. Alkyl groups include medium length alkyl groups having from 4-10 carbon atoms. Alkyl groups include long alkyl groups having more than 10 carbon atoms, particularly those having 10-25 carbon atoms. Cyclic alkyl groups include those having one or more rings. Cyclic alkyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and particularly those having a 3-, 4-, 5-, 6-, or 7-member ring.
  • the carbon rings in cyclic alkyl groups can also carry alkyl groups.
  • Cyclic alkyl groups can include bicyclic and tricyclic alkyl groups.
  • Alkyl groups optionally include substituted alkyl groups.
  • Substituted alkyl groups include among others those which are substituted with aliphatic or aryl groups, which in turn can be optionally substituted.
  • alkyl groups include methyl, ethyl, n- propyl, iso-propyl, cyclopropyl, n-butyl, s-butyl, t-butyl, cyclobutyl, n-pentyl, branched-pentyl, cyclopentyl, n-hexyl, branched hexyl, and cyclohexyl groups, all of which are optionally substituted.
  • Alkoxy groups are -O-alkyl groups, where alkyl is as defined herein.
  • Alkenyl groups include straight-chain, branched and cyclic alkenyl groups. Alkenyl groups include those having 1, 2 or more double bonds and those in which two or more of the double bonds are conjugated double bonds. Alkenyl groups include those having from 2 to 25 carbon atoms. Alkenyl groups include small alkyl groups having 2 to 3 carbon atoms. Alkenyl groups include medium length alkenyl groups having from 4-10 carbon atoms. Alkenyl groups include long alkenyl groups having more than 10 carbon atoms, particularly those having 10-25 carbon atoms. Cyclic alkenyl groups include those having one or more rings.
  • Cyclic alkenyl groups include those in which a double bond is in the ring or in an alkenyl group attached to a ring. Cyclic alkenyl groups include those having a 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-member carbon ring and particularly those having a 3-, 4-, 5-, 6- or 7-member ring. The carbon rings in cyclic alkenyl groups can also carry alkyl groups. Cyclic alkenyl groups can include bicyclic and tricyclic alkyl groups. Alkenyl groups are optionally substituted. Substituted alkenyl groups include among others those which are substituted with aliphatic, alkyl or aryl groups, which groups in turn can be optionally substituted.
  • alkenyl groups include ethenyl, prop-1- enyl, prop-2-enyl, cycloprop-l-enyl, but-l-enyl, but-2-enyl, cyclobut-l-enyl, cyclobut-2-enyl, pent-l-enyl, pent-2-enyl, branched pentenyl, cyclopent-l-enyl, hex-l-enyl, branched hexenyl, cyclohexenyl, all of which are optionally substituted.
  • Alkenoxy groups are -O-alkenyl groups, where alkenyl is as defined herein.
  • Aryl groups include groups having one or more 5- or 6-member aromatic or
  • Aryl groups can contain one or more fused aromatic rings.
  • Heteroaromatic, or heteroaryl, rings can include one or more N, O, or S atoms in the ring.
  • Heteroaromatic rings can include those with one, two or three N, those with one or two O, and those with one or two S, or any combination thereof.
  • Aryl groups are optionally substituted.
  • Substituted aryl groups include among others those which are substituted with aliphatic, alkyl or alkenyl groups, which groups in turn can be optionally substituted.
  • Specific aryl groups include phenyl groups, biphenyl groups, pyridinyl groups, and naphthyl groups, all of which are optionally substituted.
  • Arylalkyl groups are alkyl groups substituted with one or more aryl groups where the attachment to the rest of the molecule is through the alkyl group, and wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted.
  • Specific alkylaryl groups are phenyl-substituted alkyl groups, e.g., benzyl, and phenylethyl groups.
  • Alkylaryl groups are aryl groups substituted with one or more alkyl groups where the attachment to the rest of the molecule is through the aryl group, and wherein the alkyl groups optionally carry additional substituents and the aryl groups are optionally substituted.
  • Specific alkylaryl groups are alkyl-substituted phenyl groups such as methylphenyl.
  • the rings that may be formed from two or more of any R (e.g., Rl and R2) groups herein together can be optionally substituted cycloalkyl groups, optionally substituted cycloalkenyl groups or aromatic groups.
  • the rings may contain 3, 4, 5, 6, 7 or more carbons.
  • the rings may be heteroaromatic in which one, two or three carbons in the aromatic ring are replaced with N, O or S.
  • the rings may be heteroalkyl or heteroalkenyl, in which one or more CH 2 groups in the ring are replaced with O, N, NH, or S.
  • Optional substitution of any aliphatic, alkyl, alkenyl and aryl groups includes substitution with one or more of the following substituents: halogens, -CN, -COOR, -OR, -COR, -OCOOR, -CON(R) 2 , -OCON(R) 2 , -N(R) 2 , -N0 2 , -SR, -S0 2 R, -S0 2 N(R) 2 or -SOR groups.
  • Optional substitution of aliphatic or alkyl groups includes substitution with one or more alkenyl groups, aryl groups or both, wherein the alkenyl groups or aryl groups are optionally substituted.
  • Optional substitution of alkenyl groups includes substitution with one or more aliphatic or alkyl groups, aryl groups, or both, wherein the aliphatic, alkyl or aryl groups are optionally substituted.
  • Optional substitution of aryl groups includes substitution of the aryl ring with one or more aliphatic, alkyl, alkenyl groups, or both, wherein the aliphatic, alkyl or alkenyl groups are optionally substituted.
  • Optional substituents for aliphatic, alkyl, alkenyl and aryl groups include among others:
  • R is a hydrogen or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which are optionally substituted;
  • R is a hydrogen, aliphatic, or an alkyl group or an aryl groups and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted;
  • -CON(R) 2 where each R, independently of each other R, is a hydrogen, aliphatic, or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted, or both R groups together with the nitrogen to which they are attached, can form a ring which may contain one or more double bonds and/or one or more additional heteroatoms such as O, S or N;
  • each R independently of each other R, is a hydrogen, aliphatic or an alkyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl groups all of which groups are optionally substituted, or both R groups together with the nitrogen to which they are attached, can form a ring which may contain one or more double bonds and/or one or more additional heteroatoms such as O, S or N;
  • each R independently of each other R, is a hydrogen, aliphatic, or an alkyl group, acyl group or an aryl group and more specifically where R is methyl, ethyl, propyl, butyl, or phenyl or acetyl groups all of which are optionally substituted, or both R groups together with the nitrogen to which they are attached, can form a ring which may contain one or more double bonds and/or one or more additional heteroatoms such as O, S or N;
  • R is an aliphatic, alkyl or aryl group and more specifically where R is methyl, ethyl, propyl, butyl, phenyl groups all of which are optionally substituted; for -SR, R can be hydrogen;
  • R is an aliphatic, alkyl or aryl group
  • R is a hydrogen, aliphatic, alkyl or aryl group, or both R groups together with the nitrogen to which they are attached, can form a ring which may contain one or more double bonds and/or one or more additional heteroatoms such as O, S or N;
  • R H, aliphatic, alkyl, aryl, or acyl
  • R can be an acyl yielding -OCOR* where R* is a hydrogen, aliphatic or alkyl group or an aryl group and more specifically where R* is methyl, ethyl, propyl, butyl, or phenyl groups, all of which groups are optionally substituted;
  • Specific substituted aliphatic and alkyl groups include haloalkyl groups, particularly trihalomethyl groups and specifically trifluoromethyl groups.
  • Specific substituted aryl groups include mono-, di-, tri, tetra- and pentahalo-substituted phenyl groups; mono-, di-, tri-, tetra-, penta-, hexa-, and hepta-halo-substituted naphthalene groups; 3- or 4-halo-substituted phenyl groups, 3- or 4- alkyl- substituted phenyl groups, 3- or 4-alkoxy-substituted phenyl groups, 3- or
  • substituted aryl groups include acetylphenyl groups, particularly 4-acetylphenyl groups; fluorophenyl groups, particularly 3-fluorophenyl and 4-fluorophenyl groups; chlorophenyl groups, particularly 3-chlorophenyl and 4-chlorophenyl groups; methylphenyl groups, particularly 4-methylphenyl groups, and methoxyphenyl groups, particularly 4-methoxyphenyl groups.
  • the term “medium length side-chain” can include side chains that have from about 7 carbon elements to about 13 carbon elements. In other embodiments, the term “short length side-chains” can include side chains having less than about 7 carbon elements. In still other embodiments, the term “long length side-chains” can include side chains having greater than 13 carbon elements.
  • any of compound described herein, which contains one or more substituents it is understood, of course, that such groups do not contain any substitution or substitution patterns that are sterically impractical and/or synthetically non-feasible.
  • the compounds of the disclosure can contain asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials or by the use of enantioselective catalytic reactions. All chiral, diastereomeric, racemic forms and all geometric isomeric forms and tautormers of a compound are intended as part of this disclosure.
  • One diastereomer may display superior activity compared to another.
  • separation of racemic materials can be achieved by high performance liquid chromatography (HPLC) using a chiral column or by a resolution using a resolving agent such as camphonic chloride, as in Thomas J. Tucker et al., /. Med. Chem. 1994, 37, 2437-2444.
  • HPLC high performance liquid chromatography
  • a chiral compound may also be directly synthesized using a chiral catalyst or a chiral ligand; see, for example, Mark A. Huffman et al., /. Org. Chem. 1995, 60, 1590-1594.
  • Certain molecules disclosed herein contain one or more ionizable groups [groups from which a proton can be removed (e.g., -COOH) or added (e.g., amines)]. All possible ionic forms of such molecules and salts thereof are intended to be included in the disclosure herein. With regard to salts of the compounds herein, one of ordinary skill in the art will know how to select from among a wide variety of available counterions those that are appropriate for preparation of salts of this disclosure for a given application. In specific applications, the selection of a given anion or cation for preparation of a salt may result in increased or decreased solubility of that salt. Compounds of the disclosure can have prodrug forms. Prodrugs of the compounds of the disclosure are useful in the methods of this disclosure.
  • prodrug Any compound that will be converted in vivo to provide a biologically, pharmaceutically or therapeutically active form of a compound of the disclosure is a prodrug.
  • Various examples and forms of prodrugs are well known in the art. Examples of prodrugs are found, inter alia, in Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985), Methods in Enzymology, Vol. 42, at pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985); A Textbook of Drug Design and Development, edited by Krosgaard- Larsen and H. Bundgaard, Chapter 5, "Design and Application of Prodrugs," by H. Bundgaard, at pp.
  • the wavyline “ ⁇ " indicates the point of attachment for a group or moiety.
  • Ar is a 5-membered heteroaromatic ring
  • X is hydrogen, aliphatic, hydroxyl, SH, halogen, or alkoxy
  • each M is independently hydrogen, -(CH2) P -0-CO-R, - (CH 2 )p-CO-R, or aliphatic, such as alkyl, where p is 1 to 6, and R is hydrogen, aryl, or aliphatic, such as alkyl
  • each q is independently 0 or 1
  • R 11 and R 12 are independently H, or aliphatic, such as alkyl
  • r is from 1 to 6.
  • X may be alkyl.
  • -OM can also be a salt of the form -0 " A + , where A + is a cation.
  • Suitable cations include, but are not limited to, sodium, lithium, potassium, magnesium, manganese, zinc, copper, iron, ammonium or N(R') 4 + , where each R' independently is hydrogen or aliphatic, particularly alkyl or substituted alkyl. The aliphatic or alkyl groups can be the same or different.
  • Exemplary cations include trimethyl ammonium, triethyl ammonium, and triethanol ammonium cations.
  • the cation may be associated with more than one oxygen on a phosphonate, such as , or the cation
  • ° p r— M ++ -0 ⁇ p '° may be associated with two different phosphonate groups, such as ⁇ ⁇ ⁇ , which may or may not be on the same molecule.
  • the compound is not
  • the compound is not
  • the compound has a formula II
  • Ar is imidazole, pyrazole, pyrole, furan, thiophene, oxazole, isoxazole, thiazole, isothiazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, 1,2,3-oxadiazole, 1,2,4- oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, 1,2,3-thiadiazole, 1,2,4-thiadiazole, 1,2,5- thiadiazole or 1,3,4-thiadiazole.
  • Ar may be a nitrogen-containing 5-membered ring, and in some examples, the attachment point of the bisphosphonate moiety to the ring is at a ring nitrogen. In certain examples, where at least one M is a negative charge, Ar is a nitrogen-containing, 5- membered heteroaromatic ring with at least one positive charge.
  • Ar is an unsubstituted 5-membered heteroaromatic ring.
  • Ar is substituted with from 1 to as many substituents as can be accommodated by Ar, such as at least upto 4 substituents.
  • the substituent is a lipophilic substituent.
  • substituents for Ar include, but are not limited to, aliphatic, such as alkyl or alkenyl; alkoxy; alkenoxy; hydroxyl; halogen, such as fluoro, chloro bromo or iodo; nitro; haloalkyl; aryl; alkylaryl; arylalkyl; or a combination thereof.
  • Ar is an imidazole, leading to compounds having formulas III- VII
  • R 4 , R 6 and R 7 are each independently H, alkoxy, alkenoxy, haloalkyl, aryl, alkylaryl, arylalkyl, nitro, halogen or aliphatic, particularly alkyl or alkenyl;
  • R 5 is alkoxy, alkenoxy, haloalkyl, aryl, alkylaryl, arylalkyl or aliphatic, particularly alkyl or alkenyl;
  • R 8 is a lone pair of electrons, H, haloalkyl, aryl, alkylaryl, arylalkyl or aliphatic, particularly alkyl or alkenyl.
  • the imidazole ring has a positive charge.
  • R 4 , R 6 , R 7 are all H, one q is 0 and the remainder are 1, each M is H, and X is OH, then R 5 is not a lone pair of electrons.
  • R 5 is alkyl, and may be a CMS alkyl. In particular embodiments R 5 is a C 8 alkyl.
  • X is H or hydroxyl, and in particular examples, X is hydroxyl.
  • one q is 0 and the rest are 1, and each M is H.
  • R 5 and/or R 8 is aryl, and may be phenyl. In some embodiments, R 5 and/or R 8 is a moiety having a structure
  • each R 9 independently is aliphatic, alkyl, alkoxy, haloalkyl, N0 2 , OH, aryl, alkenyl, alkenyloxy, SO2R 10 , or N(R 10 ) 2 where each R 10 independently is alkyl, and n is from 0 to 5.
  • the com ound has a formula XA1:
  • n independently is hydrogen, -(CH 2 ) P -0-CO-R or -(CH 2 ) P -CO-R, or aliphatic, such as alkyl, where p is 1 to 6, and R is hydrogen, aryl or aliphatic, such as alkyl; -OM can also be a salt of form -O " A + , where A + is a cation; and Z is hydrogen, aryl, aliphatic, such as alkyl.
  • Z may be an alkyl group having a structure X-(CH 2 ) n - where n is an integer from 0 to 20, such as from 5 to 15, or 8 to 12, and X is hydrogen or halogen. In certain examples, n is 8.
  • R 1 is hydrogen or aliphatic, such as alkyl as described above and R 2 is independently hydrogen, hydroxyl, or halogen. In certain embodiments, R 2 can be fluorine or chlorine.
  • the compound is selected from:
  • the compounds disclosed herein are useful in the treatment of diseases and conditions, such as cancer. Such compounds can be administered by any routine method, in therapeutically effective amounts. In addition, more than one dose of the compound can be administered, such as every other day, every day, weekly, bi-weekly, monthly, or bi-monthly.
  • adenocarcinomas i.e. carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • Adenocarcinomas can be classified according to the predominant pattern of cell arrangement, as papillary, alveolar, etc., or according to a particular product of the cells, as mucinous adenocarcinoma.
  • Adenocarcinomas arise in several tissues, including the colon, kidney, breast, cervix, esophagus, gastric, pancreas, prostate and lung.
  • the disclosed compounds can be used in the treatment or prevention of lung cancer, such as a lung cancer with a KRAS mutation, such as one at amino acid position 12, 13 or 61.
  • a KRAS mutation such as one at amino acid position 12, 13 or 61.
  • Lung adenocarcinomas harboring KRAS mutations in contrast to those with EGFR and EML4-ALK mutations, have not yet been successfully targeted.
  • One or more of the disclosed lipophilic bisphosphonate compounds in combination with an autophagy inducer, such as rapamycin, can be used to treat such tumors (or other KRAS mutant tumors).
  • Lipophilic bisphosphonates inhibit both farnesyl and geranylgeranyldiphosphate synthases, effectively blocking prenylation of the KRAS and other small G-proteins critical for tumor growth and cell survival.
  • Bisphosphonate treatment of cells initiated autophagy, and rapamycin, in addition to inhibiting the mTOR pathway, facilitated autophagy and prevented p62 accumulation-induced NF-KB activation and tumor cell proliferation.
  • Lung adenocarcinomas account for about 50% of all non-small cell lung cancers (NSCLC), the most common type of human malignancy and a leading cause of cancer-related mortality worldwide.
  • NSCLC non-small cell lung cancers
  • KRAS mutations which are commonly found in smokers and Caucasian patients, are not effectively targeted by currently available therapeutics and have low survival rates, as well as frequent drug resistance (4).
  • KRAS mutations at amino acid positions 12, 13 or 61 are widely found in human pancreatic, thyroid, lung and colorectal cancers (5). They typically impair GTPase activity and lead to constitutive activation of downstream signaling pathways. It is therefore difficult to develop potent KRAS mutant- specific inhibitors that can directly restore intrinsic GTPase activity, although specific inhibitors of KRAS G12C have recently been reported (6), as have attempts to interfere with mutated KRAS function by altering its membrane localization; inhibiting its downstream effectors, as well as searching for synthetic lethality (7, 8).
  • FPP farnesyldiphosphate
  • GGPP geranylgeranyl diphosphate
  • Bisphosphonates are used to treat a variety of bone resorption diseases and function by blocking FPPS activity in osteoclasts.
  • the lipophilic bisphonates disclosed herein do not bind substantially to bone mineral, but maintain inhibitory activity against both FPPS as well as GGPPS (17), both of which can provide membrane anchoring 15- and 20-carbon isoprenoid chains for KRAS post-translational modification.
  • the compounds described herein can be used to prepare therapeutic pharmaceutical compositions.
  • the compounds may be added to the compositions in the form of a salt or solvate.
  • administration of the compounds as salts may be appropriate.
  • Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a-ketoglutarate, and b- glycerophosphate.
  • Suitable inorganic salts may also be formed, including hydrochloride, halide, sulfate, nitrate, bicarbonate, and carbonate salts.
  • Pharmaceutically acceptable salts may be obtained using procedures known to persons of ordinary skill in the art, for example by reacting a sufficiently basic compound, such as an amine, with a suitable acid to provide a physiologically acceptable ionic compound.
  • Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be prepared by analogous methods.
  • the compounds of the formulas described herein can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human or veterinary patient, in a variety of forms.
  • the forms can be specifically adapted to a chosen route of administration, e.g., oral or parenteral administration, by intravenous, intramuscular, topical or subcutaneous routes.
  • the compounds described herein may be systemically administered in combination with a pharmaceutically acceptable vehicle, such as an inert diluent or an assimilable edible carrier.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • compounds can be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the food of a patient's diet.
  • Compounds may also be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations typically contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations can vary and may conveniently be from about 2% to about 60% of the weight of a given unit dosage form.
  • the amount of active compound in such therapeutically useful compositions is such that an effective dosage level can be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain one or more of the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate.
  • binders such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate.
  • a sweetening agent such as sucrose, fructose, lactose or aspartame
  • a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring
  • the unit dosage form When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and flavoring such as cherry or orange flavor. Any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and devices.
  • the active compound may be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, or mixtures thereof, or in a pharmaceutically acceptable oil. Under ordinary conditions of storage and use, preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injection or infusion can include sterile aqueous solutions, dispersions, or sterile powders comprising the active ingredient adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants.
  • microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomersal, and the like.
  • isotonic agents for example, sugars, buffers, or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by agents delaying absorption, for example, aluminum monostearate and/or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • methods of preparation can include vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • compounds may be applied in pure form, e.g., when they are liquids.
  • a dermatologically acceptable carrier which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like.
  • Useful liquid carriers include water, dimethyl sulfoxide (DMSO), alcohols, glycols, or water-alcohol/glycol blends, in which a compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses, or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • compositions for delivering active agents to the skin are known to the art; for example, see U.S. Patent Nos. 4,992,478 (Geria), 4,820,508 (Wortzman), 4,608,392 (Jacquet et al.), and 4,559,157 (Smith et al.).
  • Such dermatological compositions can be used in combinations with the compounds described herein where an ingredient of such compositions can optionally be replaced by a compound described herein, or a compound described herein can be added to the composition
  • Useful dosages of the compounds described herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Patent No. 4,938,949 (Borch et al.).
  • the amount of a compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular compound or salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will be ultimately at the discretion of an attendant physician or clinician.
  • the compound can be conveniently administered in a unit dosage form, for example, containing 5 to 1000 mg/m 2 , conveniently 10 to 750 mg/m 2 , most conveniently, 50 to 500 mg/m 2 of active ingredient per unit dosage form.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
  • compositions illustrate exemplary pharmaceutical dosage forms that may be used for the therapeutic or prophylactic administration of a compound of a formula described herein, a compound specifically disclosed herein, or a pharmaceutically acceptable salt or solvate thereof (hereinafter referred to as 'Compound X'):
  • fonnulations may be prepared by conventional procedures well known in the pharmaceutical art. It will be appreciated that the above pharmaceutical compositions may be varied according to well-known pharmaceutical techniques to accommodate differing amounts and types of active ingredient 'Compound X'. Aerosol formulation (vi) may be used in conjunction with a standard, metered dose aerosol dispenser. Additionally, the specific ingredients and proportions are for illustrative purposes. Ingredients may be exchanged for suitable equivalents and proportions may be varied, according to the desired properties of the dosage form of interest.
  • the disclosed pharmaceutical compositions can further include one or more additional therapeutic agents.
  • the disclosed methods can further utilize one or more additional therapeutic agents.
  • Exemplary therapeutic agents that can be used in the disclosed methods (which are administered at effective amounts) or compositions include, but are not limited to: antineoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents.
  • Other examples include microtubule binding agents, DNA intercalators or cross -linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators.
  • Exemplary chemotherapeutic agents are described in Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.
  • Microtubule binding agent refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division.
  • microtubule binding agents that can be used in conjunction with the methods and compositions provided herein include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and are known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No.
  • Taxoids such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Patent Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.
  • the additional therapeutic agent includes one or more of: DNA and/or RNA transcription regulators, including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof.
  • DNA intercalators and cross-linking agents that can be used include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof.
  • DNA synthesis inhibitors suitable for use include, without limitation, methotrexate, 5- fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof.
  • suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof.
  • Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5- azacytidine, 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.
  • Kinase inhibitors include Gleevac, Iressa, and Tarceva that prevent phosphorylation and activation of growth factors.
  • the chemotherapy drug is epirubicin, topotecan, irinotecan, gemcitabine, iazofurine, valspodar, mitoxantrone, or Doxil (liposome encapculated doxiorubicine).
  • the drug is adriamycin, apigenin, zebularine, cimetidine, theophylline, or a derivative or analogs thereof.
  • the additional therapeutic agent is a biologic agent (e.g. , mAb) or a small molecule, such as those shown in the table below:
  • CD33 Acute myelogenous leukemia Gemtuzumab (Mylotarg, for example in combination with calicheamicin therapy)
  • CEA colorectal cancer some CEA-scan (Fab fragment, approved
  • Alpha-fetoprotein hepatocellular carcinoma ab75705 available from Abeam
  • AFP other commercially available AFP
  • TAG72 adenocarcinomas including B72.3 FDA-approved monoclonal
  • Vascular Colorectal cancer Bevacizumab (Avastin®)
  • the additional therapeutic agent is one or more of the following:
  • antibiotic e.g. , penicillin, ampicillin, metronidazole, tetracycline, chloramphenicol, tobramycin, cipro, and the like
  • anti-hypertensive drug e.g. , thiazide diuretics, ACE inhibitors, calcium channel blockers, beta blockers, and angiotensin II receptor antagonists
  • antidepressant e.g.
  • SSRIs selective serotonin reuptake inhibitors
  • SNRIs serotonin-norepinephrine reuptake inhibitors
  • TCA tricyclic antidepressant
  • MAOI monoamine oxidase inhibitor
  • buprenorphine tryptophan
  • antipsychotics and St John's wort, for example prozac
  • analgesics e.g.
  • acetaminophen non-steroidal anti-inflammatory drugs (NSAIDs), COX-2 inhibitor, and opioid drugs such as morphine, codeine, and oxycodone), reproductive hormone (e.g., estrogen, testosterone, and progesterone), blood thinners (e.g., warfarin), steroid (e.g. , prednisone), immunosuppressant (e.g. , rapamycin, cyclosporine, and methotrexate, azathioprine, rituximab, or a steroid), or cytokine (e.g. , GM-CSF) and other prescription drugs.
  • reproductive hormone e.g., estrogen, testosterone, and progesterone
  • blood thinners e.g., warfarin
  • steroid e.g. , prednisone
  • immunosuppressant e.g. , rapamycin, cyclosporine, and methotrexate, azathioprine
  • Exemplary additional therapeutic compounds include, but are not limited to, one or more of Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Adriamycin (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afinitor (Everolimus), Aldara (Imiquimod), Aldesleukin, Alemtuzumab, Alimta (Pemetrexed Disodium), Aloxi (Palonosetron Hydrochloride), Ambochlorin (Chlorambucil), Amboclorin (Chlorambucil), Aminolevulinic Acid, Anastrozole, Aprepitant, Arimidex (Anastrozole), Aromasin
  • Bendamustine Hydrochloride BEP, Bevacizumab, Bexarotene, Bexxar (Tositumomab and 1 131 Iodine Tositumomab), Bleomycin, Bortezomib, Bosulif (Bosutinib), Bosutinib, Brentuximab, Vedotin, Cabazitaxel, Cabozantinib-S-Malate, CAF, Campath (Alemtuzumab), Camptosar (Irinotecan Hydrochloride), Capecitabine, CAPOX, Carboplatin, CARBOPLATIN-TAXOL, Carfilzomib, CeeNU (Lomustine), Cerubidine (Daunorubicin Hydrochloride), Cervarix
  • Cosmegen Dactinomycin
  • Crizotinib CVP
  • Cyclophosphamide Cyclophosphamide
  • Cyfos Ifosfamide
  • Cytarabine Cytarabine, Liposomal, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin, Diftitox, Denosumab, DepoCyt (Liposomal Cytarabine), DepoFoam (Liposomal Cytarabine), Dexrazoxane Hydrochloride, Docetaxel, Doxil
  • Dox-SL Doxorubicin Hydrochloride Liposome
  • DTIC-Dome Dacarbazine
  • Efudex Fluorouracil
  • Elitek Rasburicase
  • Ellence Epirubicin Hydrochloride
  • Erlotinib Hydrochloride Erwinaze (Asparaginase Erwinia chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate),
  • Fludarabine Phosphate Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI, FOLFIRI-BEVACIZUMAB, FOLFIRINOX, FOLFOX, Folotyn (Pralatrexate), FU-LV, Fulvestrant, Gardasil (Recombinant HPV Quadrivalent Vaccine), Gefitinib, Gemcitabine Hydrochloride, GEMCIT ABINE-CISPLATIN, Gemtuzumab
  • Nanoparticle Formulation Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nolvadex (Tamoxifen Citrate), Nplate (Romiplostim), Ofatumumab, Omacetaxine,
  • Platinol (Cisplatin), Platinol-AQ (Cisplatin), Plerixafor, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag)
  • Vinorelbine Tartrate Vinorelbine Tartrate, Vismodegib, Voraxaze (Glucarpidase), Vorinostat, Votrient (Pazopanib Hydrochloride), Wellcovorin (Leucovorin Calcium), Xalkori (Crizotinib), Xeloda
  • Capecitabine XELOX, Xgeva (Denosumab), Xtandi (Enzalutamide), Yervoy (Ipilimumab), Zaltrap (Ziv-Aflibercept), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zinecard (Dexrazoxane Hydrochloride), Ziv-Aflibercept, Zoledronic Acid, Zolinza (Vorinostat), Zometa (Zoledronic Acid), Zytiga (Abiraterone Acetate) or an autophagy inducer.
  • Example 1 illustrates the disclosure and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the disclosure could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the disclosure.
  • Example 1 illustrates the disclosure and should not be construed as to narrow its scope. One skilled in the art will readily recognize that the Examples suggest many other ways in which the disclosure could be practiced. It should be understood that numerous variations and modifications may be made while remaining within the scope of the disclosure.
  • L2 cells were derived from primary culture of lung tumors isolated from KRASG12D/+ mice (C57B1/6 background) infected with CA2Cre-shp53 lentiviral vector.
  • 10 6 L2 cells were implanted into flank region at both sides. Treatment was started when decent tumor mass was palpable (day 18).
  • Mice were randomly divided into four treatment groups (5 mice each group): saline control, BPH-1222 (2 mg/kg in saline, every other day), rapamycin (2.5 mg/kg in 75% DMSO + 25% saline, every other day) and BPH-1222 plus rapamycin. Treatment was given for total 21 days and mice were harvested after that.
  • Tumors from saline control and Rapal222 groups were fixed and processed for pathological analysis. Tumor sections were stained with Ki67 antibody as a marker for cell proliferation (Red:ki67, blue: DAPI). Ki67 positive rate was counted at 10 independent areas. Necrotic area was quantified by Image J.
  • FIGS. 1A-1D and FIGS. 2A-2C illustrate the anticancer effects of bisphosphonates, such as BPH-1222, in a KRAS NSCLC xenograft model.
  • Bisphosphonates such as zoledronate (1) activate ⁇ T cells by inhibiting the enzyme farnesyldiphosphate synthase (FPPS). This results in accumulation of the FPPS substrates, isopentenyldiphosphate (IPP) and dimethylallyldiphosphate (DMAPP), both of which are
  • zoledronate has essentially no effect on the intra-erythrocytic form of the malaria parasites, since it is poorly membrane permeable.
  • GGPPS Plasmodium geranylgeranyldiphosphate synthase
  • This enzyme is unusual in that is structurally more similar to human FPPS than human GGPPS and, unlike human GGPPS, is potently inhibited by bisphosphonates. Inhibiting GGPPS in the parasite blocks formation of protein prenylation as well as carotenoid, menaquinone and vitamin E formation (FIG. 3), and results in "direct" parasite killing.
  • a lipophilic bisphosphonate was sought that would kill malaria parasites as well as activate ⁇ T cells, a possible route to malaria chemo-immunotherapy.
  • PvGGPPS Plasmodium vivax GGPPS
  • Vy2V52 T-cell activators that kill malaria parasites in vitro (and in vivo) were found, providing a combined chemo- immunotherapeutic approach to the development of anti-malarials in which both host innate immunity (host FPPS inhibition/ ⁇ T cell activation/ TNF-a and granulysin-mediated killing of liver stages and merozoites) (FIG. 6) as well as direct killing (via parasite GGPPS inhibition, carotenoid, menaquinone and vitamin E biosynthesis inhibition) are targeted by a single molecule.
  • host FPPS inhibition/ ⁇ T cell activation/ TNF-a and granulysin-mediated killing of liver stages and merozoites FIG. 6
  • direct killing via parasite GGPPS inhibition, carotenoid, menaquinone and vitamin E biosynthesis inhibition
  • a single colony that carried pET28a-(His)6HsFPPS 6-353 was inoculated in 100 mL LB broth with 50 ⁇ g/mL kanamycin and incubated at 37°C overnight. 10 mL of inoculated cells were added into 1L LB broth with 50 ⁇ g/mL kanamycin and incubated until the OD600 reached about 0.6-0.8. Cells were induced by ImM IPTG and incubated at 24 °C for at least 16 hours, then centrifuged, and the pellets frozen at -80 °C.
  • the cell pellets were then thawed in wash buffer (10 mM HEPES, pH 7.5, 500 mM NaCl and 35 mM imidazole) with addition of Benzonase (EMD Millipore) and EDTA-free protease cocktail (Roche). Thawed cells were sonicated (10 seconds active, 20 seconds rest, for 10 minutes) then centrifuged at 23,000 rpm for 30 minutes. The supernatant was loaded on a Ni-NTA column and eluted with 0-100 % elution buffer (10 mM HEPES, 500 mM NaCl and 500 mM imidazole).
  • Human FPPS inhibition assays Human FPPS inhibition assays were carried out using 96 well plates with 200 ⁇ ⁇ reaction mixture in each well. The condensation of geranyldiphosphate (100 ⁇ final) and isopentenyldiphosphate (100 ⁇ final) was monitored at room temperature by using a continuous spectrophotometric assay for phosphate-releasing enzymes.
  • the reaction buffer contained 50 mM Tris-HCl (pH 7.4), 1 mM MgC12, and 0.01 % Triton X100.
  • the compounds investigated were pre-incubated with enzyme for 30 minutes at room temperature. The IC50 values were obtained from fitting dose-response curve using Prism 4.0 (GraphPad Software, Inc., La Jolla, CA, www.graphpad.com). Tumor cell MTT assay
  • Human tumor cell line MCF-7 (breast adenocarcinoma) was obtained from the National Cancer Institute.
  • Lung cancer cell line #4, #6 and L2 were derived from mouse primary lung adenocarcinomas which harboring KRASG12D mutation and p53 knockdown.
  • Cell lines were cultured in RPMI-1640 or DMEM medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (Gibco, Grand Island, NY) at 37 °C in a 5% C0 2 atmosphere with 100% humidity.
  • Compound stock solutions were typically prepared in water at a concentration of 0.02 M.
  • a broth microdilution method was used to determine the bisphosphonate growth inhibition IC50 values.
  • Mouse lung cancer cell line L2 was used for xenograft model in C57B16/J mice. Eight- week old female mice were transplanted subcutaneously with 10 6 tumor cells in the flank region. Tumor sizes were measured with calipers twice a week after the transplantation and treatment was started when decent tumor mass was palpable. Mice were randomly divided into four treatment groups: saline control, BPH-1222 (2mg/kg, every other day), rapamycin (2.5mg/kg, every other day) and BPH-1222 plus rapamycin. Mice were sacrificed after 21 days of treatment and tumors were dissected, weighted and analyzed. Cell proliferation was examined by Ki-67 staining on the tumor sections (FIG. 2B). Necrotic area was quantified by Image J software on H&E staining sections.
  • P. vivax GGPPS expression A clone encoding P. vivax GGPPS (PlasmoDB gene ID:
  • P. vivax GGPPS inhibition assays The P. vivax GGPPS inhibition assays were carried out by using 96-well plates with 200 ⁇ ⁇ of reaction mixture in each well. The condensation of geranyldiphosphate ( ⁇ ) with isopentenyldiphosphate (100 ⁇ ) was monitored at room temperature by using a continuous spectrophotometric assay for phosphate -releasing enzymes in a reaction mixture containing 50 mM Tris-HCl (pH 7.4), 1 mM MgCl 2 , and 0.01% Triton X100.
  • Vy2V52T cell activation was assessed by TNF-a release as described previously. Briefly, the CD4+ Vy2V52T cell clone, JN.23, was stimulated with bisphosphonates in the presence of the antigen presenting cell line, CP.EBV (an EBV
  • P. falciparum growth inhibition assays were carried out as described in previous work. Briefly, a P. falciparum culture was adjusted to 2% hematocrit, 0.5% parasitemia, then dispensed by a WellMate (Thermo) into 384 well plates (Greiner) containing the compounds (final volume 50 and incubated for 72 hours.
  • Chloroquine, artemisinin, and DMSO were used within the assay plates to serve as controls.
  • a parasite lactate dehydrogenase (pLDH) assay was used to assess compound efficacy.
  • the plates were frozen overnight at -20 °C. After thawing, the plates were shaken for 45 seconds at 1,700 rpm in a Mix Mate (Eppendorf) of the lysate transferred into the corresponding well of another plate containing 30 alstat Reagent and incubated for 2 hours.
  • the absorbance (650 nm) was read using a Spectramax M5 (Molecular Devices).
  • IC50 values were obtained from fitting the dose-response curve using Prism 4.0 (GraphPad Software, Inc., La Jolla, CA, www.graphpad.com).
  • the compounds shown in Table 5 also have potent activity against a human breast adenocarcinoma cell line.
  • This example provides the materials and methods for the Examples below.
  • Tumor size was monitored 1-2 times every week during the treatment, by either palpation or in vivo lucif erase imaging. At least 5 mice were used for each group (either control or treatment). All the treatment experiments were repeated at least twice independently. Mice used for control and treatment were randomly grouped from a pool of model mice.
  • Lipophilic bisphosphonates were synthesized as described above.
  • Protein farnesyltransferase and geranylgeranyltransferase inhibitors (FTI-277 and GGTI-298) were purchased from Calbiochem.
  • MEK inhibitor U0126 was purchased from Cell Signaling.
  • FOH Farnesol
  • GGOH geranylgeraniol
  • ascorbic acid Trolox
  • Lipoic acid Lipoic acid
  • Morin hydrate Lipoic acid
  • chloroquine diphosphate salt was from Sigma.
  • Simvastatin was from Tokyo Chemical Industry Co. Rapamycin was from Alfa Aesar.
  • Mouse lung cancer cell lines (6#, L2 and M3L2) were derived from primary tumors of LSL-KRAS G12D/+ mice infected with CA2Cre-shp53 lentiviral vector.
  • L2 and M3L2 cells were derived from mouse tumors with pure C57B and pure FVB background respectively, so that they form syngeneic grafts in matched recipient mice.
  • L2 cells were stably infected with 5XKB-1UCI lentiviral vector.
  • Human cancer cell lines (A549, A427, Panc-1 and MiaPaCa2) were from ATCC.
  • MEFs were prepared from mouse embryo with matched genetic background and immortalized by shRNAs against p53 and Rbl. Cell survival after drug treatment was measured using Cell Proliferation Reagent (Wst-1) from Roche or MTT Cell Proliferation Assay Kit (30-1010K) from ATCC. Dose-response curves and corresponding IC50S were fitted using GraphPad Prism. Lentiviral vector mediated mouse lung cancer model and syngeneic graft model. LSL-
  • KRAS Ql2DI+ Rosa26 ladlac mice were used for the lentiviral vector (CA2Cre-shp53) mediated lung cancer model.
  • Tumor size was monitored by in vivo luciferase imaging system (IVIS 100) from Caliper Lifesciences. Syngeneic graft experiments were done by either subcutaneously transplanting 10 6 L2 tumor cells in the flank region of C57B mice, or tail vein injection of 2xl0 5 M3L2 cells into FVB mice. Subcutaneous tumor size was measured every 7 days after the transplantation. For single drug treatment, animals were given the drug every day. For combination therapies, animals were given BPH-1222 on days 1, 3, 5 and the other drug on days 2, 4, 6, alternately.
  • BPH-1222 (2 mg/kg) and chloroquine (60 mg/kg) were diluted in PBS while rapamycin (2.5 mg/kg) in 75% DMSO and 25% PBS. All drugs were given i.p. in a volume of 100 ⁇ . All mice studies were carried out according to the protocols that were approved by the Institutional Animal Care and Use Committee of Salk Institute.
  • Antibodies were purchased from Millipore (SPC, 1:2000), Cell Signaling (phos- ERK, total ERK, phos-AKT, total AKT, phos-MEK, phos-c-RAF, Cleaved Caspase-3, CHOP, BiP, phos-PERK, LC3MI, phos-p70 S6K, phos-4E-BPl, all 1: 1000), Abgent (p62, clone 2C11, 1:2000), Vector Labs (Ki-67, 1:500), and Santa Cruz Biotechnology (KRAS, HRAS, RAP1A, HDJ2, ACTIN, all 1: 1000).
  • RNA isolated from the treated cells was reverse transcribed using Superscript III system (In vitro gen) with random primers.
  • Quantitative PCR was performed in triplicate using 7900HT Fast Real-Time PCR system with SYBR green method (Applied Biosystems). Results were analyzed for the relative expression of mRNAs normalized against GAPDH and cyclophilin.
  • a list of primers used for PCR is in Table 6.
  • Pharmacokinetics test Pharmacokinetic studies were performed using 3 female SD rats (230- 240 g body weight). Plasma concentrations were measured at 1 minute, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, and 48 hours following a single i.v. injection of BPH-1222 at 5 mg/kg. 0.3 ml blood was taken each time. Data were analyzed using DAS2.0 software.
  • Bisphosphonates inhibit FPPS and GGPPS activity
  • FIG. 7A-7D A library of 30 synthetic analogs of zoledronate was tested (FIGS. 7A-7D) for growth inhibition of two KRAS mutant cell lines (6#, L2) and of control mouse embryonic fibroblasts (MEF). Most anti-growth activity was found with BPH-1222, a zoledronate analog having a C» side-chain and a 1-OH group (about 1 ⁇ ICso, FIG. 8A and FIG. 9A and 9B). Compounds with very short or very long chains inhibited growth the least while BPH-1222 and other intermediate chain length compounds had the most activity.
  • In vitro inhibitory activities against human FPPS Ki as low as approximately 1 nM
  • FPPS was an in vivo target (FIG. 8B and Table 8 and FIG. 8C).
  • the lipophilic bisphosphonates also inhibit human GGPPS (FIG. 8B and FIG. 9C and Table 4).
  • the intermediate chain length species possess the best inhibitory activity for GGPPS, a K ; of -300 nM for BPH-1222.
  • dual FPPS/GGPPS-targeting bisphosphonates (such as BPH-1222) were expected to be particularly potent since formation of the FPP substrate for GGPP biosynthesis was blocked by FPPS inhibition, with GGPP production being particularly important for cell survival. While inhibition of FPPS was expected to have effects on diverse metabolic pathways (such as sterol and steroid biosynthesis), these effects were not thought to be the major ones responsible for cell death since the BPH-1222 effects were reversible upon GGOH addition.
  • BPH-1222 was more toxic to cells harboring KRAS mutations, such as cell lines derived from a mouse model of KRAS-induced lung adenocarcinoma, as well as in mouse embryonic fibroblasts transformed by KRAS in vitro (FIGS. 8D and 8E).
  • these results indicated that blocking protein prenylation by lipophilic bisphosphonates could be used as a targeted therapy for cancer cells that carry KRAS mutations, since potentially both FPPS as well as GGPPS could be targeted; the lipophilic bisphosphonates had much better clogP values than did more conventional bisphosphonates; and furthermore, they did not bind to bone mineral, which rapidly removed them from the circulatory system (24).
  • Bisphosphonates block KRAS prenylation and induces its degradation
  • KRAS addiction has been shown in a mouse lung cancer model using inducible KRAS G12D (25), so given the observation that cells bearing KRAS mutations were more sensitive to bisphosphonate treatment, there was interest in determining if the lipophilic bisphosphonates blocked KRAS prenylation.
  • a cell fractionation assay was used to check KRAS protein prenylation status, since unprenylated KRAS proteins lose their ability to avidly associate with cell membranes and, consequently, appear in the cytosolic fraction (26).
  • BPH-1222 treatment robustly inhibited protein farnesylation as well as protein geranylgeranylation, as indicated by
  • BPH- 1222 treatment greatly reduced the amount of GTP-bound KRAS (representing the active form of the KRAS protein), and led to down-regulation of the AKT pathway as well as the activation of apoptosis as shown by increase in Caspase-3 cleavage (FIG. IOC).
  • Table 9 Summary of protein prenylation inhibited by various compounds.
  • Bisphosphonates enhance ER stress and initiate autophagy
  • Cancer cells usually exhibit a "stress phenotype” that consists of replicative stress, mitotic stress, metabolic stress, oxidative stress and proteotoxic or ER (endoplasmic reticulum) stress. They are, therefore, vulnerable to further enhancement of these stresses by chemotherapy (29).
  • Bisphosphonates as demonstrated above, potently block protein prenylation by eliminating the source of isoprenoid chains and lead to the accumulation of incorrectly folded proteins, inducing the so-called Unfolded Protein Response (UPR), or ER stress, as is also observed when cells are treated with HMG-CoA reductase inhibitors (30, 31).
  • Rapamycin but not chloroquine, sensitizes tumor cells to bisphosphonates in vivo
  • This example describes methods used to determine if the disclosed compounds can be used in combination with inhibitors of autophagy and/or with GGPPS inhibitors.
  • KRAS-driven tumor cells depend on autophagy to help reduce reactive oxygen species
  • NF-KB activity in tumors may stimulate cell proliferation (37), and this appeared to be the case in tumor samples treated with BPH-1222 plus chloroquine (FIGS. 13E and 13F), although these tumors also showed increased number of apoptotic cells (FIGS. 13E and 13G). Without being bound to a particular theory, it is suggested that the elevated NF- ⁇ activity and cell
  • FIGS. 13B and 13E indicated that a combination therapy using a lipophilic bisphosphonate together with the autophagy inducer rapamycin might be more effective in treating KRAS induced lung adenocarcinomas. It was found that in the L2 cell syngeneic graft model, the combination of BPH-1222 with rapamycin was indeed much more effective than either single agent acting alone (FIG. 13H). Additionally, rapamycin also potently blocked phosphorylation of the two mTOR substrates (p70 S6 kinase and 4E-BP1), both of which are important for boosting metabolism in cancer cells (FIG. 131).
  • Bisphosphonate and rapamycin combination therapy potently suppresses tumor growth in lung cancer models
  • the BPH-1222 plus rapamycin combination was tested for treating lung adenocarcinoma in both the syngeneic orthotopic graft model and the lentiviral vector-mediated model, which represent the lung microenvironment.
  • the orthotopic model tail veil injection of 2xl0 5 M3L2 cells reproducibly generated massive lung tumors in FVB mice, resulting in a median survival of 33 days.
  • BPH-1222 had good pharmacokinetic properties (FIG.
  • combination therapy of BPH-1222 and rapamycin was given to 12 mice around 3 months after lentiviral infection (counted as Day 1 in FIGS. 15D and IF), for a total of 16 doses of each compound.
  • the tumor load of each mouse was monitored every 9-10 days throughout the whole treatment.
  • Tumor regression was observed in most of the mice during treatment, although tumor size did increase after treatment ceased (FIGS. 15D, 15E and 17).
  • Tumors from treated mice showed a large reduction in cell proliferation as indicated by Ki-67 staining, although apoptotic cells were rarely found (FIGS. 15G and 15H).
  • IKK2 inhibitor that suppressed tumor progression by reducing ERK signaling, there was no significant change of ERK phosphorylation after the bisphosphonate plus rapamycin treatment, in vivo
  • FIGS. 15G and 15H BPH-1222 (either alone or combined with other agents) slightly increased ERK phosphorylation in cell culture conditions (FIG. 11C). A combination with an ERK inhibitor was tested to see if it might further improve treatment efficacy. However, ERK inhibitor U0126 increased KRAS protein, c-RAF, MEK and AKT phosphorylation levels under all conditions tested (FIG. 12D), perhaps due to the interruption of the ERK negative feedback loop (38).
  • zoledronate itself has been shown to potentiate the killing of osteosarcoma cells by RAD001 (a rapamycin analogue, 39) and, although less potent than BPH-1222, zoledronate still synergizes with rapamycin in vivo for killing KRAS tumors (FIGS. 14D and 14E). This indicates zoledronate plus rapamycin (or an analog) can be used for treating lung adenocarcinoma.
  • RAS mutations are commonly found in a variety of human cancers including lung, colon and pancreatic cancers, and in this study, the efficacy of treating lung adenocarcinomas carrying a KRAS mutation with a combination of a lipophilic bisphosphonate, an analog of zoledronate, with rapamycin were investigated.
  • Bisphosphonates are a class of drugs widely used for treating osteoporosis and for preventing bone metastasis of certain cancers (40). Mechanistically, they tightly bind to bone mineral and inhibit FPPS in osteoclasts. This results in impaired protein prenylation and function, inducing cell death of osteoclasts.
  • Targeting protein prenylation has been pursued for more than 20 years, ever since researchers first found that RAS requires post- translational prenylation for its malignancy-transforming activity. FTIs and GGTIs were developed early on to kill tumor cells in vitro, however, little success has been achieved using these compounds in animals. More interestingly, responses to these inhibitors did not always correspond to RAS mutation status. This observation strongly indicated the existence of other targets (27). Herein, it is shown that KRAS prenylation and activity was largely inhibited by lipophilic bisphosphonate treatment and suggested that this was one of the major mechanisms of action of this class of compounds.
  • G-proteins such as RAL, RHO, RAC and CDC42, require exclusively geranylgeranylation, and all of these proteins have been shown to be involved in RAS induced transformation in a context-dependent manner (15, 43-45).
  • KRAS tumors cells have been shown to rely on autophagy for providing metabolic intermediates and clearing excess reactive oxygen species (ROS) (32, 33).
  • ROS reactive oxygen species
  • the combination of a lipophilic bisphosphonate with rapamycin was far more effective because rapamycin not only facilitated autophagy, but also inhibited the mTOR pathway that is critical for the tumor cell survival.
  • the combination of a lipophilic bisphosphonate plus rapamycin offers a promising therapeutic lead for treating KRAS-related lung cancers.
  • Pancreatic cancer most commonly pancreatic ductal adenocarcinoma (PDAC), is the seventh cause of cancer deaths in 2012 globally and the forth in the United States. KRAS mutation is found in >90 of the PDAC patients so that anti-KRAS therapeutics is extremely attractive to this field.
  • PDAC pancreatic ductal adenocarcinoma
  • Ctrl control group receiving saline only.
  • Rapal222 mice were treated with BPH-1222 (1 mg/kg, i.p.) and rapamycin (2.5 mg/kg, i.p.) every other day, alternately, for total 3 weeks.
  • mice were treated with BPH-1222 (1 mg/kg, i.p.) and everolimus (2.5 mg/kg, i.p.) every other day, alternately, for total 3 weeks.
  • PDAC p53.2.2.1 cells were orthotopically transplanted into pancreas tail of FVB mouse. Tumors were visualized on day 14 with IVIS imaging and mice were randomized into control and treatment groups. Treatment was given for 2 weeks (7 doses of BPH-1222, 1 mg/kg and 7 doses of rapamycin, 2.5 mg/kg). Mice were then euthanized and whole pancreases were isolated and weighed (FIG. 18).
  • E. Massarelli, M. Varella-Garcia, X. Tang, A. C. Xavier, N. C. Ozburn, D. D. Liu, B. N. Bekele, R. S. Herbst, Wistuba, II, KRAS mutation is an important predictor of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in non-small- cell lung cancer. Clin Cancer Res 13, 2890-2896 (2007).
  • Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 25, 460-470 (2011).
  • the signaling adaptor p62 is an important NF-kappaB mediator in
  • Zoledronic acid potentiates mTOR inhibition and abolishes the resistance of
  • osteosarcoma cells to RAD001 (Everolimus): pivotal role of the prenylation process. Cancer Res 70, 10329-10339 (2010).

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Abstract

L'invention concerne des composés bisphosphonates lipophiles et des modes de réalisation d'un procédé de fabrication de ceux-ci. Les composés peuvent inhiber les enzymes FPPS et/ou GGPPS. Les composés sont utiles pour le traitement d'une maladie, comme le cancer, par exemple les cancers présentant une mutation KRAS. Dans certains modes de réalisation, les composés sont utilisés pour prévenir ou traiter un cancer, tel qu'un cancer du poumon présentant une mutation KRAS.
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US11918574B2 (en) 2015-03-06 2024-03-05 Beyondspring Pharmaceuticals, Inc. Method of treating cancer associated with a RAS mutation
US11857522B2 (en) 2016-02-08 2024-01-02 Beyondspring Pharmaceuticals, Inc. Compositions containing tucaresol or its analogs
US11040027B2 (en) 2017-01-17 2021-06-22 Heparegenix Gmbh Protein kinase inhibitors for promoting liver regeneration or reducing or preventing hepatocyte death
WO2018137036A1 (fr) 2017-01-26 2018-08-02 The Royal Institution For The Advancement Of Learning / Mcgill University Composés bicycliques substitués à base de pyrimidine, compositions et utilisations associées
US11279719B2 (en) 2017-01-26 2022-03-22 Youla S. Tsantrizos Substituted bicyclic pyrimidine-based compounds and compositions and uses thereof
WO2018170485A1 (fr) * 2017-03-16 2018-09-20 The Board Of Trustees Of The Leland Stanford Junior University Méthodes diagnostiques et thérapeutiques pour des cancers kras-positifs
CN110573878A (zh) * 2017-03-16 2019-12-13 小利兰·斯坦福大学托管委员会 用于kras阳性癌症的诊断和治疗方法
WO2020163637A1 (fr) 2019-02-06 2020-08-13 Oregon Health & Science University Composés liés à des bisphosphonates
EP3920906A4 (fr) * 2019-02-06 2023-07-12 Oregon Health & Science University Composés liés à des bisphosphonates
WO2020198125A1 (fr) * 2019-03-22 2020-10-01 Icahn School Of Medicine At Mount Sinai Procédés de traitement d'un cancer colorectal
WO2021225908A1 (fr) * 2020-05-04 2021-11-11 Beyondspring Pharmaceuticals, Inc. Trithérapie pour améliorer la destruction de cellules cancéreuses dans des cancers à faible immunogénicité
WO2022031952A3 (fr) * 2020-08-07 2022-03-10 City Of Hope Traitements de cancers à mutations kras

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