US20140050696A1 - Novel bisaminoquinoline compounds, pharmaceutical compositions prepared therefrom and their use - Google Patents

Novel bisaminoquinoline compounds, pharmaceutical compositions prepared therefrom and their use Download PDF

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US20140050696A1
US20140050696A1 US14/114,049 US201214114049A US2014050696A1 US 20140050696 A1 US20140050696 A1 US 20140050696A1 US 201214114049 A US201214114049 A US 201214114049A US 2014050696 A1 US2014050696 A1 US 2014050696A1
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cancer
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Ravi K. Amaravadi
Jeffrey Winkler
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University of Pennsylvania Penn
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/42—Nitrogen atoms attached in position 4
    • C07D215/46—Nitrogen atoms attached in position 4 with hydrocarbon radicals, substituted by nitrogen atoms, attached to said nitrogen atoms
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/42—Nitrogen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel bisaminoquinoline compounds, pharmaceutical compositions comprising these novel compounds and methods for inhibiting autophagy in biological systems.
  • Methods of treating cancer in patients in need using compounds and/or compositions according to the present invention alone or in combination with at least one additional anticancer agent represent additional aspects of the invention.
  • the use of radiation therapy in combination with the present compounds, alone or in combination with an additional anticancer agent as otherwise disclosed herein, represents an additional aspect of the invention.
  • Methods of treating disease states and/or conditions in which inhibition of autophagy plays a favorable treatment role including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, with compounds according to the present invention represent additional aspects of the invention.
  • Autophagy consists of the sequestration of organelles and proteins in autophagic vesicles (AV) and degradation of this cargo through lysosomal fusion (1). Autophagy allows tumor cells to survive metabolic and therapeutic stresses (2-5). Multiple publications indicate therapy-induced autophagy is a key resistance mechanism to many anti-cancer agents. Chloroquine (CQ) (Compound 1, FIG. 1 ) derivatives block autophagy by inhibiting the lysosome (3, 6, 7). A randomized phase III trial of CQ versus placebo with carmustine and radiation in patients with glioma reported a trend towards a doubling in duration of survival in the patients treated with CQ (8).
  • CQ Chloroquine
  • the present invention relates to compounds according to the chemical structure I
  • R 1 and R 1′ are each independently H, halo (F, Cl, Br or I), CN, NO 2 , optionally substituted C 1 -C 6 alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally substituted O—C 1 -C 6 alkyl (preferably, OCH 3 ), optionally substituted C 2 -C 7 acyl (preferably acetyl) or optionally substituted C 2 -C 7 ester (oxycarbonyl ester or carboxyester, preferably carboxyester);
  • R and R′ are each independently H, a C 1 -C 6 optionally substituted alkyl group, a C 1 -C 7 (preferably C 2 -C 7 ) optionally substituted acyl group, a C 2 -C 7 optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R′ is bonded);
  • Z is O or N—R Z ;
  • R Z is H or an optionally substituted C 1 -C 3 alkyl group, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvent or polymorph thereof.
  • R 1 and R 1′ are each independently H, a halo group, a nitro group or a trifluoromethyl group, preferably a chloro group.
  • R and R′ are preferably each independently H, a C 1 -C 3 optionally substituted alkyl group itself preferably substituted with at least one hydroxyl group, an alkoxy group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with one or two 7-substituted-4-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with a R 1 and R 1′ group as broadly described for generic structure I above, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxy
  • L is a
  • X is N—R′′, Y and Y′ are each independently absent or CH 2 , and R′′ is H or a C 1 -C 3 alkyl group which is optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with one or two 7-substituted-4-quinolinyl group wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with R 1 and/or R 1′ as broadly described for generic structure I above, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine
  • a pharmaceutical composition comprises a compound according to formula I above or as otherwise described herein in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional anticancer agent.
  • Methods of inhibiting autophagy in a biological system, in particular a patient or subject is a further aspects of this invention.
  • a bisaminoquinoline compound as otherwise described herein is presented to the biological system, including administration to a patient or subject in need, in order to inhibit autophagy.
  • the resulting inhibition may be monitored or applied in the biological system to effect a favorable result, including the inhibition, treatment and/or prevention of cancer, including metastasis of cancer, or the inhibition, treatment and/or prevention of one or more disease states or conditions in which the inhibition of autophagy provides a favorable result including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, among others.
  • Methods of inhibiting, treating and/or reducing the likelihood of cancer, including metastasis of cancer and drug resistant cancer comprises administering to a patient in need at least one compound according to the present invention, optionally in combination with at least one additional anticancer agent as otherwise described herein.
  • the present invention also relates to treating, inhibiting and/or preventing diseases, diseases states and/or conditions in a patient in need in which the inhibition of autophagy provides a favorable outcome, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the method comprising administering to said patient at least one compound according to the present invention.
  • FIG. 1 Chemical structure of mono and bisaminoquinolines.
  • FIG. 2 Synthesis scheme for bisaminoquinolines
  • FIG. 3 Effects of Lys01-Lys04 on LC3 immunoblotting Immunoblotting and quantification of LC3II/LC3I ratio in lysates from LN229 cells treated for 4 hours.
  • the graphs show (mean+/ ⁇ SD) LC3II/LC3I ratios of each treatment normalized to the LC3II/LC3I ratio of control treated cells for each experiment.
  • FIG. 4 Autophagy inhibition and cytotoxicity of Lys01 compared to HCQ.
  • A Representative images of LN229 GFP-LC3 cells treated as indicated for 4 hours. White arrows: small puncta; red arrows: dense puncta. Graphs show mean+/ ⁇ SEM puncta/cell.
  • B Representative electron micrographs of LN229-GFP-LC3 cells treated (4 hours) with DMSO, HCQ 10 ⁇ M, or Lys01 10 ⁇ M Arrows: Autophagic vesicles.
  • C LC3 immunoblotting of LN229 cells treated for 24 hours as indicated; calculated ratio of LC3II/LC3I ratios for bafilomycin versus control co-treatment.
  • FIG. 5 Autophagy inhibition and cytotoxicity of Lys05, the water soluble salt of Ly01.
  • A Immunoblotting against LC3 and p62 in c8161 cells treated as indicated
  • B MTT assay in c8161 cells at 72 hours.
  • HCQ Hydroxychloroquine. Values presented are mean+/ ⁇ SD with 5 replicates per treatment condition. *No remaining cells for analysis.
  • FIG. 6 In vivo autophagy inhibition and antitumor activity of Lys05.
  • A Representative electron micrographs (12,000 ⁇ ) of c8161 xenograft tumors harvested after 2 days of daily i.p. treatment with PBS, HCQ 60 mg/kg, or Lys05 76 mg/kg. Arrows: autophagic vesicles; scale bar 2 ⁇ m
  • B Quantification of mean ⁇ SEM number of autophagic vesicles/cell from two representative tumors from each treatment group.
  • C-D 1205Lu xenografts were treated with PBS (blue), HCQ 60 mg/kg ip (green), or Lys05 76 mg/kg (red) i.p.
  • C Tumor volumes over 14 days
  • D Daily tumor growth rate.
  • E-G HT29 xenografts were generated in the flanks of nude mice and treated with PBS, Lys05 10 mg/kg ip daily, Lys05 40 mg/kg ip daily, or Lys05 80 mg/kg ip every 3/5 days.
  • E Average daily tumor growth rate
  • F Tumor volumes over 14 days
  • G Weight of excised tumors, *p ⁇ 0.05.
  • FIG. 7 Autophagy inhibition and tumor necrosis in melanoma and colon cancer xenografts treated with Lys05 or HCQ.
  • A Immunoblotting against LC3 in lysates from individual c8161 tumors treated as indicated with daily i.p. injections for 48 hours. Quantification of LC3II/Lc3Iratio (mean+/ ⁇ SEM)
  • B Tumor necrosis (arrows) in H&E stained sections of 1205Lu tumor xenografts harvested after 14 days of treatment; Electron micrographs (7000-12000 ⁇ ) of melanoma tumor cells.
  • FIG. 8 Toxicity associated with Lys05 76 mg ⁇ kg ip 3/5 days.
  • A Mice were lethargic with arched backs after 3 days of dosing.
  • B 3/10 mice developed bowel obstruction.
  • C Dysmorphic paneth cells (arrows) in the terminal ileum of one mouse.
  • FIG. 9 Lys05 treatment at the highest dose reproduces the intestinal phenotype of a genetic autophagy deficiency.
  • A-F Weight and intestines were analyzed for mice bearing HT29 xenografts treated with PBS, Lys05 10-80 mg/kg.
  • A Daily weight
  • B Representative excised gastrointestinal tracts after 14 days of treatment
  • C Representative images (40 ⁇ ) of hemotoxylin and eosin stained ileal crypts from mice bearing HT29 xenografts (14 days), (40 ⁇ ) arrows: paneth cells.
  • FIG. 11 Lys05 inhibits autophagy by accumulating in and deacidifying the lysosome.
  • 1205Lu cells treated with PBS, HCQ 10 ⁇ M, or Lys05 10 ⁇ M for 24 hours
  • harvested 1205 Lu xenograft tumors treated with PBS, HCQ 60 mg/kg i.p. 3/5 days, or Lys05 76 mg/kg i.p. 3/5 days for 14 days
  • LAMP2 immunoblotting confirmed isolation of concentrated lysosomes for analysis.
  • C Fluorescence imaging of 1205 Lu cells treated as indicated for 30 minutes and stained with Lysotracker Red. Lysotracker puncta (red) per cell was scored for three high powered fields. Blue: nuclear DAPI staining. Data presented is mean ⁇ SEM.
  • D Fluorescence imaging of c8161 cells treated as indicated for 24 hours and stained with acridine orange (AO): orange: aggregated AO, green: diffuse AO.
  • FIG. 12 High performance liquid chromatography tandem mass spectrometry assay for HCQ and Lys05. 1205Lu cells (24 hours) and 1205Lu tumors (14 days).
  • WC Whole cell homogenate
  • L Lysosomal subfraction
  • HCQ hydroxychloroquine
  • FIG. 13 Impairment of lysosomal enzymes and extralysosomal leakage associated with Lys05 treatment.
  • A Acid phosphatase activity and
  • B Cathepsin D immunoblotting in whole cell (white, WC) and lysosomal (Black; L) fractions of 1205 Lu cells treated with PBS, HCQ 10 ⁇ M, Lys05 10 ⁇ M for 24 hours.
  • Graphs show the mean+/ ⁇ SEM for three independent experiments.
  • FIG. 14 Chemical structures of synthesized compounds Lys06-Lys 12; The chemical structures of Lys06-Lys 112 are shown.
  • FIG. 15 Chemical structures of synthesized compounds Lys 13-Lys18; The chemical structures of Lys 13-Lys18 are shown.
  • FIG. 15A shows a number of additional bisaminoquinoline autophagy inhibitors under investigation.
  • FIG. 16 Table 1 provides MTT IC 50 values in LN229 for select compounds of the present invention as presented.
  • Table 12 provides IC 50 (M) values of a number of compounds according to the present invention in P. falciparum.
  • patient or “subject” is used throughout the specification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided.
  • treatment including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided.
  • patient refers to that specific animal.
  • the patient or subject of the present invention is a human patient of either or both genders.
  • compound is used herein to describe any specific compound or bioactive agent disclosed herein, including any and all stereoisomers (including diasteromers), individual optical isomers (enantiomers) or racemic mixtures, pharmaceutically acceptable salts and prodrug forms.
  • compound herein refers to stable compounds. Within its use in context, the term compound may refer to a single compound or a mixture of compounds as otherwise described herein. It is understood that the choice of substituents or bonds within a Markush or other group of substituents or bonds is provided to form a stable compound from those choices within that Markush or other group.
  • bioactive agent refers to any biologically active compound or drug which may be formulated for use in the present invention.
  • exemplary bioactive agents include the compounds according to the present invention which are used to inhibit autophagy and to treat cancer as well as other compounds or agents which are otherwise described herein.
  • treat is used synonymously to refer to any action providing a benefit to a patient at risk for or afflicted with a disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc.
  • Treatment encompasses both prophylactic and therapeutic treatment, principally of cancer.
  • Compounds according to the present invention can, for example, be administered prophylactically to a mammal in advance of the occurrence of disease to reduce the likelihood of that disease.
  • Prophylactic administration is effective to reduce or decrease the likelihood of the subsequent occurrence of disease in the mammal, or decrease the severity of disease that subsequently occurs, especially including metastasis of cancer.
  • compounds according to the present invention can, for example, be administered therapeutically to a mammal that is already afflicted by disease.
  • administration of the present compounds is effective to eliminate the disease and produce a remission or substantially eliminate the likelihood of metastasis of a cancer.
  • Administration of the compounds according to the present invention is effective to decrease the severity of the disease or lengthen the lifespan of the mammal so afflicted, in the case of cancer.
  • pharmaceutically acceptable means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
  • inhibitor refers to the partial or complete elimination of a potential effect, while inhibitors are compounds that have the ability to inhibit.
  • prevention when used in context shall mean “reducing the likelihood” or preventing a disease, condition or disease state from occurring as a consequence of administration or concurrent administration of one or more compounds or compositions according to the present invention, alone or in combination with another agent. It is noted that prophylaxis will rarely be 100% effective; consequently the terms prevention and reducing the likelihood are used to denote the fact that within a given population of patients or subjects, administration with compounds according to the present invention will reduce the likelihood or inhibit a particular condition or disease state (in particular, the worsening of a disease state such as the growth or metastasis of cancer) or other accepted indicators of disease progression from occurring.
  • autophagy or “autophagocytosis” is used to describe a catabolic process in cells which involves the degradation of a cell's own components through lysosomes.
  • Autophagy is a highly regulated process of biological systems that plays a normal part in cell growth development and homeostasis helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which a cell allocates nutrients from unnecessary processes to more-essential processes.
  • a number of autophagic processes occur in nature, all of which have the degradation of intracellular components via the lysosome as a common feature.
  • a well-known mechanism of autophagy involves the formation of a membrane around a targeted region of a cell, separating the contents from the rest of the cytoplasm. The resultant vesicle then fuses with a lysosome which subsequently degrades the contents.
  • Autophagy consists of the sequestration of organelles and proteins in autophagic vesicles (AV) and degradation of this cargo through lysosomal fusion (1). Autophagy allows tumor cells to survive metabolic and therapeutic stresses (2-5). Multiple publications indicate therapy-induced autophagy is a key resistance mechanism to many anti-cancer agents.
  • cancer including the metastasis of cancer
  • rheumatoid arthritis rheumatoid arthritis
  • malaria antiphospholipid antibody syndrome
  • lupus lupus
  • chronic urticaria urticaria and Sjogren's disease.
  • neoplasms include, without limitation, morphological irregularities in cells in tissue of a subject or host, as well as pathologic proliferation of cells in tissue of a subject, as compared with normal proliferation in the same type of tissue. Additionally, neoplasms include benign tumors and malignant tumors (e.g., colon tumors) that are either invasive or noninvasive. Malignant neoplasms are distinguished from benign neoplasms in that the former show a greater degree of dysplasia, or loss of differentiation and orientation of cells, and have the properties of invasion and metastasis.
  • cancer also within context, includes drug resistant cancers, including multiple drug resistant cancers.
  • neoplasms or neoplasias from which the target cell of the present invention may be derived include, without limitation, carcinomas (e.g., squamous-cell carcinomas, adenocarcinomas, hepatocellular carcinomas, and renal cell carcinomas), particularly those of the bladder, bone, bowel, breast, cervix, colon (colorectal), esophagus, head, kidney, liver, lung, nasopharyngeal, neck, ovary, pancreas, prostate, and stomach; leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, acute promyelocytic leukemia (APL), acute T-cell lymphoblastic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, granulocytic leukemia, hairy cell leukemia
  • epithelial tumors including ovarian, breast, colon, head and neck, medulloblastoma and B-cell lymphoma, among others are shown to exhibit increased autophagy and are principal target cancers for compounds and therapies according to the present invention.
  • additional anti-cancer agent is used to describe an additional compound which may be coadministered with one or more compounds of the present invention in the treatment of cancer.
  • agents include, for example, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk
  • LY 317615 neuradiab, vitespan, Rta 744, Sdx 102, talampanel, atrasentan, Xr 311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, liposomal doxorubicin, 5′-deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan
  • alkyl is used herein to refer to a fully saturated monovalent radical containing carbon and hydrogen (up to 10 carbon atoms or as otherwise indicated), and which may be a straight chain, branched or cyclic.
  • alkyl groups are methyl, ethyl, n-butyl, n-heptyl, isopropyl, 2-methyl propyl, tert-butyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.
  • substituted as that term relates to alkyl groups which are described above include one or more functional groups such as lower alkyl groups containing 1-6 carbon atoms which are optionally substituted with 1 or 2 hydroxyl groups or between 1 and 5 (preferably 3-5) fluoro groups, acyl (C 1 -C 6 ), halogen (F, Cl, Br, I, e.g., alkyl halos, e.g., CF 3 ), amido, hydroxyl, carboxy/carboxylic acid, thioamido, cyano, nitro, alkenyl (C 2 -C 6 ) alkynyl (C 2 -C 6 ), azido, alkoxy (C 1 -C 6 ), (including alkoxy groups which are further substituted with a C 1 -C 6 alkoxy group thus producing a diether group), amino, C 1 -C 6 alkylamino and dialkyl-amino, where the alkyl groups may be
  • Preferred substituents on alkyl groups include, for example, at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine (where one or both alkyl groups is itself further optionally substituted with a dialkyl amine or an amine substituted with one or two (preferably one) 7-substituted-4-quinolinyl group(s) where the amine group is bonded to the 4-position of the quinolinyl group) or an alkoxy group (e.g. methoxy or ethoxy) which may be further substituted with an alkoxy group, preferably a methoxy group, thus forming a diether substituent.
  • an alkoxy group e.g. methoxy or ethoxy
  • aryl refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl).
  • Other examples include heterocyclic aromatic (heteroaromatic or heteroaryl) ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, in particular, quinoline groups, in particular, 7-substituted-amino quinoline groups, as well as other groups.
  • substituted as used in the term “substituted aryl, substituted aromatic, substituted heteroaryl, or substituted heteroaromatic” herein signifies that a substitution on the 7-position of 4-aminoquinoline may be present, said substituents being selected from atoms and groups, which when present enhance the activity of the compound as an inhibitor of autophagy.
  • substituents that may be present in a substituted aromatic or heteroaromatic group include, but are not limited to, groups such as H, halo (F, Cl, Br or I), CN, NO 2 , optionally substituted C 1 -C 6 alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally substituted O—C 1 -C 6 alkyl (preferably, OCH 3 ), optionally substituted C 2 -C 7 acyl (preferably acetyl) or optionally substituted C 2 -C 7 ester (oxycarbonyl ester or carboxyester, preferably carboxyester);. It is noted that each of the substituents disclosed herein may themselves be substituted.
  • co-administration shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time.
  • compounds according to the present invention may be co-administered to a patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, including sequential administration.
  • effective concentrations of all co-administered compounds or compositions are found in the subject at a given time.
  • co-administration or adjunct therapy also contemplates other bioactive agents being coadministered with pharmaceutical compositions according to the present invention, especially where a cancer has metastasized or is at risk for metastasis.
  • radiation therapy or “radiation therapy” is used to describe therapy for cancer which may be used in conjunction with the present compounds.
  • Radiation therapy uses high doses of radiation, such as X-rays, or other energy sources such as radioisotopes (gamma, beta or alpha emitters), to destroy cancer cells.
  • the radiation damages the genetic material of the cells so that they can't grow.
  • radiation damages normal cells as well as cancer cells, the normal cells can repair themselves and function, while the cancer cells cannot.
  • Radiotherapy therapy may be used in combination with the presently claimed compounds, alone or in combination with additional anticancer compounds as otherwise disclosed herein, depending on the cancer to be treated. Radiotherapy therapy is most effective in treating cancers that have not spread outside the area of the original cancer, but it also may be used if the cancer has spread to nearby tissue. Radiotherapy is sometimes used after surgery to destroy any remaining cancer cells and to relieve pain from metastatic cancer.
  • compositions useful in the inhibition of autophagy in a biological system and/or the inhibition, treatment or prevention of diseases states and/or conditions which benefit from the inhibition of autophagy including cancer (and its metastasis), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus (systemic lupus erythematosus), chronic urticaria and Sjogren's disease.
  • Pharmaceutical compositions comprise an effective amount of one or more compounds according to the present invention in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional agent, in the case of cancer, preferably an anticancer agent as otherwise described herein.
  • the compounds and method of the invention may be used to inhibit autophagy as otherwise described herein, and are useful for the inhibition (including prophylaxis) and/or treatment of cancer and its metastasis, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus (systemic lupus erythematosus), chronic urticaria and Sjogren's disease.
  • the treatment of cancer or malaria are important aspects of the present invention.
  • subjects or patients in need are treated with the present compounds, pharmaceutical compositions in order to inhibit, reduce the likelihood or treat a disease state, condition and/or infection as otherwise described herein.
  • the disease states, conditions and infections treated by the present compounds and compositions are readily recognized and diagnosed by those of ordinary skill in the art and treated by administering to the patient an effective amount of one or more compounds according to the present invention.
  • dosages and routes of administration of the compound are determined according to the size and condition of the subject, according to standard pharmaceutical practices. Dose levels employed can vary widely, and can readily be determined by those of skill in the art. Typically, amounts in the milligram up to gram quantities are employed.
  • the composition may be administered to a subject by various routes, e.g. orally, transdermally, perineurally or parenterally, that is, by intravenous, subcutaneous, intraperitoneal, or intramuscular injection, among others, including buccal, rectal and transdermal administration.
  • Subjects contemplated for treatment according to the method of the invention include humans, companion animals, laboratory animals, and the like.
  • Formulations containing the compounds according to the present invention may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, capsules, powders, sustained-release formulations, solutions, suspensions, emulsions, suppositories, creams, ointments, lotions, aerosols, patches or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • compositions according to the present invention typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, additives and the like.
  • the composition is about 0.1% to about 85%, about 0.5% to about 75% by weight of a compound or compounds of the invention, with the remainder consisting essentially of suitable pharmaceutical excipients.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
  • the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.
  • Liquid compositions can be prepared by dissolving or dispersing the compounds (about 0.5% to about 20% by weight or more), and optional pharmaceutical adjuvants, in a carrier, such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol, to form a solution or suspension.
  • a carrier such as, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol
  • the composition may be prepared as a solution, suspension, emulsion, or syrup, being supplied either in liquid form or a dried form suitable for hydration in water or normal saline.
  • the preparations may be tablets, granules, powders, capsules or the like.
  • the composition is typically formulated with additives, e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.
  • additives e.g. an excipient such as a saccharide or cellulose preparation, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, and other additives typically used in the manufacture of medical preparations.
  • An injectable composition for parenteral administration will typically contain the compound in a suitable i.v. solution, such as sterile physiological salt solution.
  • a suitable i.v. solution such as sterile physiological salt solution.
  • the composition may also be formulated as a suspension in a lipid or phospholipid, in a liposomal suspension, or in an aqueous emulsion.
  • composition to be administered will contain a quantity of the selected compound in a pharmaceutically effective amount for inhibiting autophagy in a biological system, including a patient or subject according to the present invention.
  • the inventors have examined the application of the strategy of polyvalency (11, 12) to the synthesis of novel autophagy inhibitors by preparing a dimeric chloroquine ( FIG. 1 , compound 3: Lys01), from commercially available materials. Based on literature precedent(14), we envisioned the preparation of compound 3 from one equivalent of compound 5 and two equivalents of compound 6 (14), as outlined retrosynthetically in FIG. 2 .
  • Each of the requisite starting compounds outlined in FIG. 15A (compounds 13-16) are known or commercially available, facilitating the synthesis of a family of analogs that differ from compound 3 (12 R ⁇ Cl) by incorporating different electron-withdrawing groups, based on the work of Lee (16).
  • the inventors also examine the biological activity of N-alkyated and N-acylated analogs of compound 12, compound 17 and compound 18 ( FIG. 15A ).
  • the preparation of these novel compounds would proceed directly from 12 (R ⁇ Cl), either by direct alkylation, reductive alkylation, or acylation.
  • the inventors also examine the introduction into compound 12 of the chirality that is present in CQ and HCQ ( FIG. 1 ) as outlined in compound 19 (Scheme C of FIG. 15A ).
  • the requisite linker compound 20 could be obtained using the method of Kokotos (J. Chem. Res, Synopses 1992, 12, 391).
  • R 2 further directs substitution of R 2 with a hydroxyethyl moiety, as found in hydroxychloroquine 2 (scheme 5).
  • the reaction of anilines with ethylene oxide to give the corresponding hydroxyethyl compounds is well-precedented (22), so that conversion of the lead structure 3 to either mono- or di-hydroxyethylated analogs 47 and 48 is readily achieved.
  • the inventors also examine the preparation of tri- and tetra-quinoline containing structures by oxidation of the primary alcohols in 47 and 48 to give the corresponding aldehydes, which on reductive alkylation with 7-chloro-4-aminoquinoline gives the tri- and tetraquinolines 49 and 50, based on the work of Bailey and coworkers (23), as shown in scheme 6.
  • the inventors also examine the effect of incorporation of both lipophilic groups, i.e., long chain alkyl, as well as more polar substituents in the place of R 2 in the lead structure in Scheme 7, via alkylation of the secondary amines (R 2 ⁇ H) with the commercially available alkylating agents 59-62, leading to the preparation of 51-58 via mono- and dialkylation of the two secondary amine functionalities, based on the work of Drefahl and Konig (Chem. Ber. 1954, 87, 1632-4).
  • the inventors examine the effect of changing R 3 in the lead structure 23 (see scheme 1, above). Certain substitutions for R 3 are already known, such as shown in 63-65 in Scheme 8, below.
  • the inventors prepare a series of new analogs, based on the alkylation of the known secondary amine 63 (13) with the alkylating agents shown in scheme 7 (59-62) to generate the novel structures (66-69).
  • the substrate 70 in which R 3 ⁇ CH 2 CH 2 OH, i.e., the analog of 63 that corresponds most closely to hydroxychloroquine is examined. This compound is available by the same sequence used to prepare the analogs shown in Scheme 5, or by demethylation of 66.
  • the inventors prepare the analog of the lead structure 3 containing the stereocenter present in chloroquine 1, i.e., 75/76 and 77/78, which are available from alanine and serine, respectively, using the method of Charlton and coworkers (24) via reductive alkylation.
  • a method for treating a mammalian patient or subject to inhibit autophagy in that patient or subject.
  • Compounds according to the present invention described herein may be used to inhibit autophagy in a manner consistent with inhibiting, treating and/or preventing disease states and/or conditions including cancer (including metastasis of cancer), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.
  • the present invention in patients or subjects in need thereof, are treated by administering to the patient or subject an effective amount of one or more compounds according to the present invention, optionally in combination with at least one additional bioactive agent useful for treating the same disease state or condition.
  • Compounds according to the present invention may be used to inhibit, reduce the likelihood or treat cancer, including the metastasis of cancer in a patient or subject in need of such treatment.
  • the treatment is useful for any cancer for which inhibition of autophagy represents a favorable result or for which metastasis is a risk element.
  • Therapy with at least one additional anticancer agent as otherwise described herein is also contemplated in the present methods.
  • the numerous cancers which may be treated pursuant to the present method are described hereinabove.
  • the present invention is directed to a method for treating a disease state and/or condition which benefits from the inhibition of autophagy, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjorgen's disease.
  • a patient or subject in need of treatment is administered an effective amount of a compound as otherwise described herein optionally in combination with a pharmaceutically acceptable carrier, additive or excipient in order to inhibit, treat and/or prevent the above disease states of conditions.
  • the method of treatment comprises administering to the subject in need of treatment, in a pharmaceutically acceptable carrier, an effective amount of a compound according to I below:
  • R 1 and R 1′ are each independently H, halo (F, Cl, Br or I), CN, NO 2 , optionally substituted C 1 -C 6 alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally substituted O—C 1 -C 6 alkyl (preferably, OCH 3 ), optionally substituted C 2 -C 7 acyl (preferably acetyl) or optionally substituted C 2 -C 7 ester (oxycarbonyl ester or carboxyester, preferably carboxyester);
  • R and R′ are each independently H, a C 1 -C 6 optionally substituted alkyl group, a C 1 -C 7 (preferably C 2 -C 7 ) optionally substituted acyl group, a C 2 -C 7 optionally substituted carboxy ester group (which forms a urethane group with the nitrogen atom to which R or R′ is bonded);
  • Z is O or N—R Z ;
  • R Z is H or an optionally substituted C 1 -C 3 alkyl group, or a pharmaceutically acceptable salt, enantiomer, diastereomer, solvent or polymorph thereof.
  • R 1 and R 1′ are each independently H, a halo group, a nitro group or a trifluoromethyl group, preferably a chloro group.
  • R and R′ are preferably each independently H, a C 1 -C 3 optionally substituted alkyl group itself preferably substituted with at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with a 7-substituted-4-quinolinyl group wherein the amine binds to the 4-position of the quinolinyl group, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or
  • L is a
  • X is N—R′′, Y and Y′ are each independently absent or CH 2 , and R′′ is H or a C 1 -C 3 alkyl group which is optionally substituted with at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with a 7-substituted-4-quinolinyl group wherein the amine binds to the 4-position of the quinolinyl group, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) (the 7-position of each quinolinyl group
  • the compounds described above may be coadministered with at least one additional anticancer agent including, for example, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK
  • additional anticancer agent including, for example, everolimus,
  • the compounds according to the present invention may be coadministered with additional agents which are traditionally used in therapy for these disease states and/or conditions.
  • Lys01 is a More Potent Autophagy Inhibitor than HCQ or CQ.
  • LC3 is a ubiquitin-like protein which exists as an unconjugated form (LC3I) or conjugated to AV membranes (LC3II)(25). The ratio of LC3II/LC3I reflects the accumulation of AV in cells, and therefore effective autophagy inhibition.
  • LC3 immunoblotting FIG.
  • Lys01 is a >10-fold more potent autophagy inhibitor than HCQ or CQ at a concentration of 10 ⁇ M.
  • Lys02 and Lys03 produced dose-response relationships for LC3 immunoblotting similar to HCQ or CQ, whereas Lys04, which retains the two chloroquinoline rings present in Lys01, demonstrated intermediate potency in the LC3 autophagy assay
  • LN229 GFP-LC3 cells were treated with Lys01 or HCQ ( FIG. 4A ).
  • HCQ 1 ⁇ M punctate fluorescence, indicating an accumulation of ineffective autophagic vesicles, was observed in a minority of cells.
  • HCQ 10 ⁇ M produced numerous small puncta
  • HCQ 100 ⁇ M resulted in larger dense puncta that represent fusion of accumulated autophagic vesicles.
  • Lys01 1 ⁇ M produced numerous small puncta, whereas in cells treated with Lys01 10 ⁇ M dense puncta similar in appearance to those observed in cells treated with HCQ 100 ⁇ M were apparent.
  • Lys01 produces morphological changes more pronounced than HCQ, a known lysosomal inhibitor, at 10-fold lower concentrations.
  • HCQ a known lysosomal inhibitor
  • rapamycin treatment resulted in a further increase in the LC3II/LC3 ratio in bafilomycin treated cells compared to control cells whereas HCQ- or Lys05-treated cells did not demonstrate an increase in LC3II/LC3I ratio in bafilomycin treated cells compared to control, providing further evidence that Lys01 is an autophagy inhibitor ( FIG. 4C ).
  • LN229 glioma
  • 1205Lu melanoma
  • HT-29 colon
  • c8161 melanoma
  • Lys02 which is a monofunctional CQ derivative (35-91 ⁇ M)
  • Lys03 the bisaminoquinoline with methoxy groups replacing chlorine (24-53 ⁇ M), or HCQ (15-42 ⁇ M)
  • HCQ HCQ
  • Lys05 the trihydrochloride salt of Lys01 was synthesized to enhance aqueous solubility and to enable in vivo studies. Lys01 and Lys05 produced equivalent dose-dependent increases in the LC3II/LC3I ratio, and accumulation of the autophagy cargo protein p62 (26), and identical IC50 values in the MTT assay, ( FIG. 5A , B).
  • c8161 xenografts matched for tumor size were treated with intraperitoneal (i.p.) daily PBS, or equimolar doses of HCQ or Lys05 (HCQ 60 mg/kg (138 nmoles/g), Lys05 76 mg/kg (138 nmoles/g)) for 48 hours.
  • HCQ 60 mg/kg (138 nmoles/g) HCQ 60 mg/kg (138 nmoles/g)
  • Lys05 76 mg/kg 138 nmoles/g
  • FIG. 6A Quantification of the mean number of AV/cell in two representative tumor from each treatment group found a significant >2-fold increase in the mean number of AV/cell in Lys05 treated tumors compared to control- or HCQ-treated tumors ( FIG. 6B ). Significantly higher LC3II/LC3I levels were observed in Lys05-treated tumors compared to control- or HCQ-treated tumors providing further evidence of in vivo autophagy inhibition ( FIG. 7A ). After 48 hours of treatment, cleaved caspase 3 levels indicative of apoptosis were elevated in Lys05 treated tumors compared to HCQ- or PBS-treated tumors.
  • mice bearing 1205Lu xenografts were matched for tumor volume per cohort were assigned to either PBS, HCQ 60 mg/kg i.p. or Lys05 76 mg/kg i.p. (equimolar dosing) dosed for 3 days of daily treatment with 2 days off treatment (3/5 days) for all 3 treatment groups, to allow for symptom recovery and to avoid excess toxicity. This schedule was tolerated well for a 14 day period. Tumor growth curves for each of the 3 groups indicated tumor growth was significantly impaired in Lys05 treated tumors compared to controls ( FIG. 6C ). Lys05 treatment resulted in a 53% reduction in the average daily tumor growth rate compared to vehicle treated controls (31.2 v.
  • mice bearing HT-29 colon cancer xenografts were treated with PBS, or Lys05 at 10 mg/kg i.p. daily, 40 mg/kg i.p. daily, or 80 mg/kg i.p. 3/5 days off.
  • Clinical toxicity was observed only in the 80 mg/kg cohort, with 2/8 mice euthanized early for bowel obstruction.
  • Daily dosing for the 10 mg/kg and 40 mg/kg cohorts was well tolerated.
  • the average daily tumor growth rate was significantly impaired in a dose-dependent fashion with Lys05 treatment ( FIG. 6E ).
  • Tumor growth curves demonstrated that all 3 doses of Lys05 produced significant tumor growth impairment compared to control ( FIG. 6F ).
  • Paneth cell dysfunction including reduced size and number of eosinophilic lysozyme-containing granules, has previously been described as the pathognomonic sign of autophagy deficiency in mice and a subset of Crohn's disease patients that have a genetic deficiency in the essential autophagy gene ATG16L1 (31).
  • FIG. 9A Resection of the entire gastrointestinal tract from mice bearing HT-29 tumors after 14 days of treatment demonstrated bowel thickening and obstruction was limited to 80 mg/kg dose cohort ( FIG. 9B ). Histological examination of the terminal ileum resected from mice bearing HT-29 xenografts treated with PBS ip daily, or Lys05 10 mg/kg ip daily, 40 mg/kg ip daily, and 80 mg/kg ip daily every 3/5 days for 14 days demonstrated dose dependent effects on Paneth cell morphology ( FIG. 9C ). While the number of Paneth cells/crypt did not change with treatment ( FIG. 9C ).
  • FIG. 9D the size and number of granules decreased in a dose dependent manner. Scoring on a Paneth cell dysfunction scale ( FIG. 10 ) indicated that Paneth cell dysfunction was observed at all doses tested of Lys05, despite signs and symptoms of toxicity being restricted to the 80 mg/kg dose ( FIG. 9E ). In mice treated with Lys05 40 mg/kg or 80 mg/kg, but not 10 mg/kg, lysozyme was significantly reduced or absent in Paneth cells ( FIG. 9F ). Taken together these findings indicate that Lys05-associated Paneth cell dysfunction mimics ATG16L1 deficiency, and lower doses of Lys05 produce significant antitumor activity without dose-limiting toxicity.
  • Lys05 Inhibits Autophagy by Deacidifying the Lysosome.
  • lysosomes were subfractionated from 1205Lu cells treated with PBS, HCQ 10 ⁇ M, or Lys05 10 PM, and 1205Lu tumors harvested after 14 days of treatment with PBS, HCQ 60 mg/kg i.p., or Lys05 76 mg./kg i.p. every 3/5 days.
  • Immunoblotting against the lysosomal marker LAMP2 confirmed adequate separation of the lysosomal and whole cell population in both cells and tumor samples ( FIG. 12A ).
  • HPLC tandem mass spectrometry (MS/MS) measurements FIG.
  • Lys05Lu cells were treated with vehicle, Lys05, or HCQ and stained with acridine orange (AO; a dye which aggregates in all endovesicular acidic compartments) at 24 hours ( FIG. 12D ).
  • AO acridine orange
  • Lys05 caused an accumulation of acidic vesicles at lower doses (10 ⁇ M), but at higher doses (50 ⁇ M), no acidic vesicles were observed, indicating the complete deacidification of the endovesicular system.
  • Lys01 derivatives have been synthesized and tested ( FIG. 14 , FIG. 15 ).
  • the IC50's of compounds Lys01-Lys 13 demonstrate increased or decreased activity compared to HCQ and CQ (Table 1, FIG. 16 ).
  • the Lys01 derivatives are more active than CQ or HCQ.
  • FIG. 17 shows the IC50 values for Lys01 derivative-induced cell death in the human cancer cell LN229 and a number of strains of P. Falciparum grown in in vitro in human RBC. There is a similar activity profile for anticancer activity of Lys01 derivatives and malaria cytotoxicity. Lys01 was more active than artesunate in some CQ-resistant cell lines.
  • Lys01 and Lys05 are lead compounds with great potential to be optimized further for potency as a novel autophagy inhibitor.
  • Autophagy inhibition is a new therapeutic strategy in cancer that is applicable to every cancer.
  • An optimized derivative of Lys01 could be developed as a second generation autophagy inhibitor.
  • the GI toxicity associated with Paneth cell dysfunction observed at LD30 doses of Lys05 support the mechanism of action of the drug, and also suggests that colon cancers, which often share features with Paneth cells, may be a tumor type that may be especially sensitive to Lys05 and its optimized derivatives. Additional cancers worth investigating include melanoma, and non small cell lung cancer, since melanoma cell lines demonstrated the highest difference in sensitivity to Lys01 compared to HCQ, and an EGFR mutated lung cancer cell line demonstrated sensitivity to both HCQ and Lys05. The synthesis of Lys01 was designed such that there is no overlap with other patented and/or published aminoquinoline compounds. Further mechanistic studies are planned that are to identify pharmacodynamics assays that guide drug development. Pharmacokinetic studies planned in mice establish initial in vivo profile.
  • Novel chloroquine derivatives for use as anticancer agents is an active area of investigation (16). Autophagy has been identified as one of the top ten areas of research in which the NIH will invest in the next few years. No studies to date have leveraged the potential of bivalency as the inventors provide here. In addition, most studies lack the in vivo studies and the mechanistic studies herein reported that can guide further development of optimize lead compounds for drug development.
  • the present application has shows that the disclosed series of bisaminoquinolines are potent autophagy inhibitors that have single agent antitumor activity in an in vivo tumor model.

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US20160168099A1 (en) 2016-06-16
US11591298B2 (en) 2023-02-28
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US20200071272A1 (en) 2020-03-05
CN103687853B (zh) 2017-12-08
EP2702054B1 (fr) 2018-10-24
WO2012149186A2 (fr) 2012-11-01
JP2014513101A (ja) 2014-05-29
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EP2702054A2 (fr) 2014-03-05
US20170166530A1 (en) 2017-06-15
CN103687853A (zh) 2014-03-26
CA2841452A1 (fr) 2012-11-01
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US10047052B2 (en) 2018-08-14

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