WO2016111957A1 - Chloroquinoline triazole compounds, composition and uses - Google Patents

Abstract

Chloroquinoline triazole compounds are provided, as are compositions comprising the compounds, and methods relating to them, including methods of inhibiting autophagy in biological systems and treatment of diseases, disorders or conditions in which autophagy inhibition can provide benefit, including treatment of cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

Description

CHLOROQUINOLINE TRIAZOLE COMPOUNDS, COMPOSITIONS AND USES

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/100,316, filed on January 6, 2015, the content of which is herein incorporated by reference into the subject application.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers CA163907, GM093282, TR001073, TR001072, and TR001071 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0004] Autophagy is an evolutionarily conserved, regulated catabolic process that degrades cellular proteins and organelles, allowing the recycling of their biochemical components for use in energy production and biosynthetic reactions (1-3). Autophagy has a role in a number of critical cell functions, including stress response, cellular quality control, tissue homeostasis, and energy production. Increased autophagic flux was found in a large majority of human tumors, compared to non-malignant tissue, and was associated with increased tumor proliferation, invasion and metastasis, and lower patient survival (4). Autophagy is also upregulated in tumor cells in response to most anti-cancer therapies, including cytotoxic chemotherapeutic drugs, targeted chemotherapeutic drugs, and radiation (1, 5-7).

SUMMARY OF THE INVENTION

[0005] The present disclosure provides, among other things, various useful chemical compounds that, in some embodiments, are variants of chloroquine (CQ) and/or hydroxychloroquine (HCQ). In some embodiments, provided compounds show activity as autophagy inhibitors. The present invention encompasses the recognition that there is an unmet need for effective inhibitors of autophagy. The upregulation of autophagy has been shown to be a mechanism of drug resistance, and the use of autophagy inhibitors can reverse this effect.

[0006] CQ and HCQ have activity as autophagy inhibitors. CQ and HCQ also have single agent antiproliferative activity against human cancer cells. There are currently over 30 HCQ trials in cancer patients involving nearly every tumor type.

[0007] The present invention further encompasses the recognition that, while CQ and HCQ are effective inhibitors of autophagy in vitro, their in vivo efficacy may require concentrations at the upper range of tolerability (8, 9), and, moreover, that the low potency of CQ and HCQ may limit their efficacy in vivo. The present invention also encompasses the recognition that compounds showing structural relatedness to CQ and/or HCQ have various desirable attributes including, in some embodiments, but not limited to, an ability to act as autophagy inhibitors.

[0008] Embodiments of this invention are directed to compounds for inhibiting autophagy in biological systems. For example, EAD1 (FIG. l), and/or variants or analogs thereof may provide potent and/or effective autophagy inhibition as described herein.

[0009] In certain embodiments, provide compounds are variants or analogs of chloroquine or triazole.

[0010] In certain embodiments, a pharmaceutical composition comprises a compound according to formula I or III 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.

[0011] In some aspects, the invention is directed to methods of inhibiting autophagy in a biological system. In some embodiments, a biological system is or comprises a cell, an organ, a tissue, and/or an organism. In some embodiments, an organism is or comprises a mammal, e.g., a human. In some embodiments, an organism is or comprises a patient or subject who is suffering from or susceptible to an autophagy-related disease, disorder or condition. In some embodiments, an organism is or comprises a patient or subject who is suffering from or susceptible to cancer.

[0012] For example, in some embodiments, a composition or compound as described herein is administered to a biological system. In some embodiments, one or more effects or results of such administration may be monitored or assessed. For example, in some embodiments, one or more effects or results on autophagy in the system is monitored or assessed. [0013] In certain embodiments, administration as described herein method inhibits, treats and/or reduces the likelihood of onset of one or more symptoms of a disease, disorder or condition associated with autophagy. In certain embodiments, administration as described herein method inhibits, treats and/or reduces the likelihood of onset of one or more symptoms of cancer (e.g., of metastasis).

[0014] In some aspects, the present invention provides systems (e.g., methods and/or reagents, kits, etc.) useful to identify and/or characterize analogs or variants of chloroquine and/or compounds that inhibit autophagy. For example, in some embodiments, one or a plurality of test compounds is administered to a system and its relevant effect(s) on such system are compared with that/those of a reference compound or result achieved under comparable conditions.

[0015] In some particular embodiments, the present disclosure provides a compound having a structure as set forth in Formula I:

wherein R_1; R₂, and R₃ are each independently H, halo (F, CI, Br or I), CN, SO2CH₃, acyl, alkyl (in some embodiments C1-C3), alkyl halo (in some embodiments CF3), optionally substituted 0-Ci-C₆ alkyl (preferably, OCH₃), OCF₃, OH, NRR' ; in some particular embodiments, R₃ is H

each El is independently N or CH;

each E2 is independently NH, NR, O, or CRR' ;

each E3 is independently CH, N, O, or S;

R and R' are each independently H or optionally substituted Ci-Ce alkyl group;

n is 0, 1, or 2;

R4 is substituted or optionally substituted alkyl, substituted or optionally substituted aryl, substituted or optionally substituted aromatic, substituted or optionally substituted heteroaryl, or substituted or optionally substituted heteroaromatic; in some particular embodiments, R4 is substituted or optionally substituted benzyl; or a pharmaceutically acceptable salt thereof.

[0016] In some embodiments, the present disclosure provides a composition comprising an appropriate unit dose of a compound as described herein. In some embodiments, an appropriate unit dose is described relative to a dose of CQ or HCQ. In some particular embodiments, a unit dose may be an amount (or may be equivalent to CQ or HCQ in an amount) selected from 1, 4, 5, 20, 30, 40, 50, 60 , 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 5000, 10000 mg or more mg of compound). In some embodiments, a unit dose may be an amount (or may be equivalent to CQ or HCQ in an amount) appropriate for administration in a regimen that delivers or utilizes a daily dose selected from 1, 4, 5, 20, 30, 40, 50, 60 , 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 5000, or 10000 mg.

[0017] In some embodiments, a provided composition may include a pharmaceutically acceptable carrier, additive or excipient.

[0018] In some embodiments, the present invention provides methods comprising steps of: administering to a subject suffering from or susceptible to an autophagy associated disease disorder or condition a pharmaceutical composition as described herein.

[0019] In some embodiments, the present invention provides methods of providing a compound, the methods comprising steps of:

chlorinating an alcohol in a compound of structure:

so that a chloride moiety is added,

replacing the chloride with propargylamine,

performing azide-alkyne cycloaddition.

[0020] In some embodiments, the present invention provides a compound having a structure as set forth in Formula II (intermediate):

[0021] In some embodiments, the present invention provides a collection of compounds (e.g., at least 100, 200, 300, 400, 500, 600, 700, 800, 1000, 5000, 10000 or more compounds), each of which shares a common structural element as set forth in Formula III:

[0022] In some embodiments, the present invention provides methods of identifyi characterizing a compound having a chemical structure as set forth in Formula I:

the methods comprising steps of:

contacting a compound to be tested with non-small cell lung cancer, or/and pancreatic cancer cells;

determining IC5₀; and

comparing the determined IC5₀ level with IC5₀ level from chemical structure as set forth in formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows chemical structures of chloroquine, hydroxychloroquine, and EAD1.

[0024] FIG. 2 shows growth inhibition by chloroquine (CQ), hydroxychloroquine (HCQ), and certain indicated compounds. Human non-small cell lung cancer (NSCLC) and pancreatic cancer cells were treated for 72 hours at the indicated concentrations, and cell numbers quantified with sulforhodamine B (SRB) assays. Data presented are means ± standard deviations of at least 3 experiments, each done in triplicate.

[0025] FIG. 3 shows growth inhibition results obtained with EAD1 in a colony growth assay study. Human non-small cell lung cancer (NSCLC) cell line H460 were plated at 250 cells per well in 24 well plates. After allowing for cell attachment overnight, HCQ and EAD1 were added at the indicated concentrations. After 24 hours, drug-containing medium was removed, and drug-free medium added. Cell colonies were stained with crystal violet after an additional 10-day growth in the absence of drug. A representative experiment is shown.

[0026] FIG. 4 depicts induction of apoptosis by EADl, CQ and HCQ. Human non-small cell lung cancer (NSCLC) cell line H460 was treated for 24 hours with the indicated concentrations of CQ, HCQ, and EADl . Cells were trypsinized, stained with APC-Annexin V for 15 minutes, and analyzed by flow cytometry. DAPI was used to assess cell viability. Data are means ± SD of 3 experiments.

[0027] FIG. 5 shows that EADl increases punctate LC3 expression in lung cancer cells. H3122 NSCLC cells were transfected with an LC3 -expressing vector (mCherry-EGFP- LC3B). Transfected cells were treated with HCQ or EADl at the indicated concentrations for 6 hours, fixed, and analyzed by fluorescent microscopy. An increase in fluorescent puncta are seen with drug treatment. The size bar is 50 μηι.

[0028] FIGS. 6a-6d illustrate an observed increase in autophagosome-associated proteins with EADl treatment. H460 cells were treated for 24 hours with the indicated concentrations of HCQ and EADl (FIGS. 6a and 6c), or for the indicated times with 10 μΜ compound (FIG. 6b). In panel FIG. 6d, cells were treated with the indicated compounds (10 μΜ for 24 hours). The IC5₀ values for the inhibition of proliferation for each compound are shown. Cell extracts were analyzed by immunoblots with antibodies to LC3 (FIGS. 6a, 6b and 6d) or p62 (FIG. 6c). Blots were also probed for actin.

[0029] FIG. 7 shows SCHEME1, which depicts an exemplary synthesis of certain chloroquinoline triazole analogs by late-stage diversification of alkyne 1.

[0030] FIG. 8 shows SCHEME 2, which depicts an exemplary synthetic route to certain triazole-containing chloroquine analogs.

[0031] FIG. 9 shows SCHEME 3, which depicts an exemplary synthetic route to certain truncated analogs. Scheme 3a, compound 200. Scheme 3b, compound 300.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The invention provides a compound having the structure set forth in Formula I:

wherein

Ri, R2, and R₃ are each independently H, halogen, CN, SO2CH₃, acyl, alkyl, alkyl halo, optionally substituted OCi-C₆ alkyl, OCF₃, OH, or NRR';

each El is independently N or CH;

each E2 is independently NH, NR, O, or CRR';

each E3 is independently CH, N, O, or S;

R and R' are each independently H or optionally substituted Ci-Ce alkyl;

wherein the optionally substituted OC1-C6 alkyl or optionally substituted C1-C6 alkyl is optionally substituted with one or more hydroxyl, halogen, =0, Ci-Ce alkyl, alkyl halo, amido, thioamido, carboxamido, carboxy acid, carboxylic acid, cyano, nitro, C2-C6 alkenyl, C2-C6 alkynyl, C2-C7 acyl, azido, Ci-Ce alkoxy, amino, Ci-Ce alkylamino, Ci-Ce dialkyl- amino, C2-C6 acylamino, C2-C6 oxyacylester, C2-C6 carboxy ester, aryloxy, aryloxy(Ci-C6) alkyl, thio, C2-C6 ether, C2-C6 thioether, 7-substituted-4-aminoquinolinyl, monoalkyl amine or dialkyl amine groups;

n is 0, 1, or 2; and

R4 is optionally substituted alkyl, optionally substituted aryl, optionally substituted aromatic, optionally substituted heteroaryl, or optionally substituted heteroaromatic, wherein the optionally substituted alkyl, optionally substituted aryl, optionally substituted aromatic, optionally substituted heteroaryl, or optionally substituted heteroaromatic is optionally substituted with one or more hydroxyl, halogen, =0, Ci-Ce alkyl, OC1-C6 alkyl, alkyl halo, amido, thioamido, carboxamido, cyano, nitro, C2-C7 acyl, C2-C7 oxycarbonyl ester, C2-C7 oxyacylester, or C2-C7 carboxy ester groups;

or a pharmaceutically acceptable salt thereof. [0033] In a preferred compound, each halogen is independently F, CI, Br or I. In a preferred compound, each alkyl is independently C1-C3 alkyl. In a preferred compound, one or more alkyl halo is CF₃. In a preferred compound, one or more OC1-C6 alkyl is OCH₃. In a preferred compound, Ri and R2 are each independently CI or OCH₃. In a preferred compound, R3 is H. In a preferred compound, R4 is optionally substituted benzyl.

[0034] Preferred compounds include those having the structure

or a pharmaceutically acceptable salt thereof.

[0035] Preferred compounds include those where R4 is C6H5, C6H₄(4-F), CeH₄(4-Cl), C₆H₄(3-C1), C₆H₄(2-C1), C₆H₄(4-Br), CH₂C₆H_5, CH₂C₆H₄(4-C1), CH₂C₆H₄(4-Br),

of R_t to N.

[0036] A preferred compound has the structure

or a pharmaceutically acceptable salt thereof.

[0037] Also provided are pharmaceutical compositions comprising one or more of any of the compounds disclosed herein, and a pharmaceutically acceptable carrier, additive or excipient. The pharmaceutical composition can comprise, for example, 1 to 2000 mg of the compound.

[0038] Also provided are methods for treating a subject suffering from or susceptible to an autophagy associated disease, disorder or condition comprising administering to the subject one or more of the compounds or pharmaceutical compositions disclosed herein. Preferably, the compound or pharmaceutical composition is administered in an amount effective to ameliorate a sign or symptom of the autophagy associated disease, disorder or condition in a subject. Preferably, the compound or pharmaceutical composition is administered in an amount effective to inhibit autophagy in a subject.

[0039] The autophagy associated disease, disorder or condition can be or comprise cancer, such as, for example, a carcinoma; a sarcoma; a neuroectodermal tumor; cancer of the breast, esophagus, colon, rectum, head, kidney, liver, lung, nasopharyngeal, neck, ovary, pancreas, skin, brain, CNS, prostate, or stomach; a leukemia; a malignant lymphoma; and combinations thereof.

[0040] The methods of treatment can further comprise administering to the subject at least one additional active agent, such as, for example, an anticancer agent, such as, e.g., everolimus, trabectedin, or abraxane.

[0041] The autophagy associated disease, disorder or condition can be one or more of rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

[0042] Also provided are methods of treating a disease, disorder or condition selected from the group consisting of cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the method comprising administering to the subject one or more of the compounds or pharmaceutical compositions disclosed herein in an amount effective to inhibit autophagy in a subject. [0043] Also provided are one or more of the compounds or pharmaceutical compositions disclosed herein for treating a subject suffering from or susceptible to an autophagy associated disease disorder or condition, or for treating cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria or Sjogren's disease. Still further provided are the use of one or more of the compounds or pharmaceutical compositions disclosed herein for treating a subject suffering from or susceptible to an autophagy associated disease disorder or condition, or for treating cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria or Sjogren's disease.

[0044] Also disclosed is a method of providing a compound of Formula (I), the method comprising steps of:

chlorinating an alcohol in a compound of structure

oride moiety is added,

replacing the chloride with propargylamine; and

performing azide-alkyne cycloaddition so as to provide a compound of Formula (I). The method can further comprise determining one or more characteristics or activities of the compound.

[0045] Also provided is a compound selected from the group consisting of:

, and -l i

wherein R is H or CH₃.

[0046] Also provided is collection of at least 100 compounds, wherein each compound has the following structure

or a pharmaceutically acceptable salt or salts thereof.

DEFINITIONS

[0047] The following terms shall be used throughout the specification to describe the present invention. Where a term is not specifically defined herein, that term shall be understood to be used in a manner consistent with its use by those of ordinary skill in the art.

[0048] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. In instances where a substituent is a possibility in one or more Markush groups, it is understood that only those substituents which form stable bonds are to be used.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0050] As used herein and in the appended claims, the singular forms "a," "and" and "the" include plural references unless the context clearly dictates otherwise.

[0051] Furthermore, the following terms shall have the definitions set out below.

[0052] The term "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. Such agents include, for example, one or more of 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 inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT-110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdRsub. l KRX-0402, lucanthone. 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-lH-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]- benzoyl]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258,); 3-[5-(methylsulfonylpiperadinemethyl)- indolyl]-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6,Azgly 10](pyro-Glu-His-T -Ser-Tyr-D-Ser(Bu t)-Leu-Arg-Pro-Azgly-NH.sub.2 acetate [C5₉H₈4Ni₈Oi₄-(C₂H₄0₂)_x where x=l to 2.4], goserelin acetate, leuprolide acetate, triplorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amnifostine. NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, arnsacrine, anagrelide, L-asparaginase, Bacillus Calmette- Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5 -deooxy uridine, cytosine arabinoside, 6-mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, finasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxy tamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, temsirolimus, AP- 23573, RADOOl, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa- 2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all- transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, sspegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, ipilumumab, vemurafenib among others.

[0053] As used herein, the term "administration" refers to the administration of a composition to a subject or system (e.g., to a cell, organ, tissue, organism, or relevant component or set of components thereof). Those of ordinary skill will appreciate that route of administration may vary depending, for example, on the subject or system to which the composition is being administered, the nature of the composition, the purpose of the administration, etc. For example, in certain embodiments, administration to an animal subject (e.g., to a human) may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal and/or vitreal. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0054] As used herein, the term "amelioration" refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does not require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury).

[0055] The term "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. Examples of alkyl groups include methyl, ethyl, n-butyl, n-heptyl, isopropyl, 2-methyl propyl, tert-butyl, neopentyl, hexyl, heptyl, octyl, nonyl, and decyl, etc.

[0056] As used herein, the term "analog" refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an "analog" shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g by chemical manipulation of the reference substance. In some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0057] As used herein, the term "antagonist" refers to an agent that i) inhibits, decreases or reduces the effects of another agent; and/or ii) inhibits, decreases, reduces, or delays one or more biological events. Antagonists may be or include agents of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and/or any other entity that shows the relevant inhibitory activity. An antagonist may be direct (in which case it exerts its influence directly upon its target) or indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, for example so that level or activity of the target is altered).

[0058] As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0059] The term "aryl" refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl) wherein each ring in the system contains 3 to 7 ring members. 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.

[0060] Two events or entities are "associated" with one another, as that term is used herein, if the presence, level and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of and/or susceptibility to the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0061] The term "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 involves 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.

[0062] The term "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.

[0063] The term "cancer" shall refer to a proliferation of tumor cells having the unique trait of loss of normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and/or metastasis. As used herein, 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. The term cancer, in some embodiments, includes drug resistant cancers, including multiple drug resistant cancers. Examples of 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, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, neutrophilic leukemia and stem cell leukemia; benign and malignant lymphomas, particularly Burkitt's lymphoma, Non-Hodgkin's lymphoma and B-cell lymphoma; benign and malignant melanomas; myeloproliferative diseases; sarcomas, particularly Ewing's sarcoma, hemangiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcomas, peripheral neuroepithelioma, and synovial sarcoma; tumors of the central nervous system (e.g., gliomas, astrocytomas, oligodendrogliomas, ependymomas, gliobastomas, neuroblastomas, ganglioneuromas, gangliogliomas, medulloblastomas, pineal cell tumors, meningiomas, meningeal sarcomas, neurofibromas, and Schwannomas); germ-line tumors (e.g., bowel cancer, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer (e.g., small cell lung cancer, mixed small cell and non-small cell cancer, pleural mesothelioma, including metastatic pleural mesothelioma small cell lung cancer and non-small cell lung cancer), ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma; mixed types of neoplasias, particularly carcinosarcoma and Hodgkin's disease; and tumors of mixed origin, such as Wilms' tumor and teratocarcinomas, among others. It is noted that certain epithelial tumors including ovarian, breast, colon, head and neck, medulloblastoma and B-cell lymphoma, among others are shown to exhibit increased autophagy; in some embodiments, provided compounds are used in the treatment and/or prevention of one or more such epithelial tumors. In some embodiments, provided are used in the treatment and/or prevention of breast, colorectal, pancreatic, ovarian, lung, renal, and head & neck cancers, melanomas, glioblastomas, leukemias and/or lymphomas.

[0064] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a composition is administered. In some exemplary embodiments, carriers can include sterile liquids, such as, for example, water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. In some embodiments, carriers are or include one or more solid components.

[0065] As used herein, the term "characteristic portion", in the broadest sense, refers to a portion of a substance whose presence (or absence) correlates with presence (or absence) of a particular feature, attribute, or activity of the substance. In some embodiments, a characteristic portion of a substance is a portion that is found in the substance and in related substances that share the particular feature, attribute or activity, but not in those that do not share the particular feature, attribute or activity. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, in some embodiments, a "characteristic portion" of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g. , of a protein, antibody, etc.) is one that, in addition to the sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance. In some embodiments, a characteristic portion may be biologically active.

[0066] As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more agents or may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, combination therapy may be referred to as "co-administration" or "adjunct therapy."

[0067] The term "comparable" is used herein to describe two (or more) sets of conditions, circumstances, individuals, or populations that are sufficiently similar to one another to permit comparison of results obtained or phenomena observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied. Those skilled in the art will appreciate that relative language used herein (e.g., enhanced, activated, reduced, inhibited, etc.) will typically refer to comparisons made under comparable conditions.

[0068] The term "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. The term 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.

[0069] As used herein, the term "designed" refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and/or (iii) that is distinct from natural substances and other known agents.

[0070] Certain methodologies described herein include a step of "determining". Those of ordinary skill in the art, reading the present specification, will appreciate that such "determining" can utilize or be accomplished through use of any of a variety of techniques available to those skilled in the art, including for example specific techniques explicitly referred to herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and/or manipulation of data or information, for example utilizing a computer or other processing unit adapted to perform a relevant analysis. In some embodiments, determining involves receiving relevant information and/or materials from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0071] As used herein, the term "dosage form" refers to physically discrete unit of a therapeutic agent for a subject (e.g., a human patient) to be treated. Each unit contains a predetermined quantity of active material calculated or demonstrated to produce a desired therapeutic effect when administered to a relevant population according to an appropriate dosing regimen. For example, in some embodiments, such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen). It will be understood, however, that the total dosage administered to any particular patient will be selected by a medical professional (e.g., a medical doctor) within the scope of sound medical judgment.

[0072] As used herein "dosing regimen" or "therapeutic regimen" is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regime comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, the therapeutic agent is administered continuously (e.g., by infusion) over a predetermined period. In some embodiments, a therapeutic agent is administered once a day (QD) or twice a day (BID). In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0073] The term "effective" is used herein, unless otherwise indicated, to describe an amount of a compound or component which, when used within the context of a particular regimen, produces or effects an intended result, whether that result relates to the prophylaxis and/or therapy of an infection and/or disease state or as otherwise described herein. The term effective subsumes all other effective amount or effective concentration terms (including the term "therapeutically effective") which are otherwise described or used in the present application.

[0074] As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0075] The term "inhibit" as used herein refers to the partial or complete elimination of a potential effect, while inhibitors are compounds that have the ability to inhibit.

[0076] As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can exist in a variety of structural and/or optical isomeric forms. In some embodiments, as will be clear to those skilled in the art from context, depiction of or reference to a particular compound structure herein may represent all structural and/or optical isomers thereof. In some embodiments, as will be clear to those skilled in the art from context, depiction of or reference to a particular compound structure herein is intended to encompass only the depicted or referenced isomeric form. In some embodiments, compositions including a chemical entity that can exist in a variety of isomeric forms include a plurality of such forms; in some embodiments such compositions include only a single form. For example, in some embodiments, compositions including a chemical entity that can exist as a variety of optical isomers (e.g., stereoisomers, diastereomers, etc.) include a racemic population of such optical isomers; in some embodiments such compositions include only a single optical isomer and/or include a plurality of optical isomers that together retain optical activity.

[0077] As used herein, the term "patient" or "subject" refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, farm animals and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms. In some embodiments, a patient is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disorder or condition. In some embodiments, a patient has been diagnosed with one or more disorders or conditions.

[0078] As used herein, the term "pharmaceutical composition" refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; trans dermally; or nasally, pulmonary, and/or to other mucosal surfaces.

[0079] The term "pharmaceutically acceptable" as used herein, refers to agents that, within the scope of sound medical judgment, are suitable for use in contact with tissues of human beings and/or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0080] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al, describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. [0081] As used herein, the term "physiological conditions" has its art-understood meaning referencing conditions under which cells or organisms live and/or reproduce. In some embodiments, the term refers to conditions of the external or internal mileu that may occur in nature for an organism or cell system. In some embodiments, physiological conditions are those conditions present within the body of a human or non-human animal, especially those conditions present at and/or within a surgical site. Physiological conditions typically include, e.g., a temperature range of 20 - 40°C, atmospheric pressure of 1 , pH of 6- 8, glucose concentration of 1-20 mM, oxygen concentration at atmospheric levels, and gravity as it is encountered on earth. In some embodiments, conditions in a laboratory are manipulated and/or maintained at physiologic conditions. In some embodiments, physiological conditions are encountered in an organism.

[0082] The term "prevention", as used herein, refers to a delay of onset, and/or reduction in frequency and/or severity of one or more symptoms of a particular disease, disorder or condition. In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a particular disease, disorder or condition if a statistically significant decrease in the development, frequency, and/or intensity of one or more symptoms of the disease, disorder or condition is observed in a population susceptible to the disease, disorder, or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.

[0083] The term "radiotherapy" 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 cannot grow. Although radiation damages normal cells as well as cancer cells, the normal cells can repair themselves and function, while the cancer cells cannot. Radiation 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.

[0084] As used herein the term "reference" describes a standard or control agent, animal, individual, population, sample, sequence or value against which an agent, animal, individual, population, sample, sequence or value of interest is compared. In some embodiments, a reference agent, animal, individual, population, sample, sequence or value is tested and/or determined substantially simultaneously with the testing or determination of the agent, animal, individual, population, sample, sequence or value of interest. In some embodiments, a reference agent, animal, individual, population, sample, sequence or value is a historical reference, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference agent, animal, individual, population, sample, sequence or value is determined or characterized under conditions comparable to those utilized to determine or characterize the agent, animal, individual, population, sample, sequence or value of interest.

[0085] As used herein, the term "sample" typically refers to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample is or comprises biological tissue or fluid. In some embodiments, a biological sample may be or comprise bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; cell-containing body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or broncheoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and/or excretions; and/or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy (e.g. , fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph, feces etc.), etc. In some embodiments, as will be clear from context, the term "sample" refers to a preparation that is obtained by processing (e.g., by removing one or more components of and/or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a "processed sample" may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc. [0086] As used herein, the term "small molecule" means a low molecular weight organic and/or inorganic compound. In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., is not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic.

[0087] As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can adopt a variety of different solid forms such as, for example, amorphous forms and/or crystalline forms (e.g., polymorphs, hydrates, solvates, etc). In some embodiments, such entities may be utilized in any form, including in any solid form. In some embodiments, such entities are utilized in a particular form, for example in a particular solid form.

[0088] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0089] The term "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 (Ci-Ce), halogen (F, CI, Br, I, e.g., alkyl halos, e.g., CF3), amido, hydroxyl, carboxy/carboxylic acid, thioamido, cyano, nitro, alkenyl (C2-C6) alkynyl (C₂-C₆), azido, alkoxy (Ci-Ce), (including alkoxy groups which are further optionally substituted with a C1-C6 alkoxy group thus producing a diether group), amino, C1-C6 alkylamino and dialkyl-amino, where the alkyl groups may be optionally substituted with 1 or 2 hydroxyl groups or an amine, aminoalkyl or dialkyl group which itself is optionally substituted with one or two alkyl groups or a 7-substituted-4-quinolinyl group, C2-C6 acylamino, C2-C6 oxyacylester or carboxy ester, aryloxy, aryloxy(Ci-C6)alkyl, carboxamido, thio, C2-C6 ether or thioether, a 7-substituted-4-aminoquinolinyl group (or a substitution on an amine group which forms a 7-substituted-4-aminoqunolinyl group) and the like. Preferred substituents on alkyl groups (within context, especially on the amino group of the 7- substituted-4-aminoquinoline) or a linker which contains at least one amine group, 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 optionally 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.

[0090] The term "substituted" as used in the term "substituted aryl, substituted aromatic, substituted heteroaryl, or substituted heteroaromatic" herein signifies, for example, 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. Examples of substituents that may be present in a substituted aromatic or heteroaromatic group include, but are not limited to, groups such as H, halo (F, CI, Br or I), CN, NO2, optionally substituted C1-C6 alkyl (when substituted, preferably substituted with 1 or 2 hydroxyl groups or 3-5 fluoro groups), optionally substituted O-C1-C6 alkyl (preferably, OCH₃), optionally substituted C2-C7 acyl (preferably acetyl) or optionally substituted C2-C7 ester (oxycarbonyl ester or carboxyester, preferably carboxy ester). It is noted that each of the substituents disclosed herein may themselves be substituted.

[0091] An individual who is "susceptible to" a disease, disorder, or condition (e.g. , influenza) is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition. In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., family members of individuals suffering from the disease, disorder, or condition).

[0092] According to the present invention, "symptoms are reduced" when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g. , intensity, severity, etc.) and/or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom.

[0093] As used herein, the phrase "therapeutic agent" in general refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a "therapeutic agent" is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is an agent for which a medical prescription is required for administration to humans.

[0094] A "therapeutic regimen", as that term is used herein, refers to a dosing regimen whose administration across a relevant population is correlated with a desired or beneficial therapeutic outcome.

[0095] As used herein, a "therapeutically effective amount" is meant an amount that produces the desired effect for which it is administered. In some embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.

[0096] As used herein, the term "treatment" (also "treat" or "treating"), in its broadest sense, refers to any administration of a substance (e.g. , provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, signs, features, and/or causes of a particular disease, disorder, and/or condition . In some embodiments, such treatment may be administered to a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, in some embodiments, treatment may be administered to a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0097] As used herein, the term "variant" refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give but a few examples, a small molecule may have a characteristic core structural element (e.g., a macrocycle core) and/or one or more characteristic pendent moieties so that a variant of the small molecule is one that shares the core structural element and the characteristic pendent moieties but differs in other pendent moieties and/or in types of bonds present (single vs double, E vs Z, etc.) within the core.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Autophagy

[0098] Macroautophagy (referred to as autophagy) is an evolutionarily conserved, regulated catabolic process that degrades cellular proteins and organelles, allowing the recycling of their biochemical components for use in energy production and biosynthetic reactions (1-3). Autophagy has a role in a number of critical cell functions, including stress response, cellular quality control, tissue homeostasis, and energy production. Autophagy proceeds at a low basal level in all cells, where it is used to remove damaged proteins and organelles, particularly mitochondria, whose intracellular accumulation would be toxic. Depending on the tissue type and developmental stage, autophagy has both pro- and anti- survival effects, and its role in cancer is also contextual (3, 5, 6, 17). In early tumorigenesis, autophagy can suppress the initiation and development of early tumors, and the loss or inhibition of autophagy promotes aneuploidy and the development of the transformed phenotype (18-21). However in established tumors, inhibition of autophagy causes tumor regressions, suggesting that the autophagic process provides a survival advantage to tumors, and acts as a mechanism for overcoming stress during oncogenesis (17, 21-23).

Autophagy in cancer

[0099] Increased autophagic flux and elevated punctate expression of the autophagy- associated protein LC3 was found in a large majority of human tumors, compared to non- malignant tissue, and was associated with increased tumor proliferation, invasion and metastasis, and lower patient survival (4). A detailed mechanistic study found that autophagy has a critical role in pancreatic ductal adenocarcinomas pathogenesis, where it is expressed at high basal levels in the later stages of transformation and is required for continued malignant growth in vitro and in vivo (17,21). As the pancreatic ductal adenocarcinoma cells are dependent on autophagy, genetic or pharmacologic inhibition of autophagy leads to significant growth suppression in vitro and to a robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models.

[00100] Similarly, studies in a mouse genetic model of oncogenic K-Ras-induced lung cancer reported dramatic tumor regressions when autophagy was ablated genetically in the tumor (22). In this model, knockout of the autophagy gene atg-7 caused the regression of lung adenocarcinomas to benign oncocytomas, with eventual tumor disintegration (22). In a follow-up study, profound regression of advanced lung adenocarcinomas was observed when atg-7 expression and autophagy was shut-off in the entire mouse (23). This latter study provides pre-clinical proof of the principle that strategies to systemically inhibit autophagy may be therapeutically effective in lung cancer (2). In addition to lung and pancreatic cancers, autophagy inhibitors have single agent antiproliferative effects in vitro and in mice on a variety of neoplastic cells, including melanoma, glioma, lymphoma, colon, and breast cancer cells (11, 12, 22-26).

Chloroquine

[00101] Autophagy is dramatically increased during starvation, and many reports have described a similar upregulation in tumor cells in response to chemotherapeutic agents, including cytotoxic drugs, targeted agents, and radiation (1, 6, 7). This upregulation has been shown to be a mechanism of drug resistance, and the use of autophagy inhibitors, including chloroquine (CQ) or hydroxychloroquine (HCQ) (FIG. 1), can reverse this effect.

[00102] While CQ and HCQ are effective inhibitors of autophagy in vitro, the present invention encompasses the recognition that their in vivo efficacy may require concentrations at the upper range of tolerability (8, 9). For example, a recent phase I trial of HCQ in solid tumor patients used population pharmacokinetic modeling and found that peak whole blood concentrations (Cmax) of HCQ averaged approximately 7 μΜ with daily dosing of 1200 mg, the highest dose tested (10). In contrast, the IC5₀S for growth inhibition by HCQ ranged from 16 to 42 μΜ in a series of tumor cell lines (11-12). Thus the low potency of CQ and HCQ may limit their efficacy in vivo, and it is uncertain if levels of HCQ that cause sufficient and sustained inhibition of autophagy will be achieved in the large number of current clinical trials.

Provided Compounds

[00103] The present invention provides, among other things, certain substituted chloroquinoline triazoles. In some embodiments, provided compounds inhibit cancer cell growth and induce apoptosis, and are more potent than CQ and HCQ. To address the limitations of the current autophagy inhibitors, a series of compounds that retained the 4- aminoquinoline subunit has been synthesized. Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) was used to incorporate substituted triazoles into the parent structure to form a library of novel autophagy inhibitors with increased potency compared to CQ and HCQ. [00104] As described herein, certain provided compounds were prepared via a synthesis based on the alkyne 1 (FIG. 7, SCHEME 1) and late stage diversification by triazole formation with a wide range of substituted azides (14, 15, 27). Alkyne 1 was readily prepared from known alcohol 2 (scheme 2) (28). Chlorination was achieved by treatment with thionyl chloride and th pargylamine.

Intermediate: Alkyne 1

[00105] Triazoles 3a-3v were prepared by CuAAC reaction with the azides corresponding to 3a -3v (FIG. 8, SCHEME 2). The symmetrical analog 6 was prepared from dipropargylamine and azide corresponding to 3r (FIG. 9, SCHEME 3a). Truncated analogs 6a and 6b were prepared in analogy with compounds 3 (FIG. 9, SCHEME 3b).

Table 1. Growth inhibition by chloroquinoline

triazoles "

IC₅₀ ^M) Potency *

Compound (relative to

H460 HCC827 BxPC3 HCQ)

3h (EAD1) 11 7.6 5.8 7.0 ± 1.4

3q 12 8.5 7.6 6.0 ± 1.7

3i 12 9.3 8.7 5.7 ± 1.7

3c 14 9.5 9.1 5.2 ± 1.6 Table 1. Growth inhibition by chloroquinoline

triazoles ^a

[00106] All compounds were tested for growth inhibitory activity in three human cancer cell lines, H460 and HCC827 non-small cell lung cancer (NSCLC), and BxPC3 pancreatic cancer cells. The compounds were tested over a 100-fold concentration range with treatment for 72 hours, and IC5₀S were calculated. Representative growth inhibition curves are shown in FIG. 2, illustrating the effects of CQ, HCQ, two inactive analogs, 3v and 31, and two analogs with increased potency compared to CQ and HCQ, 3r and 3h (EAD1).

[00107] Approximately half of the synthesized compounds were more potent than CQ and HCQ, and all compounds showed similar effects on all three cell lines (Table 1). The most active compound, 3h (EAD1) (4-chlorophenyl triazole polyamine chloroquinoline; FIG. 1) had IC5₀S of 11, 7.6 and 5.8 μΜ against the H460, HCC827 and BxPC3 cells, respectively, and was on average 7-fold more potent than HCQ. Treatment with concentrations of EAD1 above 25 μΜ for 72 hours caused nearly complete cell death.

[00108] Growth inhibition by EAD1 (3h) was also demonstrated when the cells were treated with the drugs for 24 hours, and then allowed to form colonies in drug-free medium for 10 days. As shown in FIG. 3, EAD1 was greater than 10-fold more potent than HCQ in this assay. EAD1 induced apoptosis in the H460 cells in a concentration-dependent manner, as assessed by the detection of cell surface phosphatidylserine, a marker of early apoptosis, by fluorescently-labeled annexin V (FIG. 4). Apoptosis induced by EAD1 was significantly greater than that induced by CQ and HCQ at drug concentrations of 25, 50 and 75 μΜ.

Structure-activity of substituted chloroquinoline triazoles

[00109] Comparing the potency of the analogs, some trends in the structure-activity relationship were noticed: compounds with halogenated benzyl or phenyl N-substituents on the triazole were generally the most potent (e.g. 3h, 3i, 3c, etc.). Only minor differences between bromine and chlorine substituents were observed, but fluorine- containing analogs exhibited diminished activity. Ortho, meta and para-Cl substituents showed similar potency (3c, 3d and 3e). While 3q showed good activity, heteroaromatic substituents were generally less potent. Aliphatic substituents on the triazole, 3k, 31, and 3m, led to significant loss of activity. A three atom truncation of the spacer unit (compare 3r and 6b) also resulted in reduced potency. Finally, all compounds lacking the triazole-unit were very poor inhibitors, and introduction of a second triazole (14-04, 5) was also found to be detrimental to inhibitory activity.

[00110] Chloroquine is a diprotic weak base and in cells it is concentrated in the acidic environment of lysosomes, as predicted by its pKs (8.1 and 10.1), where it elevates lysosomal pH through its actions as a tertiary amine (29-30). Examination of their chemical structures suggests it is unlikely that differential effects on lysosomal pH can account for the potencies of the compounds as most potent compound (3h) and the weakest inhibitor (3m) have very similar basicity (chlorobenzyl vs. propanol sidechains), yet their inhibitory activities were remarkably different.

Substituted chloroquinoline triazoles inhibit autophagy more potently than CO and HCQ

[00111] Autophagy is a dynamic multistep process which is initiated when a portion of the cytoplasm containing the intended cargo is sequestered in a double membrane vesicle that eventually closes to form an autophagosome (2-5). The outer membrane of the autophagosome then fuses with the lysosome, and the inner material is degraded by lysosomal enzymes which are then recycled into the cell. Several proteins, known as atg proteins, are required for the various steps in the formation of the autophagosome. One of these proteins, the mammalian homolog of atg8, is also known as the microtubule-associated light chain 3 (LC3). LC3 is a widely used marker of autophagy, as it is either in a free cytoplasmic form (LC3-I) or as a lipidated form inserted into the inner and outer membranes of the autophagosome (LC3-II). HCQ and CQ inhibit the autophagic process at a late stage, leading to the accumulation of autophagosomes and an increase in LC3-II in treated cells (31).

[00112] The ability of EADl to inhibit autophagy by measuring autophagosome levels in lung cancer cells was evaluated using a mCherry-GFP tandem fluorescent-tagged LC3B reporter protein (32). When LC3 is localized on the autophagosome membrane, the mCherry-GFP-LC3 forms fluorescent puncta. As seen in FIG. 5, few control cells had punctate signals, and most cells had a diffuse LC3 expression. Both HCQ and EADl increased the punctate LC3 signal, with a larger increase seen in EADl -treated cells than for HCQ at the two concentrations tested. LC3 levels were also evaluated by immunoblot of drug-treated H460 cells, which allows for the distinction between cytoplasmic LC3-I and autophagosome-bound LC3-II (31). While both HCQ and EADl caused concentration and time-dependent increases in LC3-II levels (FIGS. 6a and 6b), the increase was larger at each concentration for EADl than for HCQ. The effect of 25 μΜ HCQ on LC3-II was approximately equal to that of 5 μΜ EADl (FIG. 6a).

[00113] The effects of HCQ and EADl were also compared on a second autophagy- associated protein, p62, a substrate of the autophagic process that is selectively incorporated into autophagosomes through direct binding to LC3 and is efficiently degraded by autophagy (31). Inhibition of autophagy would be anticipated to lead to the accumulation of p62, and this was observed in a concentration-dependent manner with both HCQ and EADl, although as with LC3-II, EADl had a more potent effect than did HCQ (FIG. 6c). To determine if the inhibition of autophagy was related to the growth inhibitory effect of the compounds, cells were treated with a group of the compounds which were selected to encompass the range of activity observed. As shown in FIG. 6d, the extent of LC3-II accumulation was closely linked to the corresponding compound's IC5₀, such that compounds with little or no antiproliferative activity (3s and 3v) did not cause an increase in LC3-II, while the most active compounds (3h, 3i and 3r) produced the largest increase. There was a high degree of correlation between the inhibition of autophagy and a compound's potency as an inhibitor of cell growth, consistent with the hypothesis that growth inhibition was a consequence of the inhibitory effect on autophagy.

Pharmaceutical Compositions

[00114] Compounds according to the present invention may be readily formulated into pharmaceutical compositions, with a variety of uses including, in some embodiments, 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.

[00115] As noted above, 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.

[00116] In methods according to 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. [00117] Generally, 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.

[00118] 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.

[00119] Pharmaceutical 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. Preferably, 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. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. If desired, the composition may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, or buffers.

[00120] 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. For use in oral liquid preparation, 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.

[00121] When the composition is employed in the form of solid preparations for oral administration, the preparations may be tablets, granules, powders, capsules or the like. In a tablet formulation, 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.

[00122] An injectable composition for parenteral administration will typically contain the compound in 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.

[00123] A wide variety of dosage forms and methodologies for preparing them is known or is apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co., 1985).

[00124] In many embodiments, compositions for administration as described herein 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.

Uses

[00125] According to one aspect of the invention, compounds described herein are administered to a subject in need thereof, e.g., to inhibit autophagy and/or to inhibit, treat, and/or prevent one or more disease states and/or conditions including cancer (including metastasis of cancer), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

[00126] In certain embodiments, 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. In some embodiments, the cancer is one for which inhibition of autophagy represents a favorable result and/or for which metastasis is a risk element. In certain embodiments, the cancer is drug resistant cancer.

[00127] According to some embodiments of the present invention, 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, e.g., that is useful (and/or approved) for treating the same disease state or condition.

[00128] In some embodiments, an effective amount of a provided compound may be defined as an amount equivalent to (i.e., that shows comparable activity in a relevant assay and/or under a relevant set of conditions) to a known amount of CQ or HCQ. CQ and HCQ are typically dosed in amounts up to 1200 mg per day (e.g., for autophagy inhibition and/or for treatment of malaria), up to 600 mg per day (e.g., for rheumatoid arthritis), and up to 400 mg per day (e.g., for systemic lupus erythematosus). Lower doses are often employed for chronic treatment.

[00129] As already noted, in certain embodiments, provided compounds are administered in combination with one or more additional therapeutic agents, e.g., that is/are effective and/or approved for treatment of the same disease, disorder or condition (e.g., cancer). For example, in some embodiments, provided compounds are administered in combination with one or more anticancer agents.

[00130] Representative such anticancer agents may include, for example, one or more of 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 inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitors, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase kinase (mek) inhibitor, a VEGF trap antibody, pemetrexed, erlotinib, dasatanib, nilotinib, decatanib, panitumumab, amrubicin, oregovomab, Lep-etu, nolatrexed, azd2171, batabulin, SUBSTITUTE SHEET (RULE 26) ofatumumab, zanolimumab, edotecarin, tetrandrine, rubitecan, tesmilifene, oblimersen, ticilimumab, ipilimumab, gossypol, Bio 111, 131-I-TM-601, ALT- 110, BIO 140, CC 8490, cilengitide, gimatecan, IL13-PE38QQR, INO 1001, IPdR.sub. lKRX-0402, lucanthone, 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-fiuorouridine, vincristine, temozolomide, ZK-304709, seliciclib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-lH-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]- benzoyl]- , disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, ClHIR-258,); 3-[5- (methylsulfonylpiperadinemethyl)-indolyl]-quinolone, vatalanib, AG-013736, AVE-0005, the acetate salt of [D-Ser(Bu t) 6,Azgly 10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(Bu t)-Leu-Arg- Pro-Azgly-NH₂ acetate

where x=l to 2.4], goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxy progesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatanib, canertinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, lonafamib, BMS-214662, tipifamib; amifostine, NVP-LAQ824, suberoyl analide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU111248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L- asparaginase, Bacillus Calmette-Guerin (BCG) vaccine, bleomycin, buserelin, busulfan, carboplatin, cannustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevac, hydroxyurea, idarubicin, ifoslamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinoic acid, phenylalanine mustard, uracil mustard, estramustine, altretamine, floxuridine, 5 -deooxy uridine, cytosine arabinoside, 6- mercaptopurine, deoxycoformycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, droloxifene, idoxyfene, spironolactone, linasteride, cimitidine, trastuzumab, denileukin diftitox, gefitinib, bortezimib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, cremophor-free paclitaxel, epithilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifen, pipendoxifene, ERA-923, arzoxifene, fulvestrant, acolbifene, lasofoxifene, idoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK 222584, VX-745, PD 184352, rapamycin, 40-O-(2-hydroxyethyl)- rapamycin, temsirolimus, AP-23573, RADOOl, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepoetin, erythropoietin, granulocyte colony-stimulating factor, zolendronate, prednisone, cetuximab, granulocyte macrophage colony-stimulating factor, histrelin, pegylated interferon alfa-2a, interferon alfa-2a, pegylated interferon alfa-2b, interferon alfa-2b, azacitidine, PEG- L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-transretinoic acid, ketoconazole, interleukin-2, megestrol, immune globulin, nitrogen mustard, methylprednisolone, ibritgumomab tiuxetan, androgens, decitabine, hexamethylmelamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, liposomal daunorubicin, Edwina-asparaginase, strontium 89, casopitant, netupitant, an NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, pegfilgrastim, erythropoietin, epoetin alfa and darbepoetin alfa, among others, and mixtures thereof.

[00131] In the present invention, the method of treatment comprises administering to the subject in need of treatment, in a pharmaceutically acceptable carrier, an effective amount of a compound.

[00132] Further preferred methods relate to the use/administration of the compounds according to the present invention which are presented in the various schemes.

[00133] In addition to treatment methods as described herein, the present invention provides, among other things, various technologies for identifying and/or characterizing compounds of interest. The present disclosure describes a variety of assays and systems for assessing one or more characteristics, activities, and/or attributes of compounds; those of ordinary skill in the art will appreciate that these or other available systems may be utilized, for example to identify compounds of interest by analysis of one or more test compounds and/or to characterize compounds (and/or compositions or preparations that contain them).

EXEMPLIFICATIONS

Chemicals

[00134] Chloroquine and hydroxychloroquine sulfate were obtained from Spectrum Chemicals and Sigma, respectively. Chloroquine analogs were dissolved in H₂0 and were stored at -20° C.

[00135] All chemical reagents and solvents were obtained from commercial sources (Aldrich, Acros, Fisher) and used without further purification.

[00136] Anhydrous solvents (tetrahydrofurane and toluene,) were obtained using a Pure Solv AL-258 solvent purification system. Dimethyl formamide was dried over activated 4 A molecular sieves.

Analytics

[00137] Chromatography was performed on a Teledyne ISCO CombiFlash Rf 200i using disposable silica cartridges (4, 12, and 24 g). Solvent mixtures used for chromatography are: Ultra (75:22:3 CH₂Cl+:CH₃OH:NH₄OH) and EEA (ethyl acetate:ethyl alcohol 3: 1 + 2% NH₄OH). [00138] Analytical thin layer chromatography (TLC) was performed on Silicycle silica gel plates (250 μηι film thickness, indicator F254). Compounds were visualized using a dual wave length (254 and 365 nm) UV lamp.

[00139] NMR spectra were recorded on Bruker DRX 300 and DRX 600 spectrometers. ^XH and ¹ C chemical shifts (δ) are reported relative to tetramethyl silane (TMS, 0.00/0.00 ppm) as internal standard or to residual solvent (CD₃OD: 3.31 ppm/49.00 ppm; CDCI₃: 7.26/77.16 ppm; DMSO-d6: 2.50/39.52 ppm).

[00140] Low resolution mass spectra were recorded on a Shimadzu LCMS 2010EV (direct injection).

[00141] High resolution electrospray ionization mass spectra (ESI-MS) were obtained at the Albert Einstein College of Medicine Laboratory for Macromolecular Analysis and Proteomics. IR spectra were recorded on an Agilent Cary 630 FTIR on a ZnSe crystal. Wave numbers are reported in cm^-1.

Cell Lines

[00142] Human H460, HCC827, and BxPC3 cell lines were from ATCC (Manassas, VA), and were maintained in RPMI-1640 medium with 10% fetal bovine serum, penicillin and streptomycin, at 37° C in a humidified atmosphere with 5% CO2.

Cell Proliferation

[00143] Cells were seeded in 96 well plates in triplicate (2-4 x lOVwell) and allowed to attach for 24 hours before different concentrations of the drugs were added for an additional 48 or 72 hours. Cell proliferation was quantified by a sulforhodamine B (SRB) assay. Attached cells were fixed with 10% trichloroacetic acid and incubated for 1 hour at 5° C. The cells were stained with SRB (0.4% in 1% acetic acid) by incubating at room temperature for 30 minutes. The plate was rinsed 4x with 1% acetic acid and dried. The SRB was dissolved by adding 10 mM Tris-base. Absorbances were read at 510 nm on plate reader. Cell numbers were expressed relative to untreated controls, and IC5₀S were calculated from concentration-response graphs. Data are means ± SD of at least 3 experiments.

[00144] For the colony growth assay, H460 cells were plated at 250 cells per well in 24 well plates. After allowing for cell attachment ovemight, HCQ and EADl were added at the indicated concentrations. After 24 hours, drug-containing medium was removed, and drug- free medium added. Cell colonies were stained with crystal violet after an additional 10-day growth in the absence of drug. Apoptosis assay

[00145] The detection of apoptosis was assessed using APC-conjugated Annexin V (ebioscience) to determine phosphatidylserine exposure, and DAPI staining to assess cell viability. H460 cells were cultured in medium containing 5, 25, 50 or 75 μΜ of HCQ, CQ or EAD1 for 24 hours. For quantitative determination, cells were trypsinized, stained with Annexin V for 15 minutes at room temperature, and analyzed by flow cytometry. Each experiment was performed in triplicate.

Immunoblot analysis

[00146] Cells were scraped from culture dishes, cell lysates were prepared, and immunoblot analysis was performed. Cell extracts were prepared in cold lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM Ethylenediaminetetraacetic acid(EDTA), 1% Triton XI 00, 200 μΜ Na orthovanadate and protease inhibitor cocktail (HALT; Thermo Scientific)). Protein concentrations were determined using the Lowry reagent (Bio-Rad), and normalized cell lysates were mixed with sample buffer (Bio-Rad) containing 2- mercaptoethanol and boiled for 5 mins. The samples were run on SDS-polyacrylamide gels and transferred to nitrocellulose or PVDF membranes. The membranes were incubated overnight with primary antibodies in TBS-T buffer containing 5% non-fat milk: LC3 (Cell Signaling LC3 A/B (D3U4C) and p62 (Sigma Anti-p62/SQSTMl rabbit #P0067). After washing, the membranes were incubated with HRP-conjugated secondary antibody for 1 hour. The bands were detected with enhanced chemiluminescence reagent (Pierce).

LC3 Puncta Analysis

[00147] Generation of a stable cell line expressing mCherry-EGFP-LC3B. H3122 NSCLC cells were grown to 50% confluency and were then transfected with the pDest-mCherry- EGFP-LC3B plasmid (kindly provided by Dr. Ana Maria Cuervo) using Lipofectamine 2000 reagent (Invitrogen). Briefly, a mixture of plasmid DNA and Lipofectamine were diluted in 200 μΐ and added to cells in 3 ml of medium without antibiotics. Transfected cells were then expanded and fluorescent-positive clones isolated by two reiterated cycles of cell sorting by FACS. The steady-state expression level was confirmed with fluorescence microscopy.

Synthesis of intermediate alkyn

Intermediate: Alkyne 1

[00148] The present invention relates to the compound according to intermediate: Alkyne 1, an intermediate in the synthesis of the compounds shown in chemical structures (EAD1) and others. An efficient synthesis of Intermediate: Alkyne 1 based on a published procedure (16) has been established. N¹-(2-chloroethyl)-N²-(7-chloroquinolin-4-yl)-N¹-methylethane- 1,2-diamine 2-((2-((7-chloroquinolin-4-yl)amino)ethyl)(methyl)amino)ethan-l-ol¹ (200 mg; 0.72 mmol) and toluene (5 mL) were added to a flask and thionyl chloride (0.16 mL; 2.15 mmol; 3 equiv) was added slowly. After stirring for 18 hours the reaction was quenched by careful addition of saturated aqueous NaHCC until no more gas-evolution was observed. The mixture was transferred to a separatory funnel and the pH was adjusted to 8 by addition of aqueous NaHCCb. Extraction with ethyl acetate (4 x 5 mL), drying of the combined organic phases (MgSC^), and removal of the solvent gave the title compound (189 g; 0.63 mmol; 89%) which was used without further purification. The ^XH NMR spectrum is in agreement with the literature values (33). Dimethylformamide (30 mL), propargylamine (0.92 mL; 14.4 mmol; 10 equiv.) and sodium iodide (43 mg; 0.29 mmol; 0.2 equiv.) were added to a flask containing SIl (428 mg; 1.44 mmol). After stirring for 18 hours, a distilation head was attached and the volatiles were distilled off in vacuo. The residue was purified by column chromatography (0-20% Ultra in CH2C12) to give 1 as a pale orange oil (300 mg; 0.947 mmol; 66%).; TLC: R_f = 0.29 (CH₂Cl₂:Ultra 2: 1 ; UV).; ^XH NMR (600 MHz, CDC1₃) δ 8.49 (d, J = 5.5 Hz, 1H), 7.99 (d, J = 2.6 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.38 (dd, J = 8.8, 2.1 Hz, 1H), 6.38 (d, J = 5.5 Hz, 1H), 3.48 (d, J = 2.3 Hz, 2H), 3.37 - 3.28 (m, 2H), 2.89 (t, J = 5.9 Hz, 2H), 2.80 (t, J = 6.0, 2H), 2.65 (t, J = 5.9 Hz, 2H), 2.33 (s, 3H), 2.23 (t, J = 2.4 Hz, 1H).; ¹ C NMR (151 MHz, CDC1₃) δ 150.61, 135.45, 127.50, 125.53, 121.98, 117.14, 99.02, 81.82, 71.77, 56.51, 54.66, 45.78, 41.82, 40.19, 38.17.; HRMS (ESI) calc'd for C17H22CIN4 (M+H)⁺ 317.1528 found 217.1559.; IR (Neat): 3300 (w), 2846 (w), 2804 (w), 1610 (w), 1578 (s), 1449 (m), 1329 (m). ynthesis of chloroquinoline triazole compounds

[00149] The alkyne (intermediate alkyne 1; 0.347 mmol), azide (N₃R, 0.69 mmol; 2 equiv.), acetone (5 mL), water (2.5 mL), sodium ascorbate (1 M in H₂0; 0.347 mL; 0.347 mmol; 1 equiv.) and copper(II) sulfate (1 M in H2O; 0.035 mL; 0.035 mmol; 10 mol%) were added to a vial and stirred vigorously. When TLC analysis showed full conversion of the alkyne the acetone was remove under reduced pressure and the solution was made basic by addition of concentrated ammonia. 5 mL brine was added and the product was extracted with CH2CI2 (3 x 5 mL). The combined organic extracts were dried over MgSC^, filtered and concentrated. Column chromatography gave the desired trizole. Treatment with HCl in diethyl ether followed by evaporation of the volatiles have the corresponding HCl salt which showed improved water solubility.

A lead compound EAD1

N¹-((l-(4-chlorophenyl)-lH-l,2,3-triazol-4-yl)methyl)-N²-(2-((7-chloroquinolin-4- y l)amino)ethy l)-N²-methy lethane- 1 ,2-diamine

[00150] Column: 50-70% EEA in hexanes. Yield: 22%. Pale orange powder; TLC: R_f = 0.27 (CH₂Cl₂:Ultra 2: 1 ; UV); ^lH NMR (600 MHz, D₂0) δ 8.44 (d, J = 7.1 Hz, 1H), 8.23 (s, 1H), 8.19 (dd, J = 9.1, 3.3 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.72 - 7.56 (m, 1H), 7.36 (dd, J = 8.6, 2.8 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 7.1 Hz, 1H), 5.61 (s, 2H), 4.51 (s, 2H), 4.09 (t, J = 6.3 Hz, 2H), 3.83 - 3.69 (m, 4H), 3.65 (dd, J = 9.3, 5.7 Hz, 2H), 3.10 (s, 3H); ¹ C NMR (151 MHz, D₂0) δ 156.16, 142.95, 139.70, 138.00, 137.56, 134.00, 133.13, 129.54, 128.99, 127.96, 126.66, 124.25, 119.27, 115.56, 98.70, 54.21, 53.32, 51.62, 41.73, 40.62, 40.26, 37.81.; HRMS (ESI) calc'd for C₂4H₂₈C1₂N7 (M+H)⁺ 484.1778 found 484.1760.; IR (Neat): 3226 (w), 2937 (m), 2625 (m), 1610 (s), 1591 (s), 1452 (s). EQUIVALENTS

[00151] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow.

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Claims

structure set forth in Formula I:

wherein

Ri, R-2, and R3 are each independently H, halogen, CN, SO2CH₃, acyl, alkyl, alkyl halo, optionally substituted OCi-C₆ alkyl, OCF₃, OH, or NRR' ; each El is independently N or CH; each E2 is independently NH, NR, O, or CRR' ; each E3 is independently CH, N, O, or S;

R and R' are each independently H or optionally substituted C1-C6 alkyl; wherein the optionally substituted OC1-C6 alkyl or optionally substituted C1-C6 alkyl is optionally substituted with one or more hydroxyl, halogen, =0, C1-C6 alkyl, alkyl halo, amido, thioamido, carboxamido, carboxy acid, carboxylic acid, cyano, nitro, C2-C6 alkenyl, C2-C6 alkynyl, C2-C7 acyl, azido, C1-C6 alkoxy, amino, C1-C6 alkylamino, C1-C6 dialkyl- amino, C2-C6 acylamino, C2-C6 oxyacylester, C2-C6 carboxy ester, aryloxy, aryloxy(Ci- C6)alkyl, thio, C2-C6 ether, C2-C6 thioether, 7-substituted-4-aminoquinolinyl, monoalkyl amine or dialkyl amine groups; n is 0, 1, or 2; and R4 is optionally substituted alkyl, optionally substituted aryl, optionally substituted aromatic, optionally substituted heteroaryl, or optionally substituted heteroaromatic, wherein the optionally substituted alkyl, optionally substituted aryl, optionally substituted aromatic, optionally substituted heteroaryl, or optionally substituted heteroaromatic is optionally substituted with one or more hydroxyl, halogen, =0, C1-C6 alkyl, OC1-C6 alkyl, alkyl halo, amido, thioamido, carboxamido, cyano, nitro, C2-C7 acyl, C2-C7 oxycarbonyl ester, C2-C7 oxyacylester, or C2-C7 carboxy ester groups; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein each halogen is independently F, CI, Br or I.

3. The compound of claim 1 or 2, wherein each alkyl is independently C1-C₃ alkyl.

4. The compound of any of claims 1-3, wherein one or more alkyl halo is CF₃.

5. The compound of any of claims 1-4, wherein one or more OC1-C6 alkyl is OCH₃.

6. The compound of any of claims 1-5, wherein Ri and R2 are each independently CI or

7. The compound of any of claims 1-6, wherein R₃ is H.

8. The compound of any of claims 1-7, wherein R4 is optionally substituted benzyl.

9. The compound of claim 1 having the structure

or a pharmaceutically acceptable salt thereof.

10. The compound of claim 1 or 9, wherein R₄ is C₆H₅, C₆H₄(4-F), C₆H₄(4-C1), C₆H₄(3-C1), C₆H₄(2-C1), C₆H₄(4-Br), CH₂C₆H_5, CH₂C₆H₄(4-C1), CH₂C₆H₄(4-Br), CH₂CH₂C₆H₅, C(CH₃)₃,

of i o N.

11. The compound of claim 1 or 9 having the structure

or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition comprising one or more compounds of any of claims 1- 11, and a pharmaceutically acceptable carrier, additive or excipient.

13. A pharmaceutical composition comprising 1 to 2000 mg of a compound of any of claims 1-11, and a pharmaceutically acceptable carrier, additive or excipient.

14. A method for treating a subject suffering from or susceptible to an autophagy associated disease, disorder or condition comprising administering to the subject a compound of any of claims 1 -11 or a pharmaceutical composition of claim 12 or 13.

15. The method of claim 14 wherein the compound or pharmaceutical composition is administered in an amount effective to ameliorate a sign or symptom of the autophagy associated disease, disorder or condition in a subject.

16. The method of claim 14 or 15, wherein the compound or pharmaceutical composition is administered in an amount effective to inhibit autophagy in a subject.

17. The method of any of claims 14-16, wherein the autophagy associated disease, disorder or condition is or comprises cancer.

18. The method of claim 17, wherein the cancer is or comprises a carcinoma; a sarcoma; a neuroectodermal tumor; cancer of the breast, esophagus, colon, rectum, head, kidney, liver, lung, nasopharyngeal, neck, ovary, pancreas, skin, brain, CNS, prostate, or stomach; a leukemia; a malignant lymphoma; and combinations thereof.

19. The method of claim 17 or 18, further comprising administering to the subject at least one additional active agent.

20. The method of claim 19, wherein the at least one additional active agent is or comprises an anticancer agent.

21. The method of claim 20, wherein the anticancer agent is selected from the group consisting of everolimus, trabectedin, and abraxane.

22. The method of any of claims 14-16, wherein the autophagy associated disease, disorder or condition is one or more of rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

23. A method of treating a disease, disorder or condition selected from the group consisting of cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the method comprising administering to the subject a compound of any of claims 1-11 or a pharmaceutical composition of claim 12 or 13 in an amount effective to inhibit autophagy in a subject.

24. A compound of any of claims 1 -11 or a pharmaceutical composition of claim 12 or 13 for treating a subject suffering from or susceptible to an autophagy associated disease disorder or condition, or for treating cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria or Sjogren's disease.

25. Use of a compound of any of claims 1 -1 1 or a pharmaceutical composition of claim 12 or 13 for treating a subject suffering from or susceptible to an autophagy associated disease disorder or condition, or for treating cancer, rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria or Sjogren's disease.

26. A method of providing a compound of claim 1 , the method comprising steps of:

chlorinating an alcohol in a compound of structure

that a chloride moiety is added,

replacing the chloride with propargylamine;

performing azide-alkyne cycloaddition

so as to provide a compound of claim 1.

27. The method according to claim 26 further comprising determining one or more characteristics or activities of the compound.

A compound selected from the group consisting of:

wherein R is H or CH₃.

29. A collection of at least 100 compounds of claim 1, wherein each compound has the following structure

or a pharmaceutically acceptable salt or salts thereof.