Patent Grant
10047052
The present invention relates to novel bisaminoquinoline compounds, pharmaceutical compositions comprising these novel compounds and methods for inhibiting autophagy in biological systems. Methods of treating cancer in patients in need using compounds and/or compositions according to the present invention alone or in combination with at least one additional anticancer agent represent additional aspects of the invention. The use of radiation therapy in combination with the present compounds, alone or in combination with an additional anticancer agent as otherwise disclosed herein, represents an additional aspect of the invention. Methods of treating disease states and/or conditions in which inhibition of autophagy plays a favorable treatment role including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, with compounds according to the present invention represent additional aspects of the invention.
Autophagy consists of the sequestration of organelles and proteins in autophagic vesicles (AV) and degradation of this cargo through lysosomal fusion (1). Autophagy allows tumor cells to survive metabolic and therapeutic stresses (2-5). Multiple publications indicate therapy-induced autophagy is a key resistance mechanism to many anti-cancer agents. Chloroquine (CQ) (Compound 1,
More recently, Lee (16) has described the potentiation of AKT inhibitors by fluorinated quinoline analogs. Solomon (17) has reported the preparation of “repositioned” chloroquine dimers, based on the use of a piperazine connector. These results suggest that these chloroquine analogs could serve as bases for the development of a new group of effective cancer chemotherapeutics.
It is an object of the invention to provide novel compounds for inhibiting autophagy in biological systems, especially including patients or subjects in need.
It is another object of the invention to treat disease states and/or conditions in which inhibition of autophagy is beneficial to the disease states and/or conditions a patient or subject.
It is yet a further object of the invention to provide pharmaceutical compositions which may be used to inhibit autophagy, especially autophagy associated with disease states and/or conditions including cancer and its metastasis.
It is still a further object of the invention to inhibit, treat or prevent cancer, including the metastasis of cancer in patients or subjects in need utilizing compounds, compositions and/or methods which are presented herein.
It is still another object of the invention to inhibit, treat or prevent diseases in which the inhibition of autophagy provides a favorable effect, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, among others.
Any one or more of these and/or other objects of the invention may be readily gleaned from a description of the invention which follows.
The present invention relates to compounds according to the chemical structure I
In preferred aspects of the invention, R1 and R1′ are each independently H, a halo group, a nitro group or a trifluoromethyl group, preferably a chloro group. R and R′ are preferably each independently H, a C1-C3 optionally substituted alkyl group itself preferably substituted with at least one hydroxyl group, an alkoxy group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with one or two 7-substituted-4-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with a R1 and/or R1′ group as broadly described for generic structure I above, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with R1 and/or R1′ as broadly described for generic structure I above, and each of said alkoxy groups (e.g. methoxy or ethoxy) is optionally further substituted with an alkoxy group, preferably a methoxy group, thus forming a diether substituent.
In certain preferred aspects of the invention L is a
group, where X is N—R″, Y and Y′ are each independently absent or CH2, and R″ is H or a C1-C3 alkyl group which is optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with one or two 7-substituted-4-quinolinyl group wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with R1 and/or R1′ as broadly described for generic structure I above, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an alkoxy group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) wherein the amine binds to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably with R1 and/or R1′ as broadly described for generic structure I above, and each of said alkoxy groups (e.g. methoxy or ethoxy) is optionally further substituted with an alkoxy group, preferably a methoxy group, thus forming a diether substituent.
Further preferred compounds according to the present invention include those which are presented in the various schemes which are presented in Scheme 1 and Schemes 3-10 and
In another aspect of the invention, a pharmaceutical composition comprises a compound according to formula I above or as otherwise described herein in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional anticancer agent.
Methods of inhibiting autophagy in a biological system, in particular a patient or subject is a further aspects of this invention. In this aspect of the invention, a bisaminoquinoline compound as otherwise described herein is presented to the biological system, including administration to a patient or subject in need, in order to inhibit autophagy. The resulting inhibition may be monitored or applied in the biological system to effect a favorable result, including the inhibition, treatment and/or prevention of cancer, including metastasis of cancer, or the inhibition, treatment and/or prevention of one or more disease states or conditions in which the inhibition of autophagy provides a favorable result including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, among others.
Methods of inhibiting, treating and/or reducing the likelihood of cancer, including metastasis of cancer and drug resistant cancer, comprises administering to a patient in need at least one compound according to the present invention, optionally in combination with at least one additional anticancer agent as otherwise described herein.
The present invention also relates to treating, inhibiting and/or preventing diseases, diseases states and/or conditions in a patient in need in which the inhibition of autophagy provides a favorable outcome, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the method comprising administering to said patient at least one compound according to the present invention.
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.
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 is 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 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.
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.
It must be noted that as used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.
Furthermore, the following terms shall have the definitions set out below.
The term “patient” or “subject” is used throughout the specification within context to describe an animal, generally a mammal, especially including a domesticated animal and preferably a human, to whom treatment, including prophylactic treatment (prophylaxis), with the compounds or compositions according to the present invention is provided. For treatment of those infections, conditions or disease states which are specific for a specific animal such as a human patient, the term patient refers to that specific animal. In most instances, the patient or subject of the present invention is a human patient of either or both genders.
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 its use, 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.
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.
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.
The terms “treat”, “treating”, and “treatment”, are used synonymously to refer to any action providing a benefit to a patient at risk for or afflicted with a disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc.
Treatment, as used herein, encompasses both prophylactic and therapeutic treatment, principally of cancer. Compounds according to the present invention can, for example, be administered prophylactically to a mammal in advance of the occurrence of disease to reduce the likelihood of that disease. Prophylactic administration is effective to reduce or decrease the likelihood of the subsequent occurrence of disease in the mammal, or decrease the severity of disease that subsequently occurs, especially including metastasis of cancer. Alternatively, compounds according to the present invention can, for example, be administered therapeutically to a mammal that is already afflicted by disease. In one embodiment of therapeutic administration, administration of the present compounds is effective to eliminate the disease and produce a remission or substantially eliminate the likelihood of metastasis of a cancer. Administration of the compounds according to the present invention is effective to decrease the severity of the disease or lengthen the lifespan of the mammal so afflicted, in the case of cancer.
The term “pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.
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.
The term “prevention” when used in context shall mean “reducing the likelihood” or preventing a disease, condition or disease state from occurring as a consequence of administration or concurrent administration of one or more compounds or compositions according to the present invention, alone or in combination with another agent. It is noted that prophylaxis will rarely be 100% effective; consequently the terms prevention and reducing the likelihood are used to denote the fact that within a given population of patients or subjects, administration with compounds according to the present invention will reduce the likelihood or inhibit a particular condition or disease state (in particular, the worsening of a disease state such as the growth or metastasis of cancer) or other accepted indicators of disease progression from occurring.
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 consists of the sequestration of organelles and proteins in autophagic vesicles (AV) and degradation of this cargo through lysosomal fusion (1). Autophagy allows tumor cells to survive metabolic and therapeutic stresses (2-5). Multiple publications indicate therapy-induced autophagy is a key resistance mechanism to many anti-cancer agents.
Diseases, disease states and/or conditions which benefit from the inhibition of autophagy include cancer (including the metastasis of cancer), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.
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 also within context, 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 and are principal target cancers for compounds and therapies according to the present invention.
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, 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, panitunumab, 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-PE39QQR, INO 1001, IPdR1, 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, seliciciib; PD0325901, AZD-6244, capecitabine, L-Glutamic acid, N-[4-[2-(2-amino-4,7-dihydro-4-oxo-1H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-, disodium salt, heptahydrate, camptothecin. PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrazole, exemestane, letrozole, DES(diethylstilbestrol), estradiol, estrogen, conjugated estrogen, bevacizumab, IMC-1C11, CHIR-258); 3-[5-(methylsulfonylpiperadinemethyl)-indolylj-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 [C59H84N18Oi4-(C2H4O2)X where x=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin 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; amifostine, 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, bleomiycin, buserelin, busulfan, carboplatin, carmnustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, diethyistilbestrol, 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-deoxyuridine, cytosine arabinoside, 6-mecaptopurine, 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-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, RAD001, 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.
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 are methyl, ethyl, n-butyl, n-heptyl, isopropyl, 2-methyl propyl, tert-butyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.
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 (C1-C6), halogen (F, Cl, 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 (C1-C6), (including alkoxy groups which are further 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 substituted one or two alkyl groups or a 7-substituted-4-quinolinyl group, C2-C6 acylamino, C2-C6 oxyacylester or carboxyester, aryloxy, aryloxy(C1-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 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.
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). 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.
The term “substituted” as used in the term “substituted aryl, substituted aromatic, substituted heteroaryl, or substituted heteroaromatic” herein signifies that a substitution on the 7-position of 4-aminoquinoline may be present, said substituents being selected from atoms and groups, which when present enhance the activity of the compound as an inhibitor of autophagy. 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, Cl, 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 carboxyester). It is noted that each of the substituents disclosed herein may themselves be substituted.
The term “co-administration” or “adjunct therapy” shall mean that at least two compounds or compositions are administered to the patient at the same time, such that effective amounts or concentrations of each of the two or more compounds may be found in the patient at a given point in time. Although compounds according to the present invention may be co-administered to a patient at the same time, the term embraces both administration of two or more agents at the same time or at different times, including sequential administration. Preferably, effective concentrations of all co-administered compounds or compositions are found in the subject at a given time. The term co-administration or adjunct therapy also contemplates other bioactive agents being coadministered with pharmaceutical compositions according to the present invention, especially where a cancer has metastasized or is at risk for metastasis.
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 can't 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.
Pharmaceutical Compositions
Compounds according to the present invention may be readily formulated into pharmaceutical compositions, useful in the inhibition of autophagy in a biological system and/or the inhibition, treatment or prevention of diseases states and/or conditions which benefit from the inhibition of autophagy including cancer (and its metastasis), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus (systemic lupus erythematosus), chronic urticaria and Sjogren's disease. Pharmaceutical compositions comprise an effective amount of one or more compounds according to the present invention in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional agent, in the case of cancer, preferably an anticancer agent as otherwise described herein.
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.
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.
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.
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.
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.
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.
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.
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.
Methods for preparing such dosage forms are known or is apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (17th Ed., Mack Pub. Co., 1985). The composition to be administered will contain a quantity of the selected compound in a pharmaceutically effective amount for inhibiting autophagy in a biological system, including a patient or subject according to the present invention.
Synthesis of Compounds According to the Present Invention
Strategy for Synthesis of Bivalent Aminoquinoline Autophagy Inhibitors
The inventors have examined the application of the strategy of polyvalency (11, 12) to the synthesis of novel autophagy inhibitors by preparing a dimeric chloroquine (
While compound 4 (R═Cl) is a known compound (14), the bisquinoline compound 3 (R2=Me) has not been described in the literature. Due its putative lysosomotropism, we refer to compound 3 as Lys01. Reaction of compound 5 with two equivalents of compound 4 led to the formation of a mixture of the desired product compound 3 along with some of the monoquinoline compound 7 (
To determine the importance of the polyamine connector of compound 3, we prepared the polyether analog compound 11 (
In an effort to obtain SAR data on the lead compound 3 (Lys01), the inventors examined the systematic modification of the structure of compound 3 (i.e., 12 R═Cl), as outlined in Scheme A of
Each of the requisite starting compounds outlined in
The inventors also examine the biological activity of N-alkyated and N-acylated analogs of compound 12, compound 17 and compound 18 (
The inventors also examine the introduction into compound 12 of the chirality that is present in CQ and HCQ (
Finally, the modification of the structure of compound 12 via replacement of the N-methyl group (Scheme A of
While characterization of the second generation compounds described continue, there are additional compounds which are believed to represent more potent autophagy inhibitors. Taking the lead compound as compound 12 (scheme 1; Lys01), the inventors describe more extensive systematic modification of three parts of the structure of 12, as outlined in the generic structure 23 (see above) as the next logical step in SAR analysis. Each of the three parts of the structure (R1, R2 and R3) is modified, as outlined below.
The Role of R1
The effect of substituting the chlorine moiety in chloroquine 1 (scheme 2) has been examined
by Egan and coworkers (20), who established that electron-withdrawing groups are important for the antiplasmodial activity of these 7-substituted quinolines. The inventors therefore examined these same substitutions in the case of the generic structure 23, substituting both R1 and R1′ in 23, and also examining the effect of monosubstitution, in which the R1′ substituent is then either Cl as in 12 or hydrogen (X═H). The Egan study indicates that all of the requisite 4-chloro-7-substituted-quinolines are known, thereby facilitating the preparation of each of the compounds shown in Scheme 3. None of the compounds outlined in scheme 3 are known, although the synthesis of linked bis-quinolines (R1=H) has been described (21).
The Role of R2
The role of R2═H in lead structure 3 is examined by either acetylation (R2=MeCO—; mono- or di-) or methylation (R2=Me; mono- or di-) to give the structures 41-44 shown in Scheme 4. While the homologous analogs 45 and 46 (containing one or two propylene chains between the nitrogen atoms) are known (FR 1345573; CAN 60:68181), each of the analogs 41-44 represents a novel structure.
Well-tolerated substitution at R2 further directs substitution of R2 with a hydroxyethyl moiety, as found in hydroxychloroquine 2 (scheme 5). The reaction of anilines with ethylene oxide to give the corresponding hydroxyethyl compounds is well-precedented (22), so that conversion of the lead structure 3 to either mono- or di-hydroxyethylated analogs 47 and 48 is readily achieved.
The inventors also examine the preparation of tri- and tetra-quinoline containing structures by oxidation of the primary alcohols in 47 and 48 to give the corresponding aldehydes, which on reductive alkylation with 7-chloro-4-aminoquinoline gives the tri- and tetraquinolines 49 and 50, based on the work of Bailey and coworkers (23), as shown in scheme 6.
The inventors also examine the effect of incorporation of both lipophilic groups, i.e., long chain alkyl, as well as more polar substituents in the place of R2 in the lead structure in Scheme 7, via alkylation of the secondary amines (R2═H) with the commercially available alkylating agents 59-62, leading to the preparation of 51-58 via mono- and dialkylation of the two secondary amine functionalities, based on the work of Drefahl and Konig (Chem. Ber. 1954, 87, 1632-4).
The inventors examine the effect of changing R3 in the lead structure 23 (see scheme 1, above). Certain substitutions for R3 are already known, such as shown in 63-65 in Scheme 8, below. The inventors prepare a series of new analogs, based on the alkylation of the known secondary amine 63 (13) with the alkylating agents shown in scheme 7 (59-62) to generate the novel structures (66-69). The substrate 70 in which R3═CH2CH2OH, i.e., the analog of 63 that corresponds most closely to hydroxychloroquine is examined. This compound is available by the same sequence used to prepare the analogs shown in Scheme 5, or by demethylation of 66.
Another important difference between the lead structure 3 and chloroquine 1 (scheme 9) is the presence of the stereocenter next to the nitrogen atom in 1. The inventors also prepare hybrid structures, in which either one of both of the sides of 23 more closely resemble 1, such as 71/72 and 73/74. The requisite diamines in each case are prepared starting from (L) glutamic acid, following the procedure employed by Craig in the stereoselective synthesis of chloroquine (J. Org. Chem. 1988, 53, 1167-1170).
The inventors prepare the analog of the lead structure 3 containing the stereocenter present in chloroquine 1, i.e., 75/76 and 77/78, which are available from alanine and serine, respectively, using the method of Charlton and coworkers (24) via reductive alkylation.
Finally, the inventors examine a series of compounds that contain four nitrogen atoms in the tether connecting the quinoline rings, instead of the three nitrogen atoms that are present in the connector chain in 3 (scheme 9), as illustrated in scheme 10, below. Denny and coworkers (24) have described the synthesis of the requisite tetramines 81 and 82.
Attachment of the additional chloroquine moieties present in 80 takes place via the same methodology employed in Scheme 8 for the synthesis of 65.
Method of Treatment
According to one aspect of the invention, a method is provided for treating a mammalian patient or subject to inhibit autophagy in that patient or subject. Compounds according to the present invention described herein may be used to inhibit autophagy in a manner consistent with inhibiting, treating and/or preventing disease states and/or conditions including cancer (including metastasis of cancer), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.
According to the present invention, in patients or subjects in need thereof, are treated by administering to the patient or subject an effective amount of one or more compounds according to the present invention, optionally in combination with at least one additional bioactive agent useful for treating the same disease state or condition. Compounds according to the present invention may be used to inhibit, reduce the likelihood or treat cancer, including the metastasis of cancer in a patient or subject in need of such treatment. The treatment is useful for any cancer for which inhibition of autophagy represents a favorable result or for which metastasis is a risk element. Therapy with at least one additional anticancer agent as otherwise described herein is also contemplated in the present methods. The numerous cancers which may be treated pursuant to the present method are described hereinabove.
In another aspect the present invention is directed to a method for treating a disease state and/or condition which benefits from the inhibition of autophagy, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease. In this method, a patient or subject in need of treatment is administered an effective amount of a compound as otherwise described herein optionally in combination with a pharmaceutically acceptable carrier, additive or excipient in order to inhibit, treat and/or prevent the above disease states of conditions.
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 according to I below:
In certain preferred methods of the invention, R1 and R1′ are each independently H, a halo group, a nitro group or a trifluoromethyl group, preferably a chloro group. R and R′ are preferably each independently H, a C1-C3 optionally substituted alkyl group itself preferably substituted with at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with a 7-substituted-4-quinolinyl group wherein the amine binds to the 4-position of the quinolinyl group, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) (the 7-position of each quinolinyl group may be substituted with R1 and/or R1′ as broadly described for generic structure I above), or an alkoxy group (e.g. methoxy or ethoxy) which alkoxy group may be further substituted with an alkoxy group, preferably a methoxy group (thus forming a diether substituent).
In other preferred methods of the invention L is a
group, where X is N—R″, Y and Y′ are each independently absent or CH2, and R″ is H or a C1-C3 alkyl group which is optionally substituted with at least one hydroxyl group, an amine, monoalkyl amine or dialkyl amine group, wherein said amine group or said monoalkyl amine group is optionally substituted on the amine position with a 7-substituted-4-quinolinyl group wherein the amine binds to the 4-position of the quinolinyl group, or one or both alkyl groups of said monoalkyl amine or dialkyl amine is itself further optionally substituted with at least one hydroxyl group, an amine, a monoalkyl amine or a dialkyl amine wherein the amine or monoalkyl amine is optionally substituted on the amine position with one or two 7-substituted-quinolinyl group(s) (the 7-position of each quinolinyl group may be substituted with R1 and/or R1′ as broadly described for generic structure I above), or an alkoxy group (e.g. methoxy or ethoxy) which alkoxy group may be further substituted with an alkoxy group, preferably a methoxy group (thus forming a diether substituent).
Further preferred methods relate to the use/administration of the compounds according to the present invention which are presented in the various schemes which are presented in Scheme 1 and Schemes 3-10 and
In the methods treating or inhibiting cancer or the metastasis of cancer, the compounds described above may be coadministered with at least one additional anticancer agent including, for example, everolimus, trabectedin, abraxane, TLK 286, AV-299, DN-101, pazopanib, GSK690693, RTA 744, ON 0910.Na, AZD 6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD 1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, a FLT-3 inhibitor, a VEGFR inhibitor, an EGFR TK inhibitor, an aurora kinase inhibitor, a PIK-1 modulator, a Bcl-2 inhibitor, an HDAC inhibitor, a c-MET inhibitor, a PARP inhibitor, a Cdk inhibitor, an EGFR TK 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, IPdR1 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-1H-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)-indolylj-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 [C59H84N18Oi4-(C2H4O2)X where x=1 to 2.4], goserelin acetate, leuprolide acetate, triptorelin 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; amifostine, NVP-LAQ924, 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-deooxyuridine, cytosine arabinoside, 6-mecaptopurine, 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-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, RAD001, 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.
In methods involving infections, disease states and/or conditions caused by rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the compounds according to the present invention may be coadministered with additional agents which are traditionally used in therapy for these disease states and/or conditions.
The following examples illustrate and describe the present invention but are not intended to limit the invention in any way.
Synthesis of Compound 3 (Lys01). A round-bottom flask was charged with the 4-bromo-7-chloroquinoline (compound 5) (734 mg, 3.0 mmol), Pd(OAc)2 (23 mg, 0.1 mmol), BINAP (125 mg, 0.2 mmol), K3PO4 (1.06 g, 5.0 mmol), and triamine (compound 6) (117 mg, 1.0 mmol). Dioxane (10 mL) was introduced through the septum. The resulting suspension was stirred under argon at 90° C. for 18 h and cooled. The mixture was adsorbed onto silica gel and purified by flash chromatography (CH2Cl2/MeOH: 90/9/1) to afford compound 3 (387 mg, 88%) as a yellow solid. mp 199-200° C.; Rf=0.28 (silica gel, CH2Cl2/MeOH/NH4OH: 90/9/1); 1H NMR (500 MHz, CDCl3: δ 8.53 (d, J=5.5 Hz, 2H), 7.94 (d, J=2.0 Hz, 2H), 7.41 (d, J=9.0 Hz, 2H), 6.98 (dd, J=9.0, 2.0 Hz, 2H), 6.39 (d, J=5.0 Hz, 2H), 5.44 (s, 2H), 3.42 (q, J=5.0 Hz, 4H), 2.90 (t, J=6.0 Hz, 2H), 2.46 (s, 9H). 13C NMR (125 MHz, CDCl3): δ 152.1, 149.5, 149.1, 135.1, 128.9, 125.5, 120.6, 117.1, 99.3, 55.5, 42.4, 40.3 FTIR (thin film): 3215, 2917, 1609, 1579, 1449. HRMS-ESI (m/z): calcd for C23H24N5Cl2 [M+H]+: 440.1409, found: 440.1406.
Synthesis of Compound 11 (Lys 05). To generate a water soluble salt of compound 3, a suspension of compound 3 (896 mg, 2.04 mmol) in MeOH (40 mL) was bubbled with HCl gas for 10 min at 0° C. The mixture was stirred for another 12 h at room temperature. The solvent was removed by rotary evaporation and the residue was dried under vacuum at 50° C. overnight to afford the salt 3 (1.13 g, 100%) as a yellow solid. mp 270° C. (decomp.); 1H NMR (500 MHz, D2O): δ 8.12 (d, J=7.0 Hz, 2H), 7.73 (d, J=9.0 Hz, 2H), 7.58 (d. J=2.0 Hz, 2H), 7.26 (dd, J=9.0, 2.0 Hz, 2H), 6.62 (d, J=2.0 Hz, 2H), 3.89 (br, 4H), 3.68 (br, 4H), 3.12 (s, 3H). 13C NMR (125 MHz, D2O): δ 155.8, 142.8, 140.2, 137.2, 128.1, 123.8, 119.1, 114.8, 98.7, 52.9, 42.7, 38.2. FTIR (thin film): 3376, 3019, 2914, 1631, 1612, 1215 cm−1. HRMS-ESI (m/z): calcd for C23H24N5Cl2 [M-3HCl+H]+: 440.1409, found: 440.1408.
Biological Testing
Lys01 is a More Potent Autophagy Inhibitor than HCQ or CQ.
LN229 (human glioblastoma) were treated with Lys01 and derivatives Lys02, Lys03, Lys04, HCQ and CQ. Near complete cell death of cultured cells was observed in cells treated with Lys01 at concentrations of 10 μM or higher between 4-24 hours. LC3 is a ubiquitin-like protein which exists as an unconjugated form (LC3I) or conjugated to AV membranes (LC3II)(25). The ratio of LC3II/LC3I reflects the accumulation of AV in cells, and therefore effective autophagy inhibition. LC3 immunoblotting (
To further characterize the effects of Lys01 on autophagy, LN229 GFP-LC3 cells were treated with Lys01 or HCQ (
To determine the implications of more potent autophagy inhibition on cytotoxicity, LN229 (glioma), 1205Lu (melanoma), HT-29 (colon) and c8161 (melanoma) cells were treated with Lys01, Lys02, Lys03 Lys04, and HCQ at concentrations between 0.01-100 μM (
In Vivo Autophagy Inhibition and Antitumor Efficacy Lys05.
Lys05, the trihydrochloride salt of Lys01 was synthesized to enhance aqueous solubility and to enable in vivo studies. Lys01 and Lys05 produced equivalent dose-dependent increases in the LC3II/LC3I ratio, and accumulation of the autophagy cargo protein p62 (26), and identical IC50 values in the MTT assay, (
Except for certain models of pancreatic cancer (27), in many animal tumor models, where high levels of autophagy are likely present in untreated tumors (4, 28), treatment with single agent HCQ does not impair tumor growth (29, 30). To determine if a more potent autophagy inhibitor such as Lys055 could significantly impair tumor growth as a single agent 1205Lu xenografts were generated in the flanks of nude mice. For chronic treatment experiments the 1205Lu melanoma model was chosen over the c8161 xenograft model because c8161 xenografts tend to spontaneously ulcerate confounding tumor measurements and safety analysis. Ten mice bearing 1205Lu xenografts were matched for tumor volume per cohort were assigned to either PBS, HCQ 60 mg/kg i.p. or Lys05 76 mg/kg i.p. (equimolar dosing) dosed for 3 days of daily treatment with 2 days off treatment (3/5 days) for all 3 treatment groups, to allow for symptom recovery and to avoid excess toxicity. This schedule was tolerated well for a 14 day period. Tumor growth curves for each of the 3 groups indicated tumor growth was significantly impaired in Lys05 treated tumors compared to controls (
To determine if lower doses of Lys05 could produce antitumor activity, mice bearing HT-29 colon cancer xenografts were treated with PBS, or Lys05 at 10 mg/kg i.p. daily, 40 mg/kg i.p. daily, or 80 mg/kg i.p. 3/5 days off. Clinical toxicity was observed only in the 80 mg/kg cohort, with 2/8 mice euthanized early for bowel obstruction. Daily dosing for the 10 mg/kg and 40 mg/kg cohorts was well tolerated. The average daily tumor growth rate was significantly impaired in a dose-dependent fashion with Lys05 treatment (
Intestinal Toxicity at the Maximal Administered Dose of Lys05 Resembles a Genetic Autophagy Deficiency.
In the 1205lu xenograft experiment, individual animals treated with Lys05 76 mg/kg i.p. 3/5 days appeared lethargic with arched backs (
In the HT29 dose-finding xenograft experiment there was no significant weight loss observed in any dose cohort (
Lys05 Inhibits Autophagy by Deacidifying the Lysosome.
To compare the relative lysosomal accumulation of Lys05 compared to HCQ, lysosomes were subfractionated from 1205Lu cells treated with PBS, HCQ 10 μM, or Lys05 10 μM, and 1205Lu tumors harvested after 14 days of treatment with PBS, HCQ 60 mg/kg i.p., or Lys05 76 mg./kg i.p. every 3/5 days. Immunoblotting against the lysosomal marker LAMP2 confirmed adequate separation of the lysosomal and whole cell population in both cells and tumor samples (
Having established that Lys05 more effectively accumulates in the lysosome than HCQ, the functional effects of this accumulation were investigated. 1205Lu cells were treated with vehicle, Lys05 and HCQ, and stained the Lysotracker Red (
Finally, the functional consequences of lysosomal deacidification were investigated by measuring enzymatic activity of acid phosphatase. In 1205Lu cells treated with PBS, HCQ 10 μM or Lys05 10 μM, within 24 hours there is a 43% reduction in acid phosphatase activity in the lysosomal fraction of Lys05 treated versus PBS treated cells (
Additional compounds Lys06-Lys18 Additional Lys01 derivatives have been synthesized and tested (
Activity of Lys01 derivatives in malaria. Table 2,
Potential Commercial Uses and Applications: Lys01 and Lys05 are lead compounds with great potential to be optimized further for potency as a novel autophagy inhibitor. Autophagy inhibition is a new therapeutic strategy in cancer that is applicable to every cancer. There are currently over 30 HCQ trials in cancer patients involving nearly every tumor type. Due to its low potency and poor pharmacology, in humans HCQ will likely not yield the promising augmentation of anticancer therapy observed in laboratory models. An optimized derivative of Lys01 could be developed as a second generation autophagy inhibitor. The GI toxicity associated with Paneth cell dysfunction observed at LD30 doses of Lys05, support the mechanism of action of the drug, and also suggests that colon cancers, which often share features with Paneth cells, may be a tumor type that may be especially sensitive to Lys05 and its optimized derivatives. Additional cancers worth investigating include melanoma, and non small cell lung cancer, since melanoma cell lines demonstrated the highest difference in sensitivity to Lys01 compared to HCQ, and an EGFR mutated lung cancer cell line demonstrated sensitivity to both HCQ and Lys05. The synthesis of Lys01 was designed such that there is no overlap with other patented and/or published aminoquinoline compounds. Further mechanistic studies are planned that are to identify pharmacodynamics assays that guide drug development. Pharmacokinetic studies planned in mice establish initial in vivo profile.
Other similar technologies and competing products: Novel chloroquine derivatives for use as anticancer agents is an active area of investigation (16). Autophagy has been identified as one of the top ten areas of research in which the NIH will invest in the next few years. No studies to date have leveraged the potential of bivalency as the inventors provide here. In addition, most studies lack the in vivo studies and the mechanistic studies herein reported that can guide further development of optimize lead compounds for drug development.
Advantages over other similar technologies and products: Thus, the present application has shows that the disclosed series of bisaminoquinolines are potent autophagy inhibitors that have single agent antitumor activity in an in vivo tumor model.
The present application is a continuation application of U.S. patent application Ser. No. 14/825,477, filed Aug. 13, 2015, which is a continuation application of U.S. national phase patent application Ser. No. 14/144,049, filed Oct. 25, 2013, now abandoned, which is based upon international patent application no. PCT/US12/35251 filed Apr. 26, 2012, which claims the benefit of priority of U.S. provisional application No. 61/480,641, filed Apr. 29, 2011, of identical title to the present application, the entire contents of which all applications are incorporated by reference herewith.
This invention was made with government support under the Abramson Cancer Center Pilot grant awarded by the National Cancer Institute. The government has certain rights in the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20170166530 A1 | Jun 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61480641 | Apr 2011 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14825477 | Aug 2015 | US |
| Child | 15384823 | US | |
| Parent | 14114049 | US | |
| Child | 14825477 | US |
