HK1190718A

HK1190718A - Novel bisaminoquinoline compounds, pharmaceutical compositions prepared therefrom and their use

Info

Publication number: HK1190718A
Application number: HK14103894.5A
Authority: HK
Inventors: R．K．阿玛拉瓦迪; J.温克勒
Original assignee: 宾夕法尼亚大学托管会
Priority date: 2011-04-29
Filing date: 2012-04-26
Publication date: 2014-07-11

Description

Novel bisaminoquinoline compounds, pharmaceutical compositions prepared therefrom and their use

Related applications and fund funding

This application claims priority to a provisional application, entitled U.S. provisional application having application number US 61/480,641 filed 2011, 4/29, the entire contents of which are incorporated herein by reference.

The invention was made with government support sponsored by a foundation trial by the Abramson cancer center of the national cancer institute of the United states. The government has certain rights in the invention.

Technical Field

The present invention relates to novel bisaminoquinoline compounds, pharmaceutical compositions comprising these novel bisaminoquinoline compounds, and methods for inhibiting autophagy in biological systems. As another aspect of the invention, methods of treating cancer patients according to the present invention use the compounds and/or compositions alone, or in combination with at least one additional anti-cancer agent. As another aspect of the invention, the compounds of the invention are used in combination with radiation therapy, either alone or in combination with additional anti-cancer agents as described further herein. As another aspect of the invention, methods of treating conditions and/or disease states, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, with compounds according to the invention, where inhibition of autophagy plays a good therapeutic role.

Background

Autophagy consists of sequestering organelles and proteins in Autophagic Vesicles (AV) and digesting them by lysosomal fusion (1). Autophagy allows tumor cells to survive metabolic and therapeutic stress (2-5). A number of documents indicate that treatment-induced autophagy is an important resistance mechanism for many anticancer agents.

Chloroquine (CQ) (compound 1, figure 1) derivatives blocked autophagy by inhibiting lysosomes (3, 6, 7). In randomized controlled phase III clinical trials of chloroquine and placebo with carmustine and radiotherapy treated glioma patients, a trend of doubling survival time in chloroquine treated patients was reported. Based on these conclusions, clinical trials combining cancer therapy with hydroxychloroquine (HCQ; compound 2 of FIG. 1), which is safer than chloroquine at increasing doses, have been initiated. Preliminary results show that these combinations are active (9), but it remains unclear whether activity is constant due to the addition of HCQ. Inhibition of autophagy requires high micromolar concentrations of HCQ. Although there is some proof of efficacy in cancer patients using HCQ to inhibit autophagy, it is not constant because sufficient concentrations are not obtained in all patients (10). The need to open up more potential autophagy inhibitors is not met. The design and synthesis of dimeric analogues of chloroquine has been the subject of intensive research for more than 10 years, taking advantage of the thermodynamic advantage conferred by multivalence (11, 12) (13-15). An early report of Vennerstrom (14) describes the synthesis of isoalkane-bridged bisquinolines as potential antimalarials, but none of the compounds have sufficient antimalarial activity to warrant further investigation. Subsequently, Sergheraert (13) reported that a dimer of the tetraquinoline, a bisquinoline, could provide an effective antimalarial, confirming the possibility that the use of a multivalent strategy could provide increased efficacy, at least for antimalarial activity.

Recently, Lee (16) described that the potentiation of AKT inhibitors was achieved by fluoridating quinoline analogs. Solomon (17) reported the preparation of "position-altered" chloroquine dimers based on the use of piperazine linkers. These results suggest that these quinoline analogs can serve as the basis for the development of new classes of effective cancer chemotherapy.

Objects of the invention

It is an object of the present invention to provide novel compounds for use in inhibiting autophagy in biological systems, in particular, including patients or subjects in need of assistance.

Another object of the invention is to treat conditions and/or conditions in which a patient or subject's conditions and/or conditions benefit from inhibition of autophagy.

It is a further object of the present invention to provide pharmaceutical compositions for inhibiting autophagy, in particular, autophagy associated with pathologies and/or conditions including cancer and metastases thereof.

It is a further object of the present invention to inhibit, treat or prevent cancer, including metastasis of cancer, in a patient or subject in need thereof using the compounds, compositions and/or methods of the present invention.

It is another object of the present invention to inhibit, treat or prevent diseases, including rheumatoid arthritis, malaria, antiphospholipid syndrome, lupus, chronic urticaria and Sjogren's disease, among others, in which inhibition of autophagy provides a beneficial effect.

Any one or more of these and/or other objects of the present invention can be readily gleaned from the description of the invention that follows.

Disclosure of Invention

The present invention relates to compounds of chemical structure I:

wherein R is¹And R^1′Each independently H, halogen (F, Cl, Br or I), CN, NO₂Optionally substituted C₁-C₆Alkyl (when substituted, preferably substituted with 1 or 2 hydroxy or 3-5 fluoro), optionally substituted O-C₁-C₆Alkyl (preferably OCH)₃) Optionally substituted C₂-C₇Acyl (preferably acetyl) or optionally substituted C₂-C₇Esters (oxycarbonyl esters or carboxyl esters, preferably, carboxyl esters);

r and R' are each independently H, optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₇(preferably C)₂-C₇) Acyl, optionally substituted C₂-C₇A carboxyl ester group (which forms a urethane group with the nitrogen atom to which R or R' is attached);

l is (CH)₂Y)_n―X―(Y′CH₂)_nA radical or A- (CH)₂―CH₂―Z)_nAn A 'group (A or A' may be attached to one of the two amino groups of compound I), wherein at least one CH of L₂The radicals being optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups; x is absent or is (CH)₂)_jO, S orN-R″；

Y is absent, or CH₂、O、CH₂O or N-R ', and Y' is absent, or CH₂、O、OCH₂Or N-R ", with the proviso that when one or more of X, Y and Y ' are present, each of the X and Y, X and Y ' or Y and Y ' present form a stable bond;

r' is H or optionally substituted C₁-C₆(preferably, C)₁-C₃) An alkyl group;

j is 1,2 or 3 (preferably 1 or 2);

n is 0, 1,2, 3 or 4, with the proviso that when n is 0, X is (CH)₂)_jWherein j is at least 1, and at least one CH₂The radicals being optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups;

a is absent or is (CH)₂)_jAnd A' is (CH)₂)_jWherein at least one CH in A or A₂The radicals being optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups;

z is O or N-R^z；

R^zIs H or optionally substituted C₁-C₃An alkyl group, a carboxyl group,

or a pharmaceutically acceptable salt, enantiomer, asymmetric isomer, solvent or polymorph thereof.

In a preferred aspect of the invention, R¹And R^1′Each independently is H, a halo group, nitro or trifluoromethyl, preferably a chloro group. R and R' are each independently H, optionally substituted C₁-C₃Alkyl, which is itself preferably substituted by at least one hydroxy, alkoxy, amino, monoalkylamino or dialkylamino group, wherein the ammonia is presentOptionally substituted at the amino position by one or two 7-substituted-4-quinolinyl groups, wherein said amino group is attached to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably by R as broadly described in structural formula I above¹Or R^1′Substituted, or one or both alkyl groups of said monoalkylamino or dialkylamino group are themselves further optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino, dialkylamino group, wherein said amino or said monoalkylamino group is optionally substituted at the position of the amino group by one or two 7-substituted-quinolinyl groups, wherein said amino group is attached to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably by R as broadly described in the general structural formula I above¹Or R^1′And each said alkoxy group (e.g. methoxy or ethoxy) may optionally be further substituted by an alkoxy group, preferably methoxy, to form a monoether substituent.

In certain preferred aspects of the present invention, L is- (-CH)₂Y)_n―X―(Y′CH₂)_nA radical of formula (I), wherein X is N-R ', Y and Y' are each independently absent or CH₂And, R' is H or optionally substituted C₁-C₃Alkyl, itself optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino or dialkylamino group, wherein the amino group or the monoalkylamino group is optionally substituted at the position of the amino group by one or two 7-substituted-4-quinolinyl groups, wherein the amino group is attached to the 4-position of the quinolinyl group and the 7-position of each quinolinyl group is optionally substituted, preferably by R as broadly described in the general structural formula I above¹Or R^1′Substituted, or one or both alkyl groups of said monoalkylamino or dialkylamino group are themselves further optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino, dialkylamino group, wherein said amino or said monoalkylamino group is optionally substituted at the position of the amino group by one or two 7-substituted-quinolyl groups, wherein said amino group is attached to the 4-position of the quinolyl group, and each quinolyl group has one or two alkyl groups attached theretoThe 7-position is optionally substituted, preferably by R as broadly described in the structural formula¹Or R^1′And each said alkoxy group (e.g. methoxy or ethoxy) may optionally be further substituted by an alkoxy group, preferably methoxy, to form a monoether substituent.

Further preferred compounds according to the present invention include those provided in various schemes, which are shown herein in scheme 1 and schemes 3-10 and figures 14, 15 and 15A.

In another aspect of the invention, a pharmaceutical composition comprises a compound according to formula I above or otherwise described herein in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally in combination with at least one additional anti-cancer agent.

Methods of inhibiting autophagy in a biological system, particularly in a patient or subject, are further aspects of the invention. In this aspect of the invention, the bisaminoquinoline compounds described elsewhere herein are provided to a biological system, including administration to a patient or subject in need thereof, to inhibit autophagy. The resulting inhibition can be monitored or used in biological systems to produce beneficial effects, including inhibiting, treating and/or preventing cancer, including metastasis of cancer, or inhibiting, treating and/or preventing one or more conditions or diseases, where inhibition of autophagy can provide beneficial effects, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, among others.

A method of inhibiting, treating and/or reducing the likelihood of cancer, including metastasis and drug-resistant cancer, comprising administering to a patient in need thereof at least one compound according to the invention, optionally in combination with at least one additional anti-cancer agent as otherwise described herein.

The invention also relates to the treatment, inhibition and/or prevention of diseases, conditions and/or disease states in a patient in need of assistance, wherein inhibition of autophagy provides beneficial effects, including rheumatoid arthritis, malaria, antiphospholipid syndrome, lupus, chronic urticaria and Sjogren's disease, comprising administering to said patient at least one compound according to the invention.

Drawings

FIG. 1 shows the chemical structures of monoaminoquinoline and bisaminoquinoline;

FIG. 2 is a scheme for the synthesis of bisaminoquinolines;

FIG. 3 role of Lys01-Lys04 in quantification of LC3II/LC3I ratio in LC3 immunoblots and lysates after 4 hours treatment on LN229 cells. The LC3II/LC3I ratio (mean +/-SD) for each treatment normalized to the LC3II/LC3I ratio for each experimental control-treated cell is shown;

FIG. 4 comparison of autophagy inhibition and cytotoxicity of HCQ, Lys 01. (A) Schematic representation of LN229GFP-LC3 cells after 4 hours of treatment as indicated. White arrow: small dot-like structures (small puncta); red arrow: dense dot-like structures. The graph shows the mean +/-SEM of the dots per cell. (B) Electron micrographs after treatment of LN229-GFP-LC3 cells with DMSO, 10. mu.M HCQ, or 10. mu.M Lys 01. Arrow head: autophagic vesicles. (C) LC3 immunoblots of LN229 cells after 24 hours of treatment as indicated; calculated ratio of LC3II/LC3I ratio compared to control treated with bafilomycin. Above the dotted line indicates an autophagy inducer or control and below the dotted line an autophagy inhibitor. (D) MTT assay (72 hours) for 4 cell lines. Red: lys01, blue: lys02, purple: lys03, green: lys04, orange: HCQ had 5 replicates per experiment, values are expressed as mean +/-SEM;

FIG. 5 autophagy inhibition and cytotoxicity of Lys05, water soluble salt of Lys 01. (A) As shown, immunoblots against LC3 and p62 in treated c8161 cells. (B) c8161 cells were assayed at 72 hours MTT. HCQ: hydroxychloroquine. There were 5 replicates per experiment and values are expressed as mean +/-SD. No cells left for analysis;

FIG. 6 in vivo autophagy inhibition and antitumor activity of Lys 05. (A) c8161 xenograft tumors were injected intraperitoneally (i.p.) daily with PBS, HCQ60mg/kg or Lys0576mg/kg, electron micrographs harvested 2 days later (12000X). Arrow head: (ii) autophagic vesicles; scale bar 2 μm (B) quantification of autophagic vesicles/cell number mean +/-SEM in two representative tumors per treatment group. (C-D) 1205Lu xenografts were injected intraperitoneally (i.p.) with PBS (blue), HCQ60mg/kg (green) or Lys0576mg/kg (red) for 3 days out of 5 days. (C) Tumor volume (D) over 14 days tumor daily growth rate (E-G) HT29 xenografts were generated in the flanks of nude mice with PBS, daily intraperitoneal Lys 0510 mg/kg, daily intraperitoneal Lys 0540 mg/kg, or 3 out of 5 days intraperitoneal Lys0580 mg/kg. (E) The average daily growth rate of the tumor (F) exceeds the tumor volume for 14 days (G) the tumor weight of the resection,^*p＜0.05；

FIG. 7 autophagy inhibition and tumor necrosis in melanoma and colon cancer xenografts treated with Lys05 or HCQ. (A) As shown, immunoblots against LC3 in a single c8161 tumor lysate, the tumors were injected intraperitoneally daily for 48 hours of treatment. Quantification of the LC3II/LC3I ratio (mean +/-SEM) (B) tumor necrosis in the H & E stained fraction of the harvested 1205Lu xenograft tumors after 14 days of treatment (arrows); electron micrograph of melanoma tumor cells (7000-12000X). Arrow head: autophagic vesicles (white); immunoblotting of apoptotic cells (orange) (C) against LC3 in HT29 xenografts dosed daily (10, 40 mg/kg) or 3 out of 5 days dosed (80 mg/kg) for 14 days of HT29 xenografts;

FIG. 8 toxicity associated with intraperitoneal injection of 76mg/kg Lys05 on 3 out of 5 days. (A) After 3 days of dosing, mice were stooped lethargic. (B) 3/10 mice developed ileus. (C) One mouse Pantoea cells malformed in the terminal ileum (arrow);

figure 9 treatment with Lys05 at the highest dose resulted in a reproducible intestinal phenotype of autophagy gene deficiency. (A-F) carrying PBS, Lys 0510-80 mgWeight and intestine of/kg treated HT29 xenografted mice were analyzed. (A) Daily body weight (B) represents a representative picture of ileal crypts (hematoxylin and eosin staining) of mice bearing HT29 xenografts (14 days) in gastrointestinal tract (C) excised after 14 days of treatment (40 ×), arrow: pan cells. (D) Number of Pan cells per crypt (E) Pan cell dysfunction score,^*p < 0.05 (F) score for lysozyme-positive cells,^*p =0.001. representative graph of mouse ileal lysozyme immunofluorescence (green), wherein mice were treated intraperitoneally with PBS, Lys0580mg/kg for 3 out of 5 days;

FIG. 10 Pan cell dysfunction scale. The size and number of eosinophils per Pan cell were calculated at 40 Xmagnification, with 10 Pan cells per sample: a0= normal size and number; a1= reduced, normal number; a2: normal size, reduced number; a3: the size is reduced, and the number is reduced;

figure 11 Lys05 inhibits autophagy by accumulating and deacidifying lysosomes. 1205Lu cells (treated with PBS, 10 μ MHCQ or 10 μ M Lys05 for 24 hours) and harvested 1205Lu xenograft tumors (i.p. PBS, HCQ60mg/kg on day 3 of 5 days, or 76mg/kg Lys05 on day 3 of 5 days, treated for 14 days) were homogenized and divided into Whole Cell (WC) and lysosomal (L) fractions. LAMP2 immunoblotting confirmed concentrated lysosome isolation for analysis. (B) Concentration of HCQ or Lys05 in cell and tumor whole cells and lysosome homogenates. (C) Fluorescence profiles of 1205Lu cells treated for 30 minutes and stained with lysosomal red fluorescent probe as indicated. Small spots of three lysosomal fluorescent probe (red) highlights were counted per cell. Blue color: nuclear DAPI staining. Data are presented as mean ± SEM. (D) Fluorescence profile of c8161 cells treated as indicated for 24 hours and stained with Acridine Orange (AO): orange color: (ii) an aggregated AO; green: (ii) a diffuse AO;

FIG. 12 HPLC tandem mass spectrometry analysis of HCQ and Lys 05. 1205Lu cells (24 hours) and 1205Lu tumors (14 days). WC: homogeneous whole cells; l: a small fraction of lysosomes; HCQ: hydroxychloroquine;

figure 13 damage to lysosomal enzymes and lysosomal leakage associated with Lys05 treatment. (A) Acid phosphatase activity, and (B) immunoblotting of cathepsin D in whole cells (white, WC) and in the lysosomal fraction (black; L) of 1205Lu cells treated with PBS, 10. mu.M HCQ, 10. mu.M Lys05 for 24 hours. Mean +/-SEM for three independent experiments is shown. (C) Acid phosphatase activity, and (D) 1205Lu xenograft were treated with 5 days of 3 (tumor) intraperitoneal injections of PBS, 60mg/kg HCQ, 76mg/kg Lys05, immunoblotted for cathepsin D in whole cells (white, WC) and in the lysosomal fraction (black; L). Three separate tumors were pooled together and homogeneous whole cells (white) and homogeneous lysosomes (black) were prepared therefrom.^*p＜0.05；

FIG. 14 chemical structure of the synthetic compound Lys06-Lys 12; the chemical structure of the compound Lys06-Lys12 is shown;

FIG. 15 chemical structure of the synthetic compound Lys13-Lys 18; the chemical structure of the compound Lys13-Lys18 is shown;

figure 15a shows several additional bisaminoquinoline autophagy inhibitors in the study;

FIG. 16 Table 1 provides MTT IC in LN229 cells₅₀Values for the selection of compounds of the invention;

FIG. 17 Table 2 provides the IC of several compounds of the invention in P.falciparum (P.falciparum)₅₀(M) value.

Detailed Description

The following terms used throughout the specification are used to describe the present invention. As understood by those of ordinary skill in the art, where a term is not explicitly defined, the term will take its ordinary meaning in the context in which it is used.

Unless the context clearly dictates otherwise, ranges of values provided herein are to be understood as each intervening value, in the tenth of the unit of the lower limit taken to be encompassed within the invention, between the upper and lower limit of that range and any other stated or intervening value in that stated range. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are 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 those included limits are also included in the invention. When substituents are possible in one or more markush groups, it is understood that only those substituents which form stable bonds are employed.

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. 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," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Furthermore, the following terms shall have the meanings set forth below.

The term "patient" or "subject" as used throughout the context of the specification refers to an animal, typically a mammal, particularly including a domestic animal, preferably a human being treated (including prophylactic treatment (prophylaxis)) with a composition of the invention. When treating such infections, diseases or disease states in a particular animal, such as a human patient, the term "patient" refers to that particular animal. In most cases, the patient or subject of the invention is a human patient of either sex or both sexes.

As used herein, unless otherwise indicated, the term "effective amount" is an amount of a compound or ingredient that, in the context of its use, produces or causes the intended effect, whether such effect is with respect to the prevention and/or treatment of infection and/or disease conditions, or otherwise described. All other effective amounts or effective concentration terms (including the term "therapeutically effective") are incorporated by reference into this term as if otherwise described or used in this application.

The term "compound" as used herein refers to any of the specific compounds or biologically active agents disclosed herein, including any or all stereoisomers (including asymmetric isomers), individual optical isomers (enantiomers) or racemic mixtures, pharmaceutically acceptable salts and prodrug forms. The term compound herein refers to a stable compound. In the context of their use, the compounds may refer to a single compound or a mixture of compounds, as otherwise specified herein. It is understood that the choice of substituents or bonds among markush or other groups of substituents or bonds is intended to form stable compounds from such choices among markush or other groups.

The term "bioactive agent" refers to any bioactive compound or drug that can be formulated for use in the present invention. Examples of bioactive agents include compounds according to the present invention that are useful for inhibiting autophagy and treating cancer, as well as other compounds and agents described elsewhere herein.

The terms "treating", "treating" and "treatment" are used synonymously and refer to any action that benefits a patient at risk or affliction with a disease, including improving the physical condition by alleviating or inhibiting at least one symptom, delaying the development of a disease, preventing or delaying the onset of a disease, etc.

The therapeutic methods used herein include both prophylactic (primarily cancer) and therapeutic treatments. For example, the compounds of the present invention may be prophylactically administered to a mammal to prevent the occurrence of a disease and reduce the likelihood of such a disease. Prophylactic administration is effective to reduce or reduce the likelihood of subsequent occurrence of the disease in the mammal, or to reduce the severity of subsequent occurrences of the disease, including in particular, metastasis. Alternatively, the compounds according to the invention may, for example, be administered therapeutically to a mammal already afflicted with a disease. In one embodiment of therapeutic administration, administration of a compound of the invention is effective to eliminate the disease and to produce a likelihood of alleviating or substantially eliminating metastasis. In the case of cancer, administration of a compound of the invention is effective to reduce the severity of the disease or to extend the lifespan of the afflicted mammal.

The term "pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject to obtain the treatment described herein, without undue adverse side effects depending on the severity of the disease and the nature of the treatment.

The term "inhibit" as used herein means that when the inhibitor is a compound having inhibitory ability, it is capable of partially or totally eliminating the potential effect.

The term "preventing" as used in the context herein shall mean "reducing the likelihood" or preventing a disease, disorder or condition secondary to administration of one or more compounds or compositions of the present invention, alone or in combination or concomitantly with another agent. Note that there is little 100% effective prevention; therefore, the terms prevention and reduction of the likelihood are used to denote the fact that: in a given patient or population of subjects, administration of a compound of the invention will reduce the likelihood of occurrence, or inhibition (in particular, worsening of the condition, such as cancer cell growth or metastasis), of a particular disorder or condition, or other recognized indicator of disease progression.

The term "autophagy" or "autophagy" is used to describe a catabolic process in a cell that involves degradation of its own components by lysosomes. Autophagy is a highly controlled biological system process that plays a normal role in cell growth and development and maintenance of homeostasis to help maintain a balance between synthesis and degradation of cellular products and subsequent recovery. This is the main mechanism by which cells distribute nutrients from unnecessary processes to more important processes.

In fact, many autophagy processes occur, all of which share the common feature of degrading intracellular components by lysosomes. One known autophagy mechanism involves the formation of a membrane around a target region in a cell, which isolates these contents from the rest of the cytoplasm. The resulting vesicles then fuse with lysosomes, which then degrade the contents.

Diseases, conditions and/or disorders that benefit from autophagy inhibition include cancer (including cancer metastasis), rheumatoid arthritis, malaria, antiphospholipid syndrome, lupus, chronic urticaria and Sjogren's disease.

The term "cancer" shall refer to tumor cell proliferation with the unique feature of losing normal control, resulting in uncontrolled growth, loss of differentiation, local tissue invasion and/or metastasis. Neoplasms (neoplases), as used herein, include, but are not limited to, morphologically irregular cells in a subject or host tissue, as compared to normal proliferation in the same type of tissue, and pathologically proliferating cells in a subject tissue. In addition, neoplasms include aggressive or non-aggressive benign tumors and malignant tumors (e.g., colon tumors). Malignant neoplasms are distinguished from benign neoplasms in that the former exhibit a greater degree of dysplasia, or lack of cell differentiation and orientation, as well as being invasive and metastatic in character. In the present context, the term cancer also includes drug resistant cancers, including cancers that are resistant to multiple drugs. Neoplasms or neoplasias are derived from cells of interest of the present invention, including, but not limited to, cancers (e.g., squamous cell carcinoma, adenocarcinoma, hepatocellular carcinoma, and renal cell carcinoma), specifically those of the bladder, bone, intestine, breast, cervix, rectum (colorectal), esophagus, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, and stomach; leukemias, such as acute myelogenous leukemia, acute lymphocytic leukemia, Acute Promyelocytic Leukemia (APL), acute T-cell lymphocytic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, myelogenous leukemia, hairy cell leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia, small myelogenous leukemia, monocytic leukemia, neutrophilic leukemia, stem cell leukemia; benign and malignant lymphomas, in particular, burkitt's lymphoma, non-Hodgkin's lymphoma and B-cell lymphoma; benign and malignant melanoma; myeloproliferative diseases; sarcomas, in particular ewing's sarcoma, angiosarcoma, kaposi's sarcoma, liposarcoma (liposarcomas), myosarcoma, peripheral neuroepithelial tumors and synovial sarcoma; tumors of the central nervous system (e.g., gliomas, astrocytomas, oligodendrogliomas, ependymomas, glioblastomas, neuroblastomas, ganglion cell tumors, ganglion gliomas, medulloblastomas, pinealocytomas, meningiomas, meningeal sarcomas, neurofibromas, and schwannoma); germ cell tumors (e.g., intestinal, breast, prostate, cervical, uterine, lung (e.g., small cell lung, mixed small cell and non-small cell), pleural mesothelioma, including metastatic pleural mesothelioma, small cell lung, and non-small cell lung cancers), ovarian, testicular, thyroid, astrocytoma, esophageal, pancreatic, gastric, liver, colon, and melanoma; mixed types of tumors, in particular, carcinosarcoma and hodgkin's disease; and mixed cell of origin (origin) tumors such as Wilms' tumor and teratocarcinoma. It is noted that certain epithelial cell tumors, including ovarian, breast, colon, head and neck, medulloblastoma and B-cell lymphoma, among others, exhibit enhanced autophagy and are the primary target cancers for the compounds and treatments of the present invention.

The term "additional anti-cancer agent" is used to describe additional compounds that can be administered with one or more compounds of the invention to treat cancer. Such agents include, for example, everolimus, trabectedin, abraxane, TLK286, AV-299, DN-101, pazopanib, GSK690693, RTA744, ON0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD1152, enzastaurin, vandetanib (vandetanib), ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, FLT-3 inhibitors, VEGFR inhibitors, EGFR TK inhibitors, aurora kinase inhibitors, PIK-1 modulators, Bcl-2 inhibitors, HDAC inhibitors, c-MET inhibitors, PARP inhibitors, Cdk inhibitors, EGFR TK inhibitors, IGFR-TK inhibitors, anti-HGF antibodies, PI3 kinase inhibitors, AKT inhibitors, JAK/STAT inhibitors, checkpoint-1 or 2 inhibitors, inhibitors of adhesion to spot kinase (Mamek) inhibitors, VEGF trap antibody, pemetrexed, erlotinib, dasatinib (dasatanib), nilotinib, dacatinib (decatenaib), panitumumab, amrubicin, agovacizumab, Lep-etu, loratadine (nolatrexed), azd2171, barbitulin (batabulin), ofatumumab, zalimumab, idarubicin (eotecarin), tetrandrine, rubitecan (rubitecan), tilmicorfene, oblimersen (oblimersen), tizzamulimumab (ticilimumab), yipamirumab (Ipilimumab), gossypol, Bio111, 131-I-TM-601, ALT-110, BIO140, CC8490, cerampelin, gimeracan, IL13-PE38QQR, INO1001, IPdR₁KRX-0402, thioanthrone, LY317615, neuradiab, vistepan (vitespa), Rta744, Sdx102, talampanel, atrasentan, Xr311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, doxorubicin liposomes, 5' -deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib (seliciclib); PD0325901, AZD-6244, capecitabine, L-glutamic acid, N- [4- [2- (2-amino-4, 7-dihydro-4-oxo-1H-pyrrole [2,3-d ]]Pyrimidin-5-yl) ethyl]Benzoyl radical]-, disodium salt, heptahydrateCompound, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen conjugate, bevacizumab, IMC-1C11, CHIR-258; 3- [5- (methylsulfonylpiperidylmethyl) -indole-quinolone, vatalanib (vatalanib), AG-013736, AVE-0005, [ D-Ser (but)6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂Acetate salt [ C ]₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂) x, wherein x =1 to 2.4]Goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide (nilutamide), megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, carratinib (canertinib), ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, arabinoside, dacarbazine, actinomycin D, daunomycin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevec (gleevac), hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levotetramisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, tolperistrophin, L-N-D, L-S-D, L-S-A, S-S, S, Mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis-retinolAcid, melphalan, uramustine, estramustine, altretamine, floxuridine, 5-deoxyuridine (5-deoooxyuridine), cytarabine, 6-mercaptopurine, desoxymestranol, 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, cimetidine, trastuzumab, interleukin fusion (deuterin diftitox), gefitinib, bortezomib (bortezomib), taxol, irinotecan, epothilonol, docetaxel, paclitaxel, doxorubicin, castor oil and paclitaxel (paclitaxel) Epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxyttamoxifen, pentoxifene (pipindoxifene), ERA-923, azoxifene, fulvestrant, acobiprofen, lasofoxifene, indoxifene (idoxifene), TSE-424, HMR-3339, ZK186619, PTK787/ZK222584, VX-745, PD184352, rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, temolimus (mseirolimus), AP-23573, RAD001, ABT-578, BC-210, LY294002, LY 292222223, 292696, LY293684, LY293646, LY 29222penicillin, ZM 332, L-779,450, PEG-filgrastim, darbepoetin (dapoxetine), erythropoetin, lipocalin, macrophage stimulating factor, and colony stimulating factor, PEGylated interferon alpha-2 a, PEGylated interferon alpha-2 b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab ozolomide, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, mechlorethamine, methylprednisolone, ibritumomab tiuxetan, androgen, decitabine, hexamethamine, bexagliptin, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, daunorubicin liposome, Edwina-dayAsparaginase, strontium 89, casomopritan, netupitant, NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochloraz, granisetron, ondansetron, dolasetron, tropisetron, ssPEG filgrastim (sspegfilgrastim), erythropoietin, epoetin alpha, dabepoetin alpha, damepril, vemurafenib, and the like.

The term "alkyl" as used herein refers to a fully saturated monovalent group containing carbon and hydrogen (no more than 10 carbon atoms or as otherwise specified), and can be straight-chain, branched-chain, or cyclic. Examples of alkyl groups are methyl, ethyl, n-butyl, n-heptyl, isopropyl, 2-methylpropyl, t-butyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl and the like.

The term "substituted" with respect to alkyl described above includes one or more functional groups, such as lower alkyl groups containing 1 to 6 carbon atoms, optionally substituted with: 1 or 2 hydroxyl groups or 1 to 5 fluorine (preferably, 3-5), acyl (C)₁-C₆) Halogen (fluorine, chlorine, bromine, e.g. haloalkanes, e.g. CF)₃) Amino, hydroxy, carboxy/carboxylic acid, thioamido (thioamido), cyano, nitro, alkenyl (C)₂-C₆) Alkynyl (C)₂-C₆) Azido and alkoxy (C)₁-C₆) (including further by C)₁-C₆Alkoxy substituted by alkoxy to form a diether), amino group, C₁-C₆Alkylamino and dialkylamino, wherein, optionally, the alkyl group may be substituted with: 1 or 2 hydroxy or amino (amine), aminoalkyl or dihydrocarbyl groups, which may themselves be substituted by: one or two alkyl groups, or 7-substituted-4-quinolyl, C₂-C₆Amido, C₂-C₆Oxoacyl esters (oxosylester) or carboxylic esters, aryloxy (C)₁-C₆) Alkane (I) and its preparation methodAlkyl, amido (carboxamido), mercapto, C₂-C₆Ethers or thioethers, 7-substituted-4-aminoquinolines (or substitution at the amino group to form 7-substituted-4-aminoquinolines), and the like. Preferred substituents on alkyl (in the context herein, particularly low, on the amino group of a 7-substituted-4-aminoquinoline), or a linking group (linker) comprising at least one amino group, include, for example, at least one hydroxy, amino, monoalkylamino or dialkylamino group (wherein, optionally, one or both alkyl groups are themselves further substituted with a dialkylamino group, or an amino group is substituted with one or two (preferably one) 7-substituted-4-quinolines wherein the amino group is attached at 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 methoxy, to produce a diether substituent.

The term "aryl" refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., phenyl) or multiple fused 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, particularly quinolinyl, particularly 7-substituted-aminoquinolinyl, and others.

The term "substituted" as used herein in the term "substituted aryl, substituted aromatic, substituted heteroaryl or substituted aromatic heterocycle" means a substituent present at the 7-position of the 4-aminoquinoline, said substituent being selected from the group consisting of atoms and groups, when present, that enhances the activity of the compound as an autophagy inhibitor. Examples of substituents which may be present in a substituted aromatic or heteroaromatic ring include, but are not limited to, groups such as H, halogen (F, Cl, Br or I), CN, NO₂Optionally substituted C₁-C₆Alkyl (when substituted, preferably substituted with 1 or 2 hydroxy or 3-5 fluoro), optionally substituted O-C₁-C₆Alkyl (preferably OCH)₃) Optionally substituted C₂-C₇Acyl (preferably acetyl) or optionally substituted C₂-C₇Esters (oxycarbonyl esters or carboxyl esters, preferably, carboxyl groups)Esters). It is noted that each substituent disclosed herein may be substituted on its own.

The terms "simultaneous administration" or "adjunctive therapy" shall mean that at least two compounds or compositions are administered to a patient at the same time, such that an effective amount or concentration of each of the two or more compounds is obtained in the patient at a given point in time. Although the compounds according to the invention may be administered to a patient at the same time, the term encompasses not only the administration of two or more agents at the same time, but also the administration at different times, including sequential administration. Preferably, an effective concentration of all simultaneously administered compounds or compositions is found in the subject at a given time. The terms co-administration or adjunctive therapy also contemplate other bioactive agents administered concurrently with the pharmaceutical compositions of the invention, particularly when the cancer has metastasized or is at risk of metastasis.

The term "radiotherapy" or "radiotherapy" is used to describe a therapy for treating cancer in conjunction with a compound of the invention. Radiation therapy uses high doses of radiation, e.g., X-rays, or other energy sources, such as radioisotopes (gamma, beta, or alpha emitters) to kill cancer cells. The radiation destroys the genetic material of the cells so that they cannot grow. While radiation destroys normal cells and cancer cells at the same time, normal cells repair themselves and restore function, while cancer cells do not.

Depending on the cancer to be treated, radiation therapy may be used alone in combination with the claimed compounds, or in combination with additional anti-cancer agents as otherwise described herein. Radiation therapy is most effective in treating cancers that do not spread outside the original tumor area, but may also be used if the tumor has spread to nearby tissues. After surgery, radiation therapy is typically used to kill any remaining cancer cells and to reduce the pain associated with metastatic cancer.

Pharmaceutical composition

The compounds according to the invention can be readily formulated into pharmaceutical compositions for autophagy inhibition in biological systems, and/or inhibition, treatment or prevention of pathologies and/or conditions benefiting from autophagy inhibition, including cancer (and its metastases), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus (systemic lupus erythematosus), chronic urticaria and Sjogren's disease. The pharmaceutical composition comprises an effective amount of one or more compounds according to the invention in combination with a pharmaceutically acceptable carrier, additive or excipient, optionally, in the case of cancer, in combination with at least one additional agent, preferably an anti-cancer agent as described further herein.

As mentioned above, the compounds and methods of the invention are useful for inhibiting autophagy as otherwise described herein, and are useful for the treatment and inhibition (including prevention) 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 is an important aspect of the present invention.

In the methods according to the present invention, a subject or patient in need of assistance is treated with a compound, pharmaceutical composition of the present invention to inhibit, treat, or reduce the likelihood of a condition, and/or infection as otherwise described herein. One skilled in the art can readily identify and diagnose the conditions, and infections treated by the compounds and compositions of the present invention and treat them by administering to the patient an effective amount of one or more compounds of the present invention.

Generally, the dosage and manner of administration of the compounds will be determined by the size and condition of the subject according to standard pharmaceutical practice. The dosage level employed can be wide and can be readily determined by one skilled in the art. Typically, the amount employed is between milligrams and grams. The composition may be administered to the patient by a variety of means, for example, orally, transdermally, perineurally (perineurally), or parenterally, i.e., intravenously, subcutaneously, intraperitoneally, or intramuscularly, including orally, rectally, transdermally. Subjects contemplated for treatment according to the methods of the present invention include humans, companion animals, laboratory animals, and the like.

The formulations comprising the compounds of the present invention may take the form of solid, semi-solid, lyophilized powder or liquid dosage forms, e.g., tablets, capsules, powders, sustained release dosage forms, solutions, suspensions, emulsions, suppositories, creams, ointments, lotions, aerosols, transdermal patches, and the like, preferably in unit dosage forms suitable for easy administration of precise dosages.

The pharmaceutical compositions according to the present invention typically comprise a conventional pharmaceutical carrier or excipient and may additionally comprise other pharmaceutical agents, carriers, adjuvants, additives and the like. Preferably, the compound or compounds of the present invention comprise from about 0.1% to about 85%, from about 0.5% to about 75%, by weight of the composition, with the balance consisting essentially of suitable pharmaceutical excipients. For oral administration, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, gelatin, sucrose, sodium carbonate, and the like. The compositions may also protect minor amounts of non-toxic auxiliary substances, such as wetting agents, emulsifying agents, or buffering agents, if desired.

Liquid compositions may be prepared by dissolving or dispersing the compound (about 0.5% to about 20% by weight, or more) or optional pharmaceutical adjuvants in a carrier such as, for example, an aqueous salt solution, an aqueous dextrose solution, glycerol or ethanol to form a solution or suspension. For liquid formulations for use in oral administration, the composition may be prepared as a solution, suspension, emulsion or syrup, provided in a liquid form suitable for hydration in water or physiological saline, or in a dry form.

When the composition is orally administered using a solid formulation, the formulation may be a tablet, granule, powder, capsule, or the like. In the case of tablet formulations, the compositions are generally prepared with additives, for example excipients such as sugar or cellulose preparations, a binder such as starch paste or methyl cellulose, a filler, a disintegrant or other additives commonly used in the preparation of pharmaceutical preparations.

Injectable compositions for parenteral administration typically comprise the compound in a suitable i.v. solution, for example, sterile physiological saline. The composition can also be prepared into a suspension of lipids or phospholipids, a suspension of liposomes, or an aqueous emulsion.

Methods for preparing such dosage forms are known or will be apparent to those skilled in the art, see, for example, Remington's Pharmaceutical Sciences (17th ed., Mack pub. co., 1985). The compositions to be administered comprise a pharmaceutically effective amount of some of the selected compounds for inhibiting autophagy in a biological system, including a patient or subject of the invention.

Synthesis of Compounds of the invention

Synthetic route to bivalent aminoquinoline autophagy inhibitors

The present inventors examined the strategy of using multivalence for the synthesis of novel autophagy inhibitors by preparing dimeric chloroquine (FIG. 1, compound 3: Lys 01) from the purchased material (11, 12). Based on the existing literature (14), we expected to prepare compound 3 (14) from one equivalent of compound 5 and two equivalents of compound 6, as shown in the reverse synthesis of fig. 2.

Although compound 4 (R = Cl) is known as compound (14), bisaminoquinoline compound 3 (R = Cl)₂= Me) is not described in the literature. Because of its putative lysosome, we refer to compound 3 as Lys 01. Reaction of compound 5 with two equivalents of compound 4 yielded a mixture of the desired product compound 3 and some mono-quinoline compound 7 (fig. 1, Lys02, fig. 2), a synthetic process previously reported by Higuchi (18). To examine the effect of the C-7 chloro substituent in Compound 3, we prepared the dimethoxy analog of Compound 3, Compound 9 (FIG. 1, Compound 9: Lys 03).

To determine the importance of the polyamine linkage of the compounds, we prepared the polyether analogue compound 11 of compound 3 (FIG. 1, compound 11: Lys 04) using the purchased 2, 2' - (ethylenedioxy) bis (ethylamine) 10 (see FIG. 2).

The inventors tried to obtain structure-effect (SAR) data of lead compound 3 (Lys 01) by examining the systematic modification of the structure of compound 3 (i.e., 12R = Cl), as in scheme a of fig. 15A. Initial attempts focused on studying structural changes in three different regions of compound 12: 1) modification of the C-7 chloro substituent present in Compound 3 (X in Compound 12 of FIG. 15A); 2) modification of the C-4 nitrogen substituent (i.e., N-alkylation or acylation) and the adjacent carbon atom (containing a stereocenter in CQ and HCQ (FIG. 1)); and 3) modification of the N-methyl group in Compound 12 (FIG. 15A).

Each of the necessary starting compounds shown in fig. 15A (compounds 13-16) are known or commercially available, and based on Lee (16) studies, synthesis of a family of analogs other than compound 3 (12R = Cl) can be facilitated by mixing different electron withdrawing groups.

The inventors also tested the biological activity of the N-alkylated and N-acylated analogs of compound 12, compound 17, and compound 18 (fig. 15A). These novel compounds are prepared directly from 12 (R = Cl) by direct alkylation, reductive alkylation or acylation.

The inventors also examined the chirality that occurs in the introduction of CQ and HCQ (FIG. 1) to Compound 12, as shown for Compound 19 (scheme C of FIG. 15A). The requisite linker compound 20 was obtained using the method of Kokotos (j. chem. res, Synopses1992,12,391).

Finally, structural modifications of compound 12 by replacing the N-methyl group (scheme a of fig. 15A) with other functional groups were investigated. Scheme D of fig. 15A shows two interesting possibilities, compound 21 and compound 22, in which the N-methyl group is substituted by the hydroxyethyl group of HCQ, wherein the N-methyl group is partially substituted by another quinoline, yielding the triquinoline compound 22. The synthetic routes leading to 21 and 22 are closely based on studies of Lee (19) and Solomon (17), respectively.

While continuing to describe the properties of the second generation compounds, there are a number of additional compounds that are considered to be more potent autophagy inhibitors. The inventors have used compound 12 (scheme 1; Lys 01) as a lead compound to describe a more comprehensive systematic modification of three parts of the 12 structure, as shown in general structural formula 23 (see above), as the next logical step in SAR analysis. Each of the three parts of the structure (R) is as follows¹、R²And R³) Are all modified.

R¹Function of

Egan and his colleagues (20) have studied the role of replacing the chlorine moiety in chloroquine 1 (scheme 2), and they confirmed

Electron withdrawing groups are important for the anti-malarial activity of these 7-substituted quinolines. Thus, the inventors examined these same substitutions for general structural formula 23, simultaneous substitution of R in 23¹And R^1′Then R therein^1′The substituents are Cl, as in 12, or hydrogen (X = H). The studies of Egan show that all the necessary 4-chloro-7-substituted-quinolines are known, facilitating the preparation of each of the compounds shown in scheme 3. None of the compounds shown in scheme 3 are known, but the bonded (linked) bisquinoline (R)¹H) has been described (21).

R²Function of

In scheme 4, by acetylation (R)²= MeCO-; mono or di) or methylated (R)²= Me; one or two) results in structures 41-44 to detect R in the pilot structure 3²Effect of H. While analogues 45 and 46 of the same series, containing one or two propylene chains between the nitrogen atoms, are known (FR 1345573; CAN60: 68181), each 41-44 analogue represents a new structure.

R²The well tolerated substituents are further directed to the replacement of R by the hydroxyethyl moiety of hydroxychloroquine 2²(scheme 5). The reaction of aniline with ethylene oxide has been well documented to produce the corresponding hydroxyethyl compound (22), and thus, the conversion of lead structure 3 to mono-or bis-hydroxyethylated analogs 47 and 48 is readily accomplished.

The inventors also investigated the preparation of structures containing tri-and tetraquinolines as shown in scheme 6, based on a study by Bailey and his colleagues (23) by oxidation of the primary alcohols in 47 and 48 to the corresponding aldehydes, and reductive alkylation with 7-chloro-4-aminoquinoline to give tri-and tetraquinolines 49 and 50.

Based on the study by Drefahl and Konig (chem. ber.1954,87,1632-4), the inventors also investigated scheme 7 for the incorporation of lipophilic groups, i.e., long chain alkyl groups, in the lead structure, and more polar substituents replacing R²By the action of

59-62 para secondary amine (R) with a commercially available alkylating agent²= H) so that the preparation of 51-58 is by mono-and dialkylation of the two secondary amine functions.

The inventors have investigated varying R in the pilot structure 23³(see scheme 1 above). R³Certain substitutions of (a) are known, for example as shown in schemes 63-65 below in scheme 8. The inventors generated new structures (66-69) based on alkylation of known secondary amines 63 (13) using alkylating agents (59-62) shown in scheme 7, thereby preparing new analogs. Investigation of substrate 70, where R³=CH₂CH₂OH, i.e. the corresponding 63 analogue closest to hydroxychloroquine. This compound can be obtained by using the same sequence as for the preparation of the analogue in scheme 5, or by demethylation of 66.

Another important difference between the lead structure 3 and chloroquine 1 (scheme 9) is that the stereocentre is located next to the nitrogen atom in 1. The inventors have also prepared hybrid structures where both sides or one side of 23 more closely resemble 1, e.g., 71/72 and 73/74. Each kind of

The diamines which are necessary in this case are prepared starting from (L) glutamic acid and then using the procedure employed by Craig in the stereoselective synthesis of chloroquine (J.org.chem.1988, 53, 1167-1170).

The inventors prepared analogues of leader 3 containing the stereocenter of chloroquine 1, namely 75/76 and 77/78, which were obtained from alanine and serine, respectively, by reductive alkylation using Charlton and his colleagues method (24).

Finally, the inventors investigated a series of compounds containing four nitrogen atoms in the chain connecting the quinoline rings, as shown in scheme 10 below, rather than connecting the three nitrogen atoms in the chain in scheme 3 (scheme 9). Denny and his colleagues (24) describe the synthesis of the essential tetraamines 81 and 82. The attachment of the additional chloroquine moiety presented in 80 was performed by the same method used for synthesis 65 in scheme 8.

Method of treatment

According to one aspect of the present invention, a method is provided for treating a mammalian patient or subject to inhibit autophagy in the patient or subject. In accordance with the present invention, the compounds described herein are useful for inhibiting autophagy in a manner that inhibits, treats and/or prevents pathologies and/or conditions including cancer (including metastasis of cancer), rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease.

In accordance with the present invention, in a patient or subject in need of assistance, treatment is effected by administering to the patient or subject an effective amount of one or more compounds of the present invention, optionally in combination with at least one additional bioactive agent useful for treating the same condition or disease state. The compounds of the invention are useful for the inhibition, reduction of the likelihood, or treatment of cancer, including metastasis of cancer in a patient or subject in need of such treatment. Such treatment is useful for any cancer where inhibition of autophagy indicates a favorable outcome, or metastasis is a risk factor. Treatment with at least one additional anti-cancer agent as otherwise described herein is also understood to be a method of the invention. Various cancers that may be treated according to the methods of the invention are described above.

Another aspect of the invention is directed to methods of treating conditions and/or disease states that benefit from autophagy inhibition, including rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease. In this method, an effective amount of a compound described further herein is administered to a patient or subject in need of therapeutic assistance, optionally in combination with a pharmaceutically acceptable carrier, additive or excipient, to inhibit, treat and/or prevent the above-described condition and/or disorder.

In the present invention, the method of treatment comprises administering to a subject in need of therapeutic assistance an effective amount of a compound of formula I:

l is (CH)₂Y)_n―X―(Y′CH₂)_nA radical or A- (CH)₂―CH₂―Z)_nAn A' group (A orA' may be linked to one of the two amino groups of compound I), wherein at least one CH in L₂The radicals being optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups;

x is absent or is (CH)₂)_jO, S or N-R';

j is 1,2 or 3 (preferably 1 or 2);

a is absent or is (CH)₂)_jAnd A' is (CH)₂)_jWherein at least one CH2 group in A or A' is optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups;

z is O or N-R^z；

R^zIs H or optionally substituted C₁-C₃An alkyl group, a carboxyl group,

In the inventionIn a preferred process, R¹And R^1′Each independently is H, a halo group, nitro or trifluoromethyl, preferably a chloro group. R and R' are each independently H, optionally substituted C₁-C₃An alkyl group which itself is optionally substituted with at least one hydroxy, amino, monoalkylamino or dialkylamino group, wherein the amino group or the monoalkylamino group is optionally substituted at the position of the amino group with a 7-substituted-4-quinolyl group, wherein the amino group is attached to the 4-position of the quinolyl group, or one or both alkyl groups of the monoalkylamino or dialkylamino group are itself further optionally substituted with at least one hydroxy, amino, monoalkylamino or dialkylamino group, wherein the amino group or the monoalkylamino group is optionally substituted at the position of the amino group with one or two 7-substituted-quinolyl groups (as generally described above for structural formula I, the 7-position of each quinolyl group may be substituted with R¹And/or R^1′Substituted), or alkoxy (e.g., methoxy or ethoxy) substituted, preferably methoxy (thereby forming a monoether substituent), wherein the alkoxy may be further substituted with a monoalkoxy group.

In a further preferred process of the invention, L is (CH)₂Y)_n―X―(Y′CH₂)_nA radical of formula (I), wherein X is N-R ', Y and Y' are each independently absent or CH₂And R' is H or optionally substituted C₁-C₃An alkyl group which itself is optionally substituted with at least one hydroxy, amino, monoalkylamino or dialkylamino group, wherein the amino group or the monoalkylamino group is optionally substituted at the position of the amino group with a 7-substituted-4-quinolyl group, wherein the amino group is attached to the 4-position of the quinolyl group, or one or both alkyl groups of the monoalkylamino or dialkylamino group are itself further optionally substituted with at least one hydroxy, amino, monoalkylamino or dialkylamino group, wherein the amino group or the monoalkylamino group is optionally substituted at the position of the amino group with one or two 7-substituted-quinolyl groups (as generally described above for structural formula I, the 7-position of each quinolyl group may be substituted with R¹And/or R^1′Substituted), or alkoxy (e.g. methoxy or ethoxy) substituted, preferablyAnd (b) a methoxy group (thereby forming a diether substituent), wherein the alkoxy group may be further substituted with an alkoxy group.

A further preferred method involves the use/administration of a compound of the invention, which is provided in a variety of regimens, shown herein in scheme 1 and schemes 3-10, and figures 14, 15 and 15A.

In methods of treating or inhibiting cancer or metastasis thereof, the above compounds may be administered with AT least one additional anti-cancer agent, including, for example, everolimus, trabectedin, abraxane, TLK286, AV-299, DN-101, pazopanib, GSK690693, RTA744, ON0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD1152, enzastaurin, vandetanib (vandetanib), ARQ-197, MK-0457, MLN8054, PHA-9358, R-763, AT-927363, FLT-3 inhibitors, VEGFR inhibitors, TK inhibitors, aurora kinase inhibitors, PIK-1 modulators, Bcl-2 inhibitors, HDAC inhibitors, c-MET inhibitors, PARP inhibitors, Cdk inhibitors, TKR inhibitors, TK inhibitors, IGPI-3 inhibitors, AKT-1 inhibitors, and the like, JAK/STAT inhibitors, checkpoint-1 or 2 inhibitors, focal adhesion kinase inhibitors, Map kinase (mek) inhibitors, VEGF trap antibodies, pemetrexed, erlotinib, dasatinib, nilotinib, dacatinib, panitumumab, amrubicin, ogovazumab, Lep-etu, nolatrexed, azd2171, barbitulin, ofatumumab, zafirumab, zamoxan, idakaclin (eotecarin), tetrandrine, rubitecan, tilmiconazene, orlimerson (oblimersen), tizimumab (ticilimumab), yilimumab (Ipilimumab), gossypol, Bio111, 131-I-TM-601, ALT-110, BIO140, CC 90, Ciceripin, IMITUMB, IRE 13, IRQRQ 8432, QRQ-8432, and QRdR₁KRX-0402, lucanthone, LY317615, neuradiab, vistepan (vitespan), Rta744, Sdx102, talampanel, atrasentan, Xr311, romidepsin, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, adriamycinPlain, irinotecan, doxorubicin liposomes, 5' -deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib (seliciclib); PD0325901, AZD-6244, capecitabine, L-glutamic acid, N- [4- [2- (2-amino-4, 7-dihydro-4-oxo-1H-pyrrole [2,3-d ]]Pyrimidin-5-yl) ethyl]Benzoyl radical]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen conjugate, bevacizumab, IMC-1C11, CHIR-258; 3- [5- (methylsulfonylpiperidylmethyl) -indole-quinolone, vatalanib (vatalanib), AG-013736, AVE-0005, [ D-Ser (but)6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂Acetate salt [ C ]₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂) x, wherein x =1 to 2.4]Goserelin acetate, leuprolide acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide (nilutamide), megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, carratinib (canertinib), ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, arabinoside, dacarbazine, actinomycin D, daunomycin, diethylstilbestrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevec (gleevac), hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levotetramisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, tolperistrophin, L-N-D, L-S-D, L-S-A, S-S, S, Mitoxantrone, nilutamideNitratamide (nilutamide), octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium (porfimer), procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis retinoic acid, melphalan, uramustine, estramustine, hexamethylmelamine, floxuridine, 5-deoxyuridine (5-deooxyuridine), cytarabine, 6-mercaptopurine, deoxysyndiomycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razoxin, marimastat, COL-3, neovastat, BMS-275291, squalane, endostatin, SU 16, SU6668, EMD oxifene 974, interleukin 862, viloxazine, troxine, troxidine, valtrexine, troxidine, and other pharmaceutically acceptable salts thereof, Spironolactone, finasteride, cimetidine, trastuzumab, interleukin fusion toxin (denileukin diftotox), gefitinib, bortezomib (bortezimib), paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, paclitaxel without polyoxyethylated castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifene, pefoxifene (pimesdoxifene), ERA-923, azoxifene, fulvestrant, acobiprofen, lasofoxifene, indoxifene (idoxifene), TSE-33424, HMR-3339, ZK186619, PTK787/ZK222584, VX-745, PD184352, rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, cetolimus (AP-001), ABIRUS-23573, ABMST-23573, ABMSE-A, BC-210, LY294002, LY292223, LY292696, LY293684, LY293646, wortmannin, ZM336372, L-779,450, PEG-filgrastim, darbepotin (darbepoetin), erythropoietin, granulocyte colony stimulating factor, zoledronic acid, prednisone, cetuximab, granulocyte-macrophage colony stimulating factor, histrelin, pegylated interferon alpha-2 a, pegylated interferon alpha-2 b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, white mesogenes2, megestrol, immunoglobulins, nitrogen mustards, methylprednisolone, ibritumomab (ibritgumomab tiuxetan), androgens, decitabine, hexamethamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, daunorubicin liposomes, Edwina-asparaginase, strontium 89, casipiptan, netupitaptan, NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochloraz, granisetron, ondansetron, dolasetron, tropisetron, filgrastim, erythropoietin, epoetin alpha, dopepidopetidine alpha, and the like, and mixtures thereof.

In methods involving infections, conditions and/or conditions caused by rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and Sjogren's disease, the compounds according to the invention may be administered with additional agents conventionally used to treat such conditions and/or conditions.

Examples

The following examples illustrate and describe the invention, but are not intended to limit the invention in any way.

Synthesis of Compound 3 (Lys 01). A round-bottomed flask was charged with 4-bromo-7-chloroquine (Compound 5) (734 mg, 3.0 mmol), Pd (OAc)₂（23mg，0.1mmol）、BINAP（125mg，0.2mmol）、K₃PO₄(1.06 g, 5.0 mmol) and triamine (compound 6) (117 mg, 1.0 mmol). Dioxane (10 mL) was added via septum. The resulting suspension was stirred under argon for 18 hours at 90 ℃ and cooled. The mixture was adsorbed onto silica gel and purified by flash column Chromatography (CH)₂Cl₂MeOH: 90/9/1) to yield Compound 3 (387 mg, 88%) as a yellow solid. Melting point 199-; r_f=0.28 (silica gel, CH)₂Cl₂/MeOH/NH₄OH:90/9/1）：¹H NMR(500MHz,CDCl₃:δ8.53(d,J=5.5Hz,2H),7.94(d,J=2.0Hz,2H),7.41(d,J=9.0Hz,2H),6.98(dd,J=9.0,2.0Hz,2H),6.39(d,J=5.0Hz,2H),5.44(s,2H),3.42(q,J=5.0Hz,4H),2.90(t,J=6.0Hz,2H),2.46(s,9H).¹³C NMR(125MHz,CDCl₃) Delta 152.1,149.5,149.1,135.1,128.9,125.5,120.6,117.1,99.3,55.5,42.4,40.3FTIR (thin film): 3215,2917,1609,1579,1449, HRMS-ESI (m/z): C₂₃H₂₄N₅Cl₂[M+H]⁺Calcdfor 440.1409 and found 440.1406.

Synthesis of Compound 11 (Lys 05). A suspension of compound 3 (896 mg, 2.04 mmol) in MeOH (40 mL) was bubbled with HCl gas for 10 min at 0 deg.C to yield the water soluble salt of compound 3. The compound was stirred at room temperature for an additional 12 hours. The solvent was removed by rotary evaporation and the residue dried under vacuum at 50 ℃ overnight to give salt 3 (1.13 g, 100%) as a yellow solid. Melting point 270 ℃ (decomposition);¹H NMR(500MHz,D₂O):δ8.12(d,J=7.0Hz,2H),7.73(d,J=9.0Hz,2H),7.58(d,J=2.0Hz,2H),7.26(dd,J=9.0,2.0Hz,2H),6.62(d,J=2.0Hz,2H),3.89(br,4H),3.68(br,4H),3.12(s,3H).¹³C NMR(125MHz,D₂o). delta. 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): 3376,3019,2914,1631,1612,1215cm^-1.HRMS-ESI(m/z):C₂₃H₂₄N₅Cl₂[M-3HCl+H]⁺Calculated value 440.1409, experimental value 440.1408.

Biological assay

Lys01 is a more potent autophagy inhibitor than HCQ or CQ.

LN229 (human glioblastoma) was treated with Lys01 and the derivatives Lys02, Lys03, Lys04, HCQ and CQ. In the cells treated with Lys01 at a concentration of 10. mu.M or more, almost complete death of the cultured cells was observed within 4 to 24 hours. LC3 is a ubiquitin-like protein that exists in an unbound form to the AV membrane (LC 3I) or bound to the AV membrane (LC 3 II) (25). The ratio of LC3II/LC3I reflects the accumulation of AV in cells, and thus can produce an autophagy-inhibiting effect. LC3 immunoblots (fig. 3) showed that Lys01 at a concentration of 10 μ M was a more potent autophagy inhibitor than HCQ or CQ by a factor of 10. Similar to HCQ or CQ, LC3 immunoblots of Lys02 and Lys03 exhibited a dose-effect relationship, however, Lys04, which retains two chloroquine rings in Lys01, demonstrated moderate potency in LC3 autophagy assays.

To further characterize the effect of Lys01 on autophagy, LN229GFP-LC3 cells were treated with Lys01 or HCQ. In cells treated with 1 μ M HCQ, fluorescent spots were observed in a few cells within 4 hours of treatment, indicating accumulation of ineffective autophagic vesicles. HCQ at 10. mu.M produced a large number of small spots, while HCQ at 100. mu.M produced a greater density of small spots, indicating fusion of accumulated autophagic vesicles. Treatment with 1 μ M Lys01 produced a large number of small spots, while cells treated with 10 μ M Lys01 produced dense spots, similar to that observed in 100 μ M HCQ treated cells. Cells treated with 100 μ M Lys01 all died within 4 hours. Quantification of the GFP-LC3 dot in each cell indicated a 5-fold increase in GFP-LC3 dot with Lys01 treatment between 10. mu.M compared to treatment with 10. mu.M HCQ. The average vesicle per cell was larger in cells treated with 10. mu.M Lys01 than in cells treated with 100. mu.M HCQ (FIG. 4A). Electron micrographs of DMSO, HCQ, or Lys 01-treated LN229GFP-LC3 cells further characterized the apparent morphological differences in vesicle size and number resulting from autophagic blockade caused by these agents. Thus, Lys01 produced a more significant morphological change using a 10-fold lower concentration than the known lysosomal inhibitor HCQ. To determine whether Lys01 treatment induced the production of new autophagic vesicles (autophagy inducer) or prevented the clearance of autophagic vesicles (autophagy inhibitor), a bavaomycin clamp test was performed (fig. 4C). LN229GFP-LC3 cells were treated with DMSO, rapamycin, 10. mu.M HCQ, 10. mu.M Lys01 in the presence or absence of bavacomycin. After 24 hours, rapamycin treatment further increased the LC3II/LC3I ratio of bavaomycin-treated cells compared to control cells, however, the LC3II/LC3I ratio of HCQ or Lys 05-treated cells did not increase in bavaomycin-treated cells compared to control cells, providing further evidence that Lys01 is an autophagy inhibitor (fig. 4C).

To determine the close relationship between more effective autophagy inhibition and 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 μ M (fig. 4D). The MTT assay is used to evaluate viable cells after 72 hours. Of the 4 cell lines tested, IC50 for Lys01 was 4-8. mu.M (appended Table 2). In 1205Lu and HCC827 (highly HCQ-tolerant cell line) cells treated with Lys01 at a concentration of 10 μ M, almost complete cell death was observed after 24 hours. In contrast, LC50 of monofunctional CQ derivatives Lys02 (35-91. mu.M), methoxy-substituted chloro bisaminoquinoline Lys03 (24-53. mu.M), or HCQ (15-42. mu.M) were consistently 9-30 times less effective than Lys 01. Lys04, which retains the divalent aminoquinoline ring but has an altered linking group, has moderate activity with an IC50 of 10-17. mu.M. These studies showed that Lys01 was consistently more cytotoxic than the other aminoquinolines or HCQ tested. Together with western blot data for LC3, these data indicate that the most potent cytotoxic autophagy inhibitor contains two aminoquinoline rings, a triamine linker is present in Lys01, and the C-7 position of the aminoquinoline ring is substituted with chlorine.

In vivo autophagy inhibition and antitumor efficacy of Lys 05.

The trihydrochloride salt of Lys01, Lys05, was synthesized to improve water solubility and make it useful for in vivo studies. Lys01 and Lys05 showed equal dose-dependent increases in the LC3II/LC3I ratio, as well as accumulation of autophagic cargo protein p62 (26), and had the same IC50 value in the MTT assay (fig. 5A, B). To investigate the safety of Lys05 and its effect on autophagy in vivo, c8161 xenografts matching tumor size were treated with daily intraperitoneal injections of (i.p.) PBS or equimolar amounts of HCQ or Lys05 (HCQ 60mg/kg (138 nmoles/g), Lys0576mg/kg (138 nmoles/g)) for 48 hours. None of the mice died under this high dose, short duration treatment, however, mice treated with intraperitoneal injection of 76mg/kgLys05 were observed to develop dorsum arcus and lethargy after 2 days. Mice treated for 48 hours were euthanized and tumors were examined by Electron Microscopy (EM). Morphologically, EM examination showed that in Lys 05-treated tumors, cells had intact nuclei and cytoplasmic membranes containing large AV (fig. 6A). The mean number of AV/cells in two representative tumors in each treatment group was quantitatively found to increase significantly more than 2-fold in Lys 05-treated tumors compared to control or HCQ-treated tumors (fig. 6B). Significantly higher LC3II/LC3I levels were observed in Lys 05-treated tumors compared to control or HCQ-treated tumors, providing further evidence of autophagy inhibition in vivo (fig. 7A). After 48 hours of treatment, levels of cleaved caspase3 (cleared caspase 3) indicative of apoptosis were elevated in Lys05 treated tumors compared to HCQ or PBS treated tumors.

In addition to certain models of pancreatic cancer (27), in many animal tumor models, high levels of autophagy are likely to occur in untreated tumors (4, 28), and treatment with a single agent, HCQ, did not impair tumor growth (29, 30). To determine whether a more potent autophagy inhibitor, such as Lys05, as a single agent significantly attenuated tumor growth, 1205Lu xenografts were formed on both sides of nude mice. For chronic treatment experiments, the 1205Lu melanoma model was selected over the c8161 xenograft model because c8161 xenografts tend to spontaneously form ulcers, confounding tumor measurements and safety analysis. For 3 treatment groups, 10 mice carrying 1205Lu xenografts matching the tumor volume per group were assigned intraperitoneal injections of PBS, HCQ60mg/kg or Lys0576mg/kg (equimolar dosing), 3 days of daily treatment, 2 days of no treatment (3/5 days) to allow symptom recovery and avoid excessive toxicity. This treatment was scheduled to be well tolerated over a 14 day period. The tumor growth curves for each of the 3 groups showed that tumor growth was significantly disrupted in Lys 05-treated tumors compared to controls. Lys05 treatment reduced the average daily growth rate of tumors by 53% (31.2 pairs) compared to solvent-treated controls14.6 mm³A/day; p = 0.002; fig. 6D). In HCQ and Lys05 treated tumors, significant accumulation of AV was observed after 14 days of treatment, but the AV/cell number of Lys05 treated tumors was increased 6-fold compared to control treated tumors, while the AV/cell number of HCQ treated tumors was increased 3-fold (fig. 7B). Extensive tumor necrosis was observed in Lys 05-treated tumor centers (fig. 7B).

To determine whether low doses of Lys05 could produce anti-tumor activity, mice bearing HT-29 colon cancer xenografts were PBS treated or intraperitoneally injected daily with 10mg/kg Lys05, 40mg/kg Lys05, or 80mg/kg Lys05 on 3 of 5 days. Clinical toxicity was observed only in the group of 80mg/kg, 2 out of 8 mice were euthanized at an early stage due to ileus. The groups with daily doses of 10mg/kg and 40mg/kg tolerated well. Treatment with Lys05 clearly destroyed the average daily growth rate of the tumor in a dose-dependent manner (fig. 6E). The tumor growth curves showed that all 3 doses of Lys05 produced significant tumor growth disruption compared to controls. At the end of the experiment, the excised tumor weights indicated that significant antitumor activity was observed in the group at the 10mg/kg daily dose (fig. 6G). Immunoblotting of the harvested tumor lysate against LC3 after 14 days of treatment showed a significant increase in the LC3II/LC3I ratio in all Lys05 treated tumors, including the 10mg/kg dose treated tumor, compared to the control (fig. 7C).

Enterotoxicity was similar to autophagy gene deficiency when Lys05 was administered at the maximum dose.

In the 1205Lu xenograft trial, single animals treated with 76mg/kgLys05 intraperitoneally on day 3 out of 5 presented lethargy and an arch back (fig. 8A). 3 of 10 mice treated with Lys05 developed signs of ileus (fig. 8B). Intestinal examination revealed a dilated proximal small intestine with a pseudostenotic (pseudostricture) terminal ileum. Histological examination of the ileum showed no signs of excessive inflammation, fibrosis or mechanical obstruction, indicating that the signs of ileus observed in rats were due to either pseudo-stenosis or functional ileus. Although the small intestine villus and crypt architecture were intact, aberrant panne cells were observed (fig. 8C). Panne cell insufficiency includes a reduction in particle size and number of eosinophilic lysozyme, which has been previously described as a special sign of mouse autophagy deficiency, while a proportion of crohn's disease patients have genetic defects in the essential autophagy gene ATG16L 1.

In the HT-29 dose-exploratory xenograft trial, no significant weight loss was observed in any of the dose groups (FIG. 9A). After 14 days of treatment, the entire gastrointestinal tract was excised from HT-29 bearing mice, indicating that bowel wall thickening and ileus were limited to the 80mg/kg dose group (FIG. 9B). 3 of 5 days HT-29 xenografts were treated with daily intraperitoneal injections of PBS, or 10mg/kg Lys05, 40mg/kg Lys05 and 80mg/kg Lys05, and 14 days after treatment, the ileal ends were excised from mice carrying the HT-29 xenografts for histological examination, with a dose-dependent effect on surface Pan cell morphology (FIG. 9C). After treatment, the panne cells/crypts were unchanged (fig. 9D), while the size and number of particles decreased in a dose-dependent manner. Although signs and symptoms of toxicity were limited to the 80mg/kg dose (fig. 9E), the scale of panecell dysfunction scoring (fig. 10) showed that panecell dysfunction was observed in all dose tests of Lys 05. In mice treated with 40mg/kg or 80mg/kg, but not 10mg/kg Lys05, lysozyme was significantly reduced or absent in Pan cells (FIG. 9F). Taken together, these findings indicate that Lys 05-associated pangolin cell insufficiency is visualized as ATG16L1 deficiency, and that lower doses of Lys05 produce significant anti-cancer activity without dose limiting toxicity.

Lys05 inhibits autophagy by de-acidification of lysosomes.

To compare the relative lysosomal accumulation of Lys05 versus HCQ, lysosomal fractions were separated from 1205Lu cells treated with PBS, 10 μ M HCQ, or 10 μ MLys05, and where 1205Lu tumors were harvested after 14 days using daily intraperitoneal injections of PBS, 60mg/kg HCQ, or 76mg/kg Lys05 on 3 of 5 days. Immunoblotting against the lysosomal marker LAMP2 confirmed that the lysosomes and the whole cell population were well separated in the cell and tumor samples (fig. 12A). After 24 hours of treatment of the whole cell homogenate with 10. mu.MLys 05 or 10. mu.M HCQ, the concentrations of Lys05 and HCQ therein, measured by HPLC tandem mass spectrometry (MS/MS) (FIG. 11), were 57. mu.M and 8. mu.M, respectively, indicating a 6-fold higher concentration of Lys05 in the cells compared to HCQ. After treatment of the cell lysate fraction with 10. mu.M Lys05 or 10. mu.M HCQ, the Lys05 and HCQ concentrations were 105. mu.M and 13. mu.M, respectively, indicating that the Lys05 concentration in the lysate was 8 times higher than that of HCQ. The difference in the accumulation of Lys05 and HCQ was more pronounced in cells and lysosomes than in tumor tissues. Lys05 concentrations were 11-fold and 34-fold higher in the whole cell homogenate and lysosome in tumors than in HCQ, respectively (fig. 12B).

It has been determined that Lys05 accumulates more efficiently in lysosomes than HCQ, and the functional effects of this accumulation were examined. 1205Lu cells were treated with solvent, Lys05, and HCQ and stained with lysosomal red fluorescent probe (fig. 12C). Few positive spots of lysosomal fluorescent probes were observed in Lys 05-treated cells at concentrations of 10 μ M and 100 μ M within a treatment time of 30 minutes. In contrast, a significant reduction in positive specks of the lysosomal fluorescent probe was observed in cells treated with 100 μ M HCQ, but no positive specks of the lysosomal fluorescent probe were observed in cells treated with 10 μ M HCQ. To understand the effect of this more potent and complete lysosomal inhibition, 1205Lu cells were treated with solvent, Lys05 or HCQ, and treated with acridine orange (AO; a dye that accumulates in all acidic compartments in the blood vessel) at 24 hours (FIG. 12D). HCQ produces a dose-dependent accumulation of acidic vesicles. In contrast, Lys05 resulted in accumulation of acidic vesicles at the lower dose (10 μ M), but at the higher dose (50 μ M), no acidic vesicles were observed, indicating complete de-acidification of the intravascular system.

Finally, the functional results of lysosome deacylation were examined by measuring the enzymatic activity of the acid phosphatase. 1205Lu cells were treated with PBS, 10 μ M HCQ, or 10 μ M Lys05 for 24 hours, with a 43% decrease in acid phosphatase activity in Lys 05-treated lysosomal fractions compared to PBS-treated cells (fig. 13A). Leakage of certain lysosomal enzymes, such as activated cathepsins, can lead to autophagy-independent cell death. Acid-dependent progression of immature cathepsin D to the mature, activated form in Lys 05-treated cytopolysosomes was reduced compared to HCQ or PBS-treated cells within 24 hours of treatment of 1205Lu cells with PBS, HCQ, or Lys05 (fig. 13B). After 14 days of treatment, Lys 05-treated tumors showed a 1.75-fold increase in lyso-insoluble (exolysomal) acid phosphatase activity in 1205Lu xenograft tumors, suggesting that chronic treatment resulted in enzymatic lyso-insoluble leakage (fig. 13C), but no acid-dependent increase in cathepsin D was observed in Lys 05-treated tumor whole cell homogenates (fig. 13D). These results indicate that high doses of Lys05 lead to lysosomal insufficiency by lysosome deacidification, leading to lysosomal enzyme destruction, and effective autophagy inhibition, while high doses of HCQ do not completely deacidify lysosomes, leading to incomplete autophagy inhibition associated with less cell death.

Another compound Lys06-Lys 18. Additional Lys01 derivatives were synthesized and tested (fig. 14, fig. 15). IC50 of compound Lys01-Lys13 compound exhibited increased or decreased activity compared to HCQ or CQ in the MTT assay for 72 hours (table 1, fig. 16). In most cases, derivatives of Lys01 are more potent than CQ or HCQ. These findings further perfected the starting point for drug development into Lys01 derivatives.

The activity of Lys01 derivatives in malaria. Table 2, figure 17 shows IC50 values for Lys01 derivatives inducing cell death in human cancer cell LN229, and IC50 values in several plasmodium Falciparum (p. Falciparum) grown in vitro in human RBC. Lys01 derivatives have similar anti-cancer activity and malaria cytotoxicity. Lys01 was more potent than artesunate in many CQ-resistant cell lines.

Conclusion

Potential commercial uses and applications: lys01 and Lys05 are lead compounds whose potency has great potential for further optimization of novel autophagy inhibitors. Autophagy inhibition is a novel therapeutic strategy for cancer, which can be used for any cancer. There are more than 30 HCQ trials in cancer patients, involving almost all tumor types. Due to the low potency and poor pharmacological action of HCQ, the promising enhancement of anticancer therapy observed in laboratory models may not be obtained in humans. An optimized Lys01 derivative enables the development of second-generation autophagy inhibitors. The GI toxicity associated with pancreatin insufficiency observed with LD30 dose of Lys05 supports the mechanism of action of the drug and also suggests that colon cancer, which usually presents the same features as pancreatin, may be a type of cancer that is particularly sensitive to Lys05 and its optimized derivatives. Additional cancers worth investigation included melanoma as well as non-small cell lung cancer, as melanoma cell lines exhibited the most differential sensitivity to Lys01 relative to HCQ, while EGFR-mutated lung cancer cell lines showed sensitivity to both HCQ and Lys 05. The synthesis of Lys01 was designed to be non-overlapping with other patented and/or published aminoquinoline compounds. The next mechanistic study planned was to determine pharmacodynamic tests to guide drug development. Pharmacokinetic studies were initially conducted in mice.

Other similar technologies and competing products: novel chloroquine derivatives useful as anticancer agents are a hotspot of research (16). Autophagy has been identified as one of ten areas of research, where NIH will be the direction of research in the next few years. To date, no study has been conducted to evaluate the potential of bivalency as provided herein by the inventors (leviaged). Furthermore, most studies lack in vivo studies and mechanistic studies as reported herein, which can guide further development of optimized lead compounds for drug development.

Advantages over other similar technologies and products: thus, the present application shows that a series of bisaminoquinolines disclosed are potent autophagy inhibitors with single agent antitumor activity in an in vivo tumor model.

Reference to the literature

1.Lum JJ,DeBerardinis RJ,Thompson CB.Autophagy in metazoans:cell survival in the land ofplenty.Nat Rev Mol Cell Biol 2005;6:439-48.

2.Amaravadi RK,Thompson CB.The roles of therapy-induced autophagy and necrosis in cancertreatment.Clin Cancer Res 2007;13:7271-9.

3.Amaravadi RK,Yu D,Lum JJ,et al.Autophagy inhibition enhances therapy-induced apoptosisin a Myc-induced model of lymphoma.J Clin Invest2007;117:326-36.

4.Degenhardt K,Mathew R,Beaudoin B,et al Autophagy promotes tumor cell survival andrestricts necrosis,inflammation,and tumorigenesis.Cancer Cell2006;10:51-64.

5.Amaravadi RK.Autophagy-induced tumor dormancy in ovarian cancer.J Clin Invest2008.

6.Carew JS,Nawrocki ST,Kahue CN,et al.Targeting autophagy augments the anticancer activityof the histone deacetylase inhibitor SAHA to overcome Bcr-Abl-mediated drug resistance.Blood2007,

7.Degtyarev M,De Maziere A,Orr C,et al.Akt inhibition promotes autophagy and sensitizesPTEN-null tumors to lysosomotropic agents.J Cell Biol2008;183:101-16.

8.Sotelo J,Briceno E,Lopez-Gonzalez MA.Adding chloroquine to conventional treatment forglioblastoma multiforme:a randomized,double-blind,placebo-controlled trial.Ann Intern Med2006;144:337-43,

9.Amaravadi RX,Lippincott-Schwartz J,Yin XM,et al Principles and Current Strategies forTargeting Autophagy for Cancer Treatment.Clin Cancer Res2011;17:654-66.

10.Rosenfeld MR GS,Brem S,Mikkelson T,Wang D,Piao S,Davis L,O′Dwyer PJ,AmaravadiRK Pharmacokinetic analysis and pharmacodynamic evidence of autophagy inhibition in patientswith newly diagnosed glioblastoma treated on a phase I trial of hydroxychloroquine in combinationwith adjuvant temozolomide and radiation(ABTC0603).J Clin Oncol2010;28:Abstract#3086.

11.Vance D,Shah M,Joshi A,Kane RS.Polyvalency:a promising strategy for drugdesign.Biotechnol Bioeng2008;101:429-34.

12.Shrivastava A,Nunn AD,Tweedle MF,Designer peptides:learning from nature.CurrPharm Des2009;15:675-81.

13.Girault S,Grellier P,Berecibar A,et al Antiplasmodial activity and cytotoxicity of bis-,tris-,and tetraquinolines with linear or cyclic amino linkers.J Med Chem2001;44:1658-65.

14.Vennerstrom JL,Ager AL,Jr.,Dorn A,et al.Bisquinolines.2.Antimalarial N5N-bis(7-chloroquinolin-4-yl)heteroalkanediamines.JMed Chem1998;41:4360-4.

15.Burnett JC,Schmidt JJ,Stafford RG,et al.Novel small molecule inhibitors of botulinumneurotoxin A metalloprotease activity.Biochem Biophys Res Commun2003;310:84-93.

16.Hu C,Raja Solomon V,Cano P,Lee H.A4-aminoquinoline derivative that markedly sensitizestumor cell killing by Akt inhibitors with a minimum cytotoxicity to non-cancer cells.Eur J MedChem2010;45:705-9.

17.Solomon VR,Hu C,Lee H.Design and synthesis of chloroquine analogs with anti-breastcancer property.Eur J Med Chem2010;45:3916-23.

18.T.Higuchi HO,M.Umezawa,H.Kim,Y.Wataya,.Compound with Antimalarial Activity andAntimalarial Drug Containing the Same as Active Ingredient.WO/2007/097450.

19.Lee WW,Berridge BJ,Jr.,Ross LO,Goodman L.Synthesis of Mustards from Putrescine,Cadaverinej and1,3-Diaminopropane.JMed Chem1963;6:567-9.

20.Kaschula CH,Egan TJ,Hunter R,et al Structure-activity relationships in4-aminoquinolineantiplasmodials.The role of the group at the7-position.J Med Chem2002;45:3531-9.

21.Adams A,Jarrott B,Elmes BC,Denny WA,Wakelin LP.Interaction of DNA-intercalatingantitumor agents with adrenoceptors.Mol Pharmacol1985;27:480-91.

22.Gourdie TA,Valu KK,Gravatt GL,et al.DNA-directed alkylating agents.1.Structure-activityrelationships for acridine-linked aniline mustards:consequences of varying the reactivity of themustard.J Med Chem1990;33:1177-86.

23.Bailey DM,DeGrazia CG,Hoff SJ,et al.Bispyridinamines:a new class of topical antimicrobialagents as inhibitors of dental plaque.J Med Chem1984;27:1457-64.

24.Vicker N,Burgess L,Chuckowree IS,et al.Novel angular benzophenazines:dualtopoisomerase I and topoisomerase II inhibitors as potential anticancer agents.J Med Chem2002;45:721-39.

25.Tanida I,Ueno T,Kominami E,LC3conjugation system in mammalian autophagy.Int JBiochem Cell Biol2004;36:2503-18.

26.Pankiv S,Clausen TH,Lamark T,et al.p62/SQSTM1binds directly to Atg8/LC3to facilitatedegradation of ubiquitinated protein aggregates by autophagy.J Biol Chem2007;282:24131-45.

27.Yang S,Wang X,Contino G,et al.Pancreatic cancers require autophagy for tumorgrowth.Genes Dev2011;25:717-29.

28.Ma X,Piao S,Wang DW,et al.Measurements of tumor cell autophagy predict invasiveness,resistance to chemotherapy,and survival in melanoma.Clin Cancer Res2011.

29.Fan QW,Cheng C,Hackett C,et al.Akt and autophagy cooperate to promote survival ofdrug-resistant glioma.Sci Signal2010;3;ra81.

30.Saleem A,Dvorzhinski D,Santanam U,et al.Effect of dual inhibition of apoptosis andautophagy in prostate cancer.Prostate2012.

31.Cadwell K,Liu JY,Brown SL,et al.A key role for autophagy and the autophagy gene Atg 1611in mouse and human intestinal Paneth cells.Nature2008;456;259-63.

Claims

1. A compound having the following chemical structure I:

wherein R is¹And R^1′Each independently H, halogen (F, Cl, Br or I), CN, NO₂Optionally substituted C₁-C₆Alkyl (when substituted, preferably substituted with 1 or 2 hydroxy or 3-5 fluoro), optionallySubstituted O-C₁-C₆Alkyl (preferably OCH)₃) Optionally substituted C₂-C₇Acyl (preferably acetyl) or optionally substituted C₂-C₇Esters (oxycarbonyl esters or carboxyl esters, preferably, carboxyl esters);

l is (CH)₂Y)_n―X―(Y′CH₂)_nA radical or A- (CH)₂―CH₂―Z)_nAn A 'group (A or A' may be attached to one of the two amino groups of compound I), wherein at least one CH of L₂The radicals being optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups;

x is absent or is (CH)₂)_jO, S or N-R';

j is 1,2 or 3 (preferably 1 or 2);

a is a chemical bond (absent) or (CH)₂)_jAnd A' is a chemicalBond (absent) or (CH)₂)_jWherein at least one CH in A or A₂The radicals being optionally substituted by C₁-C₃Alkyl substitution of the C₁-C₃Alkyl itself is optionally substituted by one or two hydroxy groups;

z is O or N-R^z；

R^zIs H or optionally substituted C₁-C₃An alkyl group, a carboxyl group,

or a pharmaceutically acceptable salt, enantiomer, asymmetric isomer, solvate or polymorph thereof.

2. The compound of claim 1, wherein R¹And R^1′Each independently is H, a halo group, a nitro group, or a trifluoromethyl group.

3. The compound of claim 1 or 2, wherein R and R' are each independently H, optionally substituted C₁-C₃Alkyl which is itself optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino or dialkylamino group, wherein the amino group or the monoalkylamino group is optionally substituted at the position of the amino group by a 7-substituted-4-quinolyl group, wherein the amino group is attached to the 4-position of the quinolyl group and the 7-position of the quinolyl group is substituted by an R as described in claim 1¹Or R^1′Substituted, or one or both alkyl groups of said monoalkylamino or dialkylamino group are themselves further optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino or dialkylamino group, wherein said amino or said monoalkylamino group is optionally substituted at the amino position by one or two 7-substituted-quinolinyl groups, wherein said amino group is attached to the 4-position of the quinolinyl group and the 7-position of said quinolinyl group is substituted by R according to claim 1¹Or R^1′And each said alkoxy group may be further substituted by a monoalkoxy group, preferably methoxy (thereby forming a monoether substituent).

4. The compound of any one of claims 1-3, wherein L is

―(CH₂Y)_n―X―(Y′CH₂)_nA radical of formula (I), wherein X is N-R'; n is 1,2 or 3; y and Y' are each independently absent or CH₂(ii) a And R' is H or optionally substituted C₁-C₃Alkyl, itself optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino, dialkylamino group, wherein said amino or said monoalkylamino group is optionally substituted at the position of the amino group by a 7-substituted-4-quinolyl group, wherein said amino group is attached to the 4-position of the quinolyl group and the 7-position of said quinolyl group is substituted by an R as described in claim 1¹Or R^1′Substituted, or one or both alkyl groups of said monoalkylamino or dialkylamino group are themselves further optionally substituted by at least one hydroxy, alkoxy, amino, monoalkylamino, dialkylamino group, wherein said amino or said monoalkylamino group is optionally substituted at the amino position by one or two 7-substituted-quinolinyl groups, wherein said amino group is attached to the 4-position of the quinolinyl group and the 7-position of said quinolinyl group is substituted by R according to claim 1¹Or R^1′And each said alkoxy group may be further substituted with a second alkoxy group to form a diether substituent.

5. A compound according to claim 3 or 4, wherein C is₁-C₃The alkyl group is substituted with an amino group which is substituted with one 7-substituted-4-quinoline group.

6. The compound of claim 5, wherein the alkyl is C₂An alkyl group.

7. The compound of claim 3 or 4, wherein C is₁-C₃The alkyl group is substituted with a first alkoxy group, which is substituted with a second alkoxy group, thereby forming a diether group.

8. The compound of claim 7, wherein the second alkoxy group is methoxy or ethoxy.

9. Compounds as depicted in scheme 1 or 3-10.

10. A compound of claim 9 which is any one of compounds 28-40 of scheme 3.

11. A compound of claim 9 which is any one of compounds 41-46 of scheme 4.

12. A compound of claim 9 which is one of compounds 47-48 of scheme 5.

13. A compound of claim 9 which is one of compounds 49-50 of scheme 6.

14. A compound of claim 9 which is any one of compounds 51-58 of scheme 7.

15. A compound of claim 9 which is any one of compounds 63-70 of scheme 8.

16. A compound of claim 9 which is any one of compounds 71-78 of scheme 9.

17. A compound of claim 9 which is any one of compounds 79 to 82 of scheme 10.

18. The compound of claim 9, which is compound 3 of scheme 1.

19. A compound as depicted in figure 14, 15 or 15A.

20. A compound:

N¹- (7-chloroquinolin-4-yl) -N²- (2- ((7-chloroquinolin-4-yl) amino) ethyl) -N²-methyl ethane-1, 2-diamine;

N¹- (7-chloroquinolin-4-yl) -N²- (2- ((7-chloroquinolin-4-yl) amino) ethyl) -ethane-1, 2-diamine;

n, N' - ((ethane-l, 2-diylbis (oxy)) bis (ethane-2, 1-diyl)) bis (7-chloroquinolin-4-amine);

N¹- (7-methoxyquinolin-4-yl) -N²- (2- ((7-methoxyquinolin-4-yl) amino) ethyl) -N²-methyl ethane-1, 2-diamine;

N¹- (7-chloroquinolin-4-yl) -N²- (2- ((7-chloroquinolin-4-yl) amino) ethyl) -N²-methyl ethane-1, 2-diamine trihydrochloride;

N¹- (7-chloroquinolin-4-yl) -N²- (2- ((7-chloroquinolin-4-yl) (methyl) amino) ethyl) -N¹,N²-dimethylethane-1, 2-diamine;

n, N' - ((methylazanediyl) bis (ethane-2, 1-diyl)) bis (N- (7-chloroquinolin-4-yl) acetamide);

(S)-N²- (7-chloroquinolin-4-yl) -N¹- ((S) -2 ((7-chloroquinolin-4-yl) amino) propyl) -N¹-methylpropane-1, 2-diamine;

2- (bis (2- ((7-chloroquinolin-4-yl) amino) ethyl) amino) ethanol;

N¹- (7-chloroquinolin-4-yl) -N²,N²Bis (2- ((7-chloroquinolin-4-yl) amino) ethyl) -ethane-1, 2-diamine, or

Their pharmaceutically acceptable salts.

21. The compound of claim 1, being N¹- (7-chloroquinolin-4-yl) -N²- (2- ((7-chloroquinolin-4-yl) amino) ethyl) -N²-methyl ethane-1, 2-diamine or a pharmaceutically acceptable salt thereof.

22. The compound of claim 1, being N¹- (7-chloroquinolin-4-yl) -N²- (2- ((7-chloroquinolin-4-yl) amino) ethyl) -N²-methyl ethane-1, 2-diamine trihydrochloride.

23. A pharmaceutical composition comprising an effective amount of at least one compound according to any one of claims 1-22 in combination with a pharmaceutically acceptable carrier, additive or excipient, and optionally in combination with at least one additional anti-cancer agent.

24. A method of inhibiting autophagy in a biological system, wherein inhibition of autophagy is desired, comprising exposing the biological system to an effective amount of at least one compound according to any one of claims 1-22.

25. A method of inhibiting or treating cancer in a patient in need thereof, comprising administering to said patient an effective amount of at least one compound of any one of claims 1-22, optionally in combination with at least one additional anti-cancer agent.

26. The method of claim 25, wherein the cancer is metastatic.

27. The method of claim 26, wherein the cancer is a drug-resistant cancer.

28. A method of reducing the likelihood of the occurrence of cancer in a patient or the occurrence of cancer metastasis in a patient, comprising administering an effective amount of at least one compound according to any one of claims 1-22, optionally in combination with at least one additional anti-cancer agent.

29. The method of claim 25, wherein the cancer is cancer, cancer of the esophagus, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, and stomach; leukemia, malignant lymphoma, malignant melanoma; myeloproliferative diseases; sarcoma, central nervous system tumor, germ cell tumor, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, melanoma, and mixed type tumor.

30. The method of claim 28, wherein the leukemia is acute myelogenous leukemia, acute lymphocytic leukemia, Acute Promyelocytic Leukemia (APL), acute T-cell lymphocytic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, myelogenous leukemia, hairy cell leukemia, cytopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia, small myelogenous leukemia, monocytic leukemia, neutrophilic leukemia, stem cell leukemia.

31. The method of claim 28, wherein the lymphoma is burkitt's lymphoma, non-hodgkin's lymphoma, and B-cell lymphoma.

32. The method of claim 28, wherein the sarcoma is ewing's sarcoma, angiosarcoma, kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelial tumor, or synovial sarcoma.

33. The method of claim 28, wherein the tumor of the central nervous system is a glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma, ganglionic cytoma, ganglioglioma, medulloblastoma, pinealocytoma, meningioma, meningiosarcoma, neurofibroma, or schwannoma.

34. The method of claim 28, wherein the germ cell tumor is an intestinal cancer, breast cancer, prostate cancer, cervical cancer, or uterine cancer.

35. The method of claim 28, wherein the lung cancer is small cell lung cancer, a mixture of small cell and non-small cell cancers, pleural mesothelioma, metastatic pleural mesothelioma, small cell lung cancer or non-small cell lung cancer.

36. The method of claim 28, wherein the mixed type of tumor is a carcinosarcoma and hodgkin's disease and the mixed cell of origin tumor is a wilms tumor and a teratocarcinoma.

37. The method of claim 28, wherein the cancer is ovarian cancer, breast cancer, colon cancer, head and neck cancer, medulloblastoma, and B-cell lymphoma.

38. The method of claim 28, wherein the cancer is melanoma or non-small cell lung cancer.

39. The method of any one of claims 25-38, wherein the additional anti-cancer agent is selected from the group consisting of everolimus, trabectedin, abraxane, TLK286, AV-299, DN-101, pazopanib, GSK690693, RTA744, on0910.na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatinibNib, nilotinib, dacatinib, panitumumab, amrubicin, ogovazumab, Lep-etu, Loratrex, azd2171, Bartabulin, Aframumab, Zamodumab, Itocarpine, tetrandrine, Lupyriticn, Timelifene, Orimerson, Timmumab, Yiprimma, gossypol, Bio111, 131-I-TM-601, ALT-110, BIO140, CC8490, CerenJigein, Gemasecan, IL13-PE38QQR, INO1001, IPdR₁KRX-0402, thioanthrone, LY317615, neuroadiab, vistelpan, Rta744, Sdx102, talampanel, atrasentan, Xr311, romidepeptide, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, doxorubicin liposomes, 5' -deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib; PD0325901, AZD-6244, capecitabine, L-glutamic acid, N- [4- [2- (2-amino-4, 7-dihydro-4-oxo-1H-pyrrole [2,3-d ]]Pyrimidin-5-yl) ethyl]Benzoyl radical]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen conjugate, bevacizumab, IMC-1C11, CHIR-258; 3- [5- (methylsulfonylpiperidylmethyl) -indole-quinolone, vartanib, AG-013736, AVE-0005, [ D-Ser (but)6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂Acetate salt [ C ]₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂) x, wherein x =1 to 2.4]Goserelin acetate, leuprorelin acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, carratinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-Asparaginase, Bacille (BCG), bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, arabinoside, dacarbazine, actinomycin D, daunomycin, ethylentrol, epirubicin, fludarabine, fludrocortisone, fluoxymesterone, flutamide, gemcitabine, gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, nitrogen mustard, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfipronin, procarbazine, rituximab, streptozocin, teniposide, testosterone, pentostatin, and other drugs, Thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis retinoic acid, melphalan, uramustine, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytarabine, 6-mercaptopurine, desoxymesmycin, 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, cimetidine, trastuzumab, interleukin fusion, gefitinib, bortezomib, paclitaxel, irinotecan, topotecan, doxorubicin, vinorelbine, docetaxel, and paclitaxel (monoclonal antibody) Paclitaxel without polyoxyethylated castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxyttamoxifen, perhexiline, ERA-923, azoxifene, fulvestrant, acobiprofen, lasofoxifene, indoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK 584, VX-745, PD184352, rapamycin, 40-O- (2-hydroxyethyl) -rapamycin, temsirolimus, AP-23573, RAD001, ABT-578, BC-210, LY294002, LY292223, LY292696, LY293684, ZM293646, wortmannin, 637 33LY 2, L-779,450, PEG-filgrastim, tregramicin, and doxorubin,Darbepotin, erythropoietin, granulocyte colony stimulating factor, zoledronic acid, prednisone, cetuximab, granulocyte-macrophage colony stimulating factor, histrelin, pegylated interferon alpha-2 a, pegylated interferon alpha-2 b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab ozolomide, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, all-trans retinoic acid, ketoconazole, interleukin-2, megestrol, immunoglobulin, mechlorethamine, methylprednisolone, temozolomide, androgen, decitabine, hexamethamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, daunorubicin liposome, Edwina-asparaginase enzyme, Strontium 89, casomoitan, netupitan, NK-1 receptor antagonists, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochloraz, granisetron, ondansetron, dolasetron, tropisetron, ssPEG filgrastim, erythropoietin, epoetin α, dabigatran α, yipima, vemurafenib, and mixtures thereof.

40. The method of any one of claims 25-38, wherein the additional anti-cancer agent is 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 inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase (mek) inhibitor, a VEGF trap antibody, or a mixture thereof.

41. A method of treating a condition or disease state in a patient in need thereof, wherein said condition or disease state is advantageously responsive to inhibition of autophagy, comprising administering to said patient an effective amount of a compound according to any one of claims 1-22.

42. The method of claim 41, wherein the condition or disease is rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and xerosis.

43. The method of claim 42, wherein the condition is malaria.

44. Use of a compound of any one of claims 1-22 in the manufacture of a medicament for inhibiting autophagy in a biological system, wherein inhibition of autophagy is desired.

45. Use of a compound according to any one of claims 1-22 in the manufacture of a medicament for the inhibition and treatment of cancer in a patient in need thereof, optionally in combination with at least one additional anti-cancer agent.

46. The use of claim 45, wherein the cancer is metastatic.

47. The use of claim 45, wherein the cancer is drug resistant.

48. Use of a compound according to any one of claims 1-22 in the manufacture of a medicament for reducing the likelihood of the patient developing cancer or developing metastasis of cancer in a patient, optionally in combination with at least one additional anti-cancer agent.

49. The use of claims 45-48, wherein the cancer is cancer, cancer of the esophagus, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, and stomach; leukemia, malignant lymphoma, malignant melanoma; myeloproliferative diseases; sarcoma, central nervous system tumor, germ cell tumor, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, esophageal cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, melanoma, and mixed type tumor.

50. The use of claim 49, wherein the leukemia is acute myelogenous leukemia, acute lymphocytic leukemia, Acute Promyelocytic Leukemia (APL), acute T-cell lymphocytic leukemia, adult T-cell leukemia, basophilic leukemia, eosinophilic leukemia, myelogenous leukemia, hairy cell leukemia, cytopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, megakaryocytic leukemia, small myelogenous leukemia, monocytic leukemia, neutrophilic leukemia, stem cell leukemia.

51. The use of claim 49, wherein the lymphoma is Burkitt's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma.

52. The use of claim 49, wherein the sarcoma is Ewing's sarcoma, angiosarcoma, Kaposi's sarcoma, liposarcoma, myosarcoma, peripheral neuroepithelial tumors, or synovial sarcoma.

53. The use of claim 49, wherein the tumor of the central nervous system is a glioma, astrocytoma, oligodendroglioma, ependymoma, glioblastoma, neuroblastoma, ganglionic cytoma, ganglioglioma, medulloblastoma, pinealocytoma, meningioma, meningosarcoma, neurofibroma, or schwannoma.

54. The use of claim 49, wherein the germ cell tumor is an intestinal cancer, breast cancer, prostate cancer, cervical cancer, or uterine cancer.

55. The use of claim 49, wherein the lung cancer is small cell lung cancer, a mixture of small cell and non-small cell cancers, pleural mesothelioma, metastatic pleural mesothelioma, small cell lung cancer or non-small cell lung cancer.

56. The use of claim 49, wherein the mixed type of tumor is carcinosarcoma and Hodgkin's disease, and the mixed cell of origin tumor is Wilms' tumor and teratocarcinoma.

57. The use of claim 49, wherein the cancer is ovarian cancer, breast cancer, colon cancer, head and neck cancer, medulloblastoma and B-cell lymphoma.

58. The use of claim 49, wherein the cancer is melanoma or non-small cell lung cancer.

59. The use of any one of claims 45 to 59, wherein the additional anti-cancer agent is selected from the group consisting of everolimus, trabectedin, abraxane, TLK286, AV-299, DN-101, pazopanib, GSK690693, RTA744, ON0910.Na, AZD6244 (ARRY-142886), AMN-107, TKI-258, GSK461364, AZD1152, enzastaurin, vandetanib, ARQ-197, MK-0457, MLN8054, PHA-739358, R-763, AT-9263, pemetrexed, erlotinib, dasatinib, nilotinib, dacatinib, panitumumab, amrubicin, ogovalizumab, Lep-etu, Loratrexed, AZD2171, Bartambulin, ofamumab, zakifun, Dudoburin, tetrandrine, Lupyriticon, timiline, Olissenb, Tizerumumab, Yipriima, gossypol, Bio111, 131-I-TM-601, ALT-110, BIO 6390, Zernike peptide, IMEI 8438, IPD R-8438, IPD-L R52, IPD-L-I-D-K3, and its sequence₁KRX-0402, thioanthrone, LY317615, neuroadiab, vistelpan, Rta744, Sdx102, talampanel, atrasentan, Xr311, romidepeptide, ADS-100380, sunitinib, 5-fluorouracil, vorinostat, etoposide, gemcitabine, doxorubicin, irinotecan, doxorubicin liposomes, 5' -deoxy-5-fluorouridine, vincristine, temozolomide, ZK-304709, celecoxib; PD0325901, AZD-6244, capecitabine, L-glutamic acid, N- [4- [2- (2-amino-4, 7-dihydro-4-oxo-1H-pyrrole [2,3-d ]]Pyrimidin-5-yl) ethyl]Benzoyl radical]-, disodium salt, heptahydrate, camptothecin, PEG-labeled irinotecan, tamoxifen, toremifene citrate, anastrozole, exemestane, letrozole, DES (diethylstilbestrol), estradiol, estrogen conjugate, bevacizumab, IMC-1C11, CHIR-258; 3- [5- (methylsulfonylpiperidylmethyl) -indole-quinolone, vartanib, AG-013736, AVE-0005, [ D-Ser (But)6, Azgly10](pyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-Azgly-NH₂Acetate salt [ C ]₅₉H₈₄N₁₈Oi₄-(C₂H₄O₂) x, wherein x =1 to 2.4]Goserelin acetate, leuprorelin acetate, triptorelin pamoate, medroxyprogesterone acetate, hydroxyprogesterone caproate, megestrol acetate, raloxifene, bicalutamide, flutamide, nilutamide, megestrol acetate, CP-724714; TAK-165, HKI-272, erlotinib, lapatinib, kalatinib, ABX-EGF antibody, erbitux, EKB-569, PKI-166, GW-572016, G-B-EGF-B-569, and a pharmaceutically acceptable salt thereof,Ionafarnib, BMS-214662, tipifarnib; amifostine, NVP-LAQ824, suberoylanilide hydroxamic acid, valproic acid, trichostatin A, FK-228, SU11248, sorafenib, KRN951, aminoglutethimide, amsacrine, anagrelide, L-asparaginase, BCG, bleomycin, buserelin, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, cyproterone, arabinoside, dacarbazine, actinomycin D, daunomycin, vinylestrol, epirubicin, fludarabine, fludrocortisone, fluoromethandronate, flutamide, gemcitabine, gleevec, hydroxyurea, idarubicin, ifosfamide, imatinib, leuprolide, levamisole, lomustine, mechlorethamine, melphalan, 6-mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, tolmetin, danamycin, and L-N-D, Nilutamide, octreotide, oxaliplatin, pamidronate, pentostatin, plicamycin, porfimer sodium, procarbazine, raltitrexed, rituximab, streptozocin, teniposide, testosterone, thalidomide, thioguanine, thiotepa, tretinoin, vindesine, 13-cis retinoic acid, melphalan, uramustine, estramustine, altretamine, floxuridine, 5-deoxyuridine, cytarabine, 6-mercaptopurine, desoxyhelpomycin, calcitriol, valrubicin, mithramycin, vinblastine, vinorelbine, topotecan, razin, marimastat, COL-3, neovastat, BMS-275291, squalamine, endostatin, SU5416, SU6668, EMD121974, interleukin-12, IM862, angiostatin, vitaxin, lophanolide, spironolactone, spiroxamine, troxidine, troxine, tioxadine, thioredoxin, troxine, trexadine, trex, Cimetidine, trastuzumab, interleukin fusion toxin, gefitinib, bortezomib, paclitaxel, irinotecan, topotecan, doxorubicin, docetaxel, vinorelbine, bevacizumab (monoclonal antibody) and erbitux, paclitaxel without polyoxyethylene castor oil, epothilone B, BMS-247550, BMS-310705, droloxifene, 4-hydroxytamoxifene, pefoxifene, ERA-923, azoxifene, fulvestrant, acobiprofen, lasofoxifene, indoxifene, TSE-424, HMR-3339, ZK186619, PTK787/ZK222584, VX-745, PD184352, leprosolMycin, 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, darbepotine, erythropoietin, granulocyte colony stimulating factor, zoledronic acid, prednisone, cetuximab, granulocyte-macrophage colony stimulating factor, histrelin, pegylated interferon alpha-2 a, pegylated interferon alpha-2 b, azacitidine, PEG-L-asparaginase, lenalidomide, gemtuzumab ozolomide, hydrocortisone, interleukin-11, dexrazoxane, alemtuzumab, ketoconazole, interleukin-2 b, Megestrol, immunoglobulin, mechlorethamine, methylprednisolone, temab, androgen, decitabine, hexamethamine, bexarotene, tositumomab, arsenic trioxide, cortisone, editronate, mitotane, cyclosporine, daunorubicin liposome, Edwina-asparaginase, strontium 89, casipitan, netupitant, NK-1 receptor antagonist, palonosetron, aprepitant, diphenhydramine, hydroxyzine, metoclopramide, lorazepam, alprazolam, haloperidol, droperidol, dronabinol, dexamethasone, methylprednisolone, prochlorperazine, granisetron, ondansetron, dolasetron, tropisetron, ssPEG filgrastim, erythropoietin, epoetin alpha, darbepotein alpha, prim, vefinamide, and mixtures thereof.

60. The use of any one of claims 45-58, wherein the additional anti-cancer agent is 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 inhibitor, an IGFR-TK inhibitor, an anti-HGF antibody, a PI3 kinase inhibitor, an AKT inhibitor, a JAK/STAT inhibitor, a checkpoint-1 or 2 inhibitor, a focal adhesion kinase inhibitor, a Map kinase (mek) inhibitor, a VEGF trap antibody, or a mixture thereof.

61. Use of a compound according to any one of claims 1-22 in the manufacture of a medicament for treating a condition or disorder in a patient in need thereof, wherein the condition or disorder advantageously responds to inhibition of autophagy.

62. The use of claim 61, wherein the condition or disease is rheumatoid arthritis, malaria, antiphospholipid antibody syndrome, lupus, chronic urticaria and xerosis.

63. The use of claim 62, wherein the condition or disease state is malaria.